ChemTexts (2025) 11:4

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LECTURE TEXT

Systematic toxicological analysis in forensic and clinical laboratories:

a challenging task of analytical chemistry
Fritz Pragst1

Received: 26 September 2024 / Accepted: 9 October 2024 / Published online: 13 February 2025
© The Author(s) 2025

Abstract
This lecture text presents the chemical analysis used for the investigation of a suspected poisoning in clinical and forensic
toxicology. Following the discussion of the huge number and structural diversity of toxic substances, typical indications of
a poisoning are given, which derive from the case history, the site where the unconscious person or the corpse was found,
clinical symptoms, external examination of the corpse, or autopsy results. The different human sample materials, especially
venous blood, urine, and gastric content, used for toxicological analysis are discussed with respect to their advantages,
drawbacks, and special use. The strategy of systematic toxicology, to test for as much as possible poisons with a minimum
of analytical procedures, consists of four main directions: (1) preliminary pretests and specific single or group tests (e.g., for
alcohol, carbon monoxide, cyanide, the classic illicit drugs as well as for insulin, fluoride, and sulfide); (2) screening with
inductively coupled plasma–optical emission spectroscopy (ICP-OES) or inductively coupled plasma–mass spectrometry
(ICP-MS) for toxic metals and metalloids; (3) headspace gas chromatography (GC) in combination with MS in case of
suffocation in a gas atmosphere or toxic action of volatile substances and solvent abuse; (4) sophisticated combinations of
sample preparation, liquid chromatography (LC) with UV-diode array detection and GC or LC with different kinds of MS
for toxic nonvolatile organic substances, i.e., the largest group of poisons including therapeutic and illicit drugs, pesticides
and warfare agents, and plant and mushroom poisons. Explanations of these techniques, the toxic mechanisms, symptoms,
and analytical findings are described on the basis of literature and the personal experience of the author.

Keywords Forensic toxicology · Gas chromatography–mass spectrometry · HPLC–diode array detector · Immunoassay ·
Liquid chromatography–mass spectrometry · Poison · Systematic toxicological analysis

* Fritz Pragst
[email protected]
1
Institute of Legal Medicine, University Hospital Charité,
Turmstraße 21, Haus N, 10559 Berlin, Germany

Vol.:(0123456789)

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4 Page 2 of 95 ChemTexts (2025) 11:4

Info box: Instructions for the reader

Table of contents, page

Introduction 3
Diversity of toxic substances and toxicological relevance 5
Indications from case history, clinical symptoms, or autopsy 6
Case history 6
Site where the unconscious person or the corpse was found 7
Symptoms of the alive patient and indications seen during external postmortem examination 7
Indications for an intoxication obtained during autopsy 8
Human sample materials for toxicological analysis 9
Venous blood 9
Heart blood 9
Urine 10
Vomit, gastric lavage fluid, and gastric content 10
Liver and bile 11
Tissue from lung, kidney, and muscle 11
Bone and bone marrow 11
Brain, cerebrospinal fluid (CSF), and vitreous humor 11
Hair 11
Saliva, oral fluid 12
Sweat and skin surface lipids (sebum) 12
Strategy and performance of systematic toxicological analysis 12
Preliminary tests and specific single or group tests for frequently occurring poisons 13
Alcohol 13
Carbon monoxide, CO-Hb 14
Methemoglobin (Met-Hb)-forming substances 14
Cyanide 16
Ethylene glycol, diethylene glycol 17
Immunoassays for drugs and poisons 18
Special methods for further poisons 20
Toxic metals 21
Analytical methods 21
Arsenic 22
Mercury 24
Thallium 25
Toxic gases and volatile substances 27
Headspace gas chromatographic techniques 27
Suffocation in gas atmosphere 29
Narcotic action of volatile substances and solvent abuse 30
Headspace solid phase microextraction HS-SPME 32
Nonvolatile organic toxic substances 32
History of organic poison detection and Stas–Otto separation procedure 34
High performance liquid chromatography with photodiode array detector (HPLC–DAD) 36
Sample preparation for HPLC–DAD 37
Separation by high performance liquid chromatography 39
Photodiode array detector 41
UV spectrum and substance identification 42
Metabolites in HPLC–DAD 43
Quantification with HPLC–DAD 43
Example of application 45
Gas chromatography–mass spectrometry (GC–MS) 45
Historical role of gas chromatography in toxicological screening 45
Toxicological screening by GC–MS 47
Sample preparation for GC–MS 48
Example of application 51
Specific, sensitive, and accurate analysis by selected ion monitoring (GC–MS-SIM) 51
Liquid chromatography–mass spectrometry (LC–MS) 51
General features of LC–MS 51
Liquid chromatography with triple-quadrupole mass spectrometry, LC–QQQ or LC–MS/MS 54
Hybrid quadrupole time-of-flight mass spectrometry (LC–QTOF-MS) 57
Identification of metabolites and semiquantitative determination of concentrations by LC–QTOF-MS 61
Example of application 62
Poisonings with nonvolatile organic compounds 63
Illicit drug abuse and fatalities 64
Suicide, murder, drug-facilitated crimes, and accidental poisoning by medical drugs 68
Acute and lethal poisonings by pesticides 71
Poisons from toxic plants 73
Mushroom poisons 75
Interpretation of the results: the toxicological expertise 76
General aspects 76
Pharmacokinetic/toxicokinetic considerations 77
Postmortem changes of substance concentration 77
Comparison between different samples and consideration of metabolites 78
Pharmacodynamic/toxicodynamic considerations 78

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ChemTexts (2025) 11:4 Page 3 of 95 4

Introduction of decreased criminal responsibility under the influence of

alcohol or drugs. In this way, toxicological analysis contrib-
Acute poisoning is a serious threat for health and life. It can utes to get a profound picture of the actual case and to allow
occur accidentally, self-inflicted with recreational or suicidal a fair judgement.
intention, or be inflicted by someone else for criminal pur- Poisons were known and misused in prehistory, ancient
poses such as murder, sexual assault, or robbery. Therefore, times, the middle ages, and in the modern era. The history
a suspected intoxication must be clarified as soon as pos- of murders by poisoning was repeatedly reviewed in detail
sible and, if necessary, medically treated. The statistics of [3–7]. At first, evidence of poisoning was possible only
the eight German poison information centers (GIZs) show from the symptoms of the victim or from residues of the
that poisoning is not a seldom incident. For instance, the poison in their environment. The first medicolegal autopsy
center “GIZ-Nord” for the German federal states Bremen, was performed in Bologna in 1302 [8], and the beginning
Hamburg, Niedersachsen, and Schleswig–Holstein (together of toxicology as a science is dated at 1538 when Para-
13.5 million inhabitants) reported 42,375 suspected human celsus (1493–1541) defined the term “poison” [9]. Analyti-
cases of poisoning in 2020 [1]. This concerns mainly acci- cal methods for detection of poisons were first assembled
dental intake at home of pharmaceuticals, household chemi- and published in textbooks at the beginning of the nine-
cals and plants, but also pesticides and mushrooms. In most teenth century in Germany by Victor Heinrich Leberecht
of these cases, the harmful material is known and needs only Paldamus (1777–1814) [10, 11], Peter Joseph Schneider
the analytical confirmation and quantification. However, by (1791–1871) [12], in France by Mateo José Bonaventura
far not all poisoning cases are included in these statistics. Orfila (1787–1853) [13], and in England by Samuel Farr
Ambiguous symptoms of a patient are often misdiagnosed as (1741–1795) [14]. Already at that time, forensic toxicology
another illness. Particularly death cases by accident, suicide, and toxicological analysis were closely linked together, and
or even homicide are not registered by the GIZs. Among the progress in clearing up of poisonings was determined by
them are also the deaths by illicit drugs, 1826 men or women the progress in chemistry. The development of methods was
in Germany in 2021 [2]. often achieved in the context of spectacular poisoning mur-
In many cases, the reason for an illness or cause of death der cases. This was particularly obvious for arsenic which
is not clear. The comprehensive diagnostic investigations was the most infamous poison in the middle ages (see also
did not lead to a reasonable explanation of the symptoms Sect. “Arsenic”). The formation of the garlic-like smelling
of a patient. The situation may be even more complicated ­AsH3 discovered by Swedish chemist Carl Wilhelm Scheele
in unexpected lethal cases when the circumstances of the (1742–1786) in 1775 and the precipitation of the yellow
death are unknown and the autopsy of the deceased did ­As2S3 discovered by the German physician and pharmacist
not show a cause of death. The person should be alive and Samuel Hahnemann (1755–1843) in 1786 were already
healthy. In such cases, an intoxication must be excluded or known; however, the real breakthrough was the test devel-
must be proven by qualitative and quantitative determination oped by the English chemist James Marsh (1794–1846) in
of the poison. In view of the large number of possible toxic 1836 [15].
substances, this requires a skilled analytical approach. The Whereas inorganic poisons to a certain degree could be
terms systematic toxicological analysis and general unknown detected, there was no method for plant poisons. Autop-
analysis mean the detection or exclusion of an intoxication sies were performed at that time, but the search for poisons
without a specific suspected poison by analysis of body flu- was restricted to the contents of the gastrointestinal tract
ids or tissue samples. Only the detection of the poison in where residues of toxic plants could only seldom be identi-
human sample material (blood, urine, tissues) is sufficient fied because of decomposition by digestion. The forensic
to prove harmful effect. evidence was based only on the thorough investigation of
However, a toxicological investigation in lethal cases is death circumstances and witness reports. This situation
not only required for determination of the cause of death but changed in 1850 when the Belgian chemist Jean Servais
also in order to clarify the circumstances if another reason Stas (1813–1891) developed a method to separate nicotine
for death was ascertained. This concerns, for instance, a pos- from human remains in the famous Bocarmé murder case
sible physical or psychic impairment by drugs or poisons of [16] (see also Sect. “History of organic poison detection and
victims of accidents in traffic, at work, or at home, of drown- Stas–Otto separation procedure”).
ing victims, and in cases of suicide by hanging, shooting, or Further pioneers in toxicological analysis were the Ger-
a fall from height. In murder cases other than poisoning, it is man chemist Carl Remigius Fresenius (1818–1897) who,
important to know whether the victim was disabled by toxic among analytical contributions, characterized the position
effects and unable to fight back. In the same way, the toxico- of the forensic chemist and demanded standard methods
logical investigation of a perpetrator can clear the possibility in toxicological analyses [17], the Berlin pharmacologist

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4 Page 4 of 95 ChemTexts (2025) 11:4

Louis Lewin (1850–1929) who was famous for his lectures Diversity of toxic substances
in toxicology and contributed to various fields of forensic and toxicological relevance
toxicology in textbooks and more than 300 papers [18, 19],
the Swedish scientist Erik Widmark (1889–1945) who Poisons are chemical substances which can lead to tempo-
invented the first method for the determination of alcohol rary or permanent harm or to the death of a living organism
in blood in 1932 and developed the formula for calculation by its toxic action. In this paper, the living organism shall be
and back-calculation of blood alcohol concentrations [20], limited to human beings. The famous physician, alchemist,
and the Austrian chemist Gottfried Machata (1925–2012) and natural philosopher Paracelsus (Theophrastus Bombas-
who invented the headspace gas chromatographic method tus von Hohenheim, 1493–1541, Fig. 3) was the first who
for alcohol determination in 1962 [21]. came to the conclusion: “All things are poison, and nothing
The second half of the twentieth century and the two first is without poison; the dosage alone makes it so a thing is not
decades of the twenty-first century were characterized on a poison” [9]. Besides the dose, the biochemical mechanism
the one hand by enormous development of instrumental of action, the conditions of application (e.g., oral intake,
analytical methods, in particular different chromatographic inhalation, or injection), and the individual properties of the
techniques, molecular spectroscopy, and computerization, living organism are important for the real toxic effect.
and on the other hand by an exponential increase of possible According to Chemical Abstracts Service in April 2023,
poisons, especially synthetic therapeutic and illicit drugs, there were 204 million organic substances, alloys, coordina-
pesticides, and household chemicals. This was accompanied tion compounds, minerals, mixtures, polymers, and salts as
by a tremendous gain in knowledge of all fields of toxicol- well as about 70 million protein and nucleic acid sequences
ogy. From the toxicological point of view, this development disclosed in publications since the early 1800s [23]. How-
is determined by a race between substance abuse and toxi- ever, the vast majority were prepared, isolated, or identified
cological analysis under the pressure of scientific-technical only for research purposes. Only about 100,000 chemical
progress (Fig. 1) [22]. substances are in practical use. Therefore, although in prin-
In this lecture text, the difficulties and the methodical ciple all things can be poisonous, only a limited but neverthe-
strategies of the systematic toxicological analysis and its less large number of substances have a real chance of acting
application to real cases shall be described. Figure 2 pro- as a poison. The toxicological relevance of a compound, i.e.,
vides an overview of the essential information sources, the probability of occurring in an acute human intoxication,
investigations, and methods that are used for this purpose depends on its toxicity and its availability. Furthermore, the
and dealt with in the following sections. level of awareness as a poison in the general population plays
an essential role.
The ­LD50, a measure of the acute toxicity, is the lethal
dose which causes the death of 50% of a group of test

Fig. 1  Race between forensic

toxicology and substance abuse
under the pressure of scientific
technical progress, modified
from [22]

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ChemTexts (2025) 11:4 Page 5 of 95 4

Fig. 2  Flowchart of the essential

information sources, investiga-
tions, and methods of the sys-
tematic toxicological analysis in
a suspected poisoning case

a large difference between the effects on test animals and

humans.
Regarding accessibility, these toxic substances belong
mainly to the following groups:

– Illicit drugs. Besides the traditional drugs heroin,

cocaine, amphetamine, ecstasy, and cannabinoids, a
large and steadily increasing number of so-called new
psychoactive substances (NPS) appeared on the illegal
drug market. Between 2005 and 2022, the European
Monitoring Centre for Drugs and Drug Addiction moni-
Fig. 3  Paracelsus (1493–1541)—Portrait of Peter Paul Rubens, Royal
tored around 930 NPS, 41 of which were first reported
Museum of Fine Arts, Brussels, Photo F. Pragst 2018 in Europe in 2022 [25]. This includes mainly synthetic
cannabinoids but also cathinones, phenylethylamines,
piperazines, benzodiazepines, tryptamines, opioids, and
animals (mainly rats) after a single application. According ketamines.
to the Hodge and Sterner scale [24], there are four catego- – Therapeutic drugs. The 9th edition of the handbook
ries, which are in the case of oral application ­(LD50 oral) Organic-Chemical Drugs and Their Synonyms by Neg-
related to body weight: extremely toxic (≤ 1 mg/kg), highly wer and Scharnow comprises about 20,000 pharmaco-
toxic (1–50 mg/kg), moderately toxic (50–500 mg/kg), and logically active compounds which were or are used in
slightly toxic (500–5000 mg/kg). For dermal application medications [26]. Up-to-date comprehensive informa-
through the skin or for inhalation, the degrees of toxicity tion about all medical drugs can be obtained from the
are characterized by corresponding ranges of L ­ D50 dermal or database Pharmazeutische Stoffliste [27]. As a rule, these
of the concentration in air ­LC50 inhaled over a defined period drugs are applied in the milligram range and have toxic
of time. From the viewpoint of toxicity, mainly extremely, or even lethal effects on overdose. Although many of
highly, and moderately toxic substances are relevant candi- these drugs are obsolete, they may still be used in some
dates for acute human poisonings. However, there may be countries or present in homes.

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4 Page 6 of 95 ChemTexts (2025) 11:4

– Plant and animal poisons: Biogenic toxins are a very psychical disorders, thoughts of suicide as well as inspec-
large and heterogeneous group of poisonous substances tion of the medical record and information about health state
[28, 29]. Daily used or abused compounds such as caf- and regularly prescribed medicines can substantiate the pos-
feine and nicotine as well as illegal drugs such as cocaine sibility of a poisoning and can give indications of possible
and psilocybin and important therapeutic drugs such as poisons.
morphine and atropine belong to the different classes of
alkaloids. Examples of plant toxins frequently occur- Site where the unconscious person or the corpse
ring in poisonings are digitoxin (foxglove), aconitine was found
(monkshood), taxines (yew), amanitin (death cap), or
ricin (castor oil plant). Botulinus toxin (Botox), a pro- A thorough inspection of the place where the unconscious
tein of 1296 amino acids, is produced by the bacteria person or the corpse was found can support the suspicion
Clostridium botulinum and is believed to be the most of an intoxication. Some typical situations are shown in
toxic substance known. Examples of animal poisons are Fig. 4a–d. Residues of illegal drugs and drug utensils such
cantharidin (Spanish fly), bufotenine (bufo toad), or the as syringes, spoons blackened below from soot, cocaine sniff
proteinic toxins of bees, scorpions, and rattle snakes pipe, or crack smoking pipes suggest poisoning by drug
– Pesticides: The EU Pesticides Database contains about overdose. Empty medical drug blisters and drug packages,
1500 substances used as insecticides, fungicides, acari- tablets scattered around, and cups or glasses with adhering
cides, nematicides, or herbicides [30]. Particularly the material indicate corresponding poisoning. In these cases,
acetylcholine esterase inhibitors containing phosphoric as well as for flasks of pesticides or any chemicals, resi-
acid esters and carbamates occur frequently in intoxica- dues should be taken as evidence for analytical confirma-
tions. By far not all substances are still approved, but tion. Unusual odor of burning, smoke, town gas, solvents,
residues from previous use are still found in farms and or vomit can suggest an intoxication and must be recorded.
gardens. A farewell letter in a case without a morphologically com-
– Chemicals, chemical reagents: Many inorganic or prehensible cause of death (e.g., hanging, shooting, fall from
organic substances used in industry or the laboratory are the height) is a strong indication for a poisoning. A seldom
highly toxic. Predominant examples are compounds of example, in which the farewell letter was written during the
toxic metals such as arsenic, thallium, or mercury, alkali beginning and progressing action of the drug is shown in
cyanides, toxic gases such as carbon monoxide, hydrogen Fig. 4d.
sulfide, and phosgene.
Symptoms of the alive patient and indications seen
The accessibility of poisons depends essentially on during external postmortem examination
the education, profession, and place of employment. For
instance, a pharmacist or a chemist has easy access to a wide Poisons can disturb life processes in very different ways.
variety of poisons whereas a white-collar worker usually has In line with this, the symptoms of an intoxication are very
only contact with household chemicals and a limited number diverse, depending on the poison. In most cases, the same
of medicines. It is obvious that, in view of this large and symptoms can also arise from other illnesses, and the intoxi-
heterogeneous variety of possible poisons, any information cation is only a differential diagnosis. Frequent signs of an
about the circumstances of the illness or the death of the acute poisoning are nausea, vomiting, dizziness, confusion,
affected person can simplify the analytical process. There- disorientation, disturbances of equilibrium, very low or very
fore, close cooperation of the toxicological analyst with the high blood pressure and heart rate (bradycardia or tachycar-
attending physician in case of living victims, the forensic dia), breathlessness, low breathing frequency, unconscious-
pathologist in death cases, and the criminal investigators of ness, and coma.
the police is necessary. Abnormal clinical laboratory parameters may also point
to an intoxication. A metabolic acidosis (blood pH < 7.35,
high anion gap > 20 mmol/L) can be caused by a poison-
Indications from case history, clinical ing with methanol, ethylene glycol, or acetylsalicylic acid.
symptoms, or autopsy Low potassium concentrations in serum (hypokalemia
­K+ < 2 mmol/L, normal 3.5–5.1 mmol/L) is typical for a
Case history barium poisoning. The reason for decreased coagulation and
high bleeding tendency can be a poisoning by anticoagulants
Interrogation of the victim (if alive) and of the relatives such as warfarin and superwarfarins.
about unusual events of the past few days, their profession A strongly deviating size of the pupils can be an indi-
and occupation concerning access to poisons, diseases and cation of a poison with effect on the nervous system. For

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ChemTexts (2025) 11:4 Page 7 of 95 4

Fig. 4  Indications of an
intoxication from the site where
the unconscious person or the
corpse was found. a Typical
utensils for preparation of a
heroin concoction with heroin
powder, ascorbic acid, spoon
with cotton ball for filtration,
lighter, knife (spatula), and
syringe in a drug fatality. b Tab-
let blisters and spilled red wine
in a suicide case. c Tray with
utensils in a chloroquine suicide
case. d Farewell letter written
at the beginning of agony
after a lethal diphenhydramine
overdose

instant, extremely dilated pupils are observed for poisonings Examples of other indications seen during external post-
with atropine, cocaine, amphetamines, and botulinus toxin, mortem examination are shown in Fig. 5a–d. The color of
but also by cyanide and methanol, whereas extremely nar- livor mortis is normally gray-violet, but in case of a carbon
row pupils occur in poisonings with nicotine, opiates, and monoxide intoxication with a CO-Hb concentration above
phosphoric ester pesticides. At the same time, the pupil light 30–40% it becomes bright red (Fig. 5a). However, red livor
reflex is sluggish. Abnormal pupil size and sluggish pupil mortis occurs also in cyanide poisonings and death from
light reflex lead to the suspicion of drug use in roadside traf- cold. Careful examination for punction marks is a regular
fic control. In death cases, poison-induced abnormal pupils part of the external postmortem examination and gives evi-
are detectable at most until the end of rigor mortis. dence for drug abuse by injection (Fig. 5b). They were also

Fig. 5  Indications of a poi-

soning seen during external
postmortem examination. a
Bright-red livor mortis in a case
of a lethal carbon monoxide
intoxication. b Punction marks
after chronic drug injection in a
heroin fatality. c Foam from the
mouth can be caused by a toxic
lung edema. d “Holzer blisters”
formed during a longer agony
phase after drug overdose

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4 Page 8 of 95 ChemTexts (2025) 11:4

reported at hidden sites in murder cases [31]. Foam at the acidic substances. High doses of KCN can lead to a basic
mouth (Fig. 5c), sometimes also bloody, points to a pulmo- pH. Foaming can be caused by ingestion of surfactants.
nary edema which can be caused by aggressive gases and Drinking of formaldehyde solution leads to necrosis and
volatile substances such as ­Cl2, NO/NO2, ­SO2, ­O3, or ­COCl2, hardening of the gastric mucosa. A case of heavy erosion,
but also by chemical weapons such as yperite and lewisite. the gastric mucosa after intake of concentrated H ­ NO3 with
Alternatively, a pulmonary edema can also be caused by a an uncertain amount of ­C u 2+, is shown in Fig. 6b. The
severe left ventricular failure which has mainly natural rea- yellow and greenish coloration results from the reaction
sons but can also arise from cardiotoxic substances. “Holzer of ­HNO3 and C ­ u2+ with proteins of the mucosa (xantho-
blisters” (Fig. 5d), named after the forensic pathologist Franz proteic test and biuret test). The cause of death was not the
Josef Holzer who first described them, are formed by pres- injury of the stomach but the strong acidosis and forma-
sure of the knees against each other during a longer motion- tion of met-Hb due to the acidic and oxidative action of
less phase of agony before death, which is typical for oral ­HNO3. Unusual color of the intestines points to the intake
overdose of sleeping drugs, e.g., barbiturates. of dye stuffs. In the suicide case in Fig. 6c, it was caused
by indigo carmine which was added to the ingested benzo-
Indications for an intoxication obtained diazepine tablets (Rohypnol®, flunitrazepam) as a warning
during autopsy dye because of its frequent misuse as a means of knockout
in beverages for sexual assaults.
Autopsies are mainly performed in order to determine the cause Specific evidence for intoxications from other parts of the
of death. There are specific and nonspecific indications of an corpse are seldom. Figure 6d shows the preparation of the
intoxication seen at the autopsy. Specific indications appear forearm in order to detect punction marks by the correspond-
mostly from the inspection of the gastrointestinal tract. Some ing hemorrhages.
examples are shown in Fig. 6a–c. On opening the stomach, the Nonspecific indications can have other reasons beside an
medical examiner pays attention to unusual odor of the gastric intoxication. These are symptoms of a central death such
content such as bitter almond (HCN), garlic-like (phosphoric as blood stasis of the internal organs, liquid, non-coagu-
­ H3 from rat poisons AlP or Z
esters or P ­ n3P2), or aromatic lated blood, and cerebral edema. A large urine volume in
(organic solvents). The gastric and intestinal contents are care- the bladder points to a longer survival time in unconscious
fully examined for tablets. Numerous capsules with cocaine state before death. If other reasons for these findings can be
were found in cases of body packing for illegal transport [32]. excluded, an intoxication appears to be probable.
Residues of tablets (filler, binding material) may adhere Indications from the site, symptoms, and external and
on the gastric mucosa (Fig. 6a). Checking the pH of the internal postmortem investigation results are not sufficient
gastric content can point to ingestion of strong basic or for proving an intoxication in the forensic context. The

Fig. 6  Indications of an intoxi-

cation seen during autopsy. a
Residues of tablets adhering to
the gastric mucosa. b Erosion
and coloration of the gastric
mucosa after drinking concen-
trated nitric acid with dissolved
copper. c Coloration of the
intestines by indigo carmine, the
warning dyestuff in Rohypnol®
tablets, in a suicide case with
flunitrazepam. d Hemorrhages
from punction marks at the pre-
pared forearm of a drug fatality

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ChemTexts (2025) 11:4 Page 9 of 95 4

poison must unambiguously be identified and quantified by (exception CO-Hb, see below). The sampling occurs usually
chemical analysis. at the veins of the arm for living individuals and at the vena
femoralis (vein of the upper thigh) during autopsy.
Whole blood consists of blood cells (42–47%) and blood
Human sample materials for toxicological plasma (53–58%) which can be separated by centrifugation
analysis (Fig. 7b). Plasma contains fibrinogen which is precipitated
by coagulation leading to blood serum. Blood coagulation
A list of human sample materials for toxicological analysis can be avoided by addition of EDTA, citrate, or heparin.
from living individuals and from deceased is given in Table 1.
Whole blood = Plasma + blood cells
Figure 7a shows a typical set of samples routinely retained dur-
ing a forensic autopsy. After analysis, the samples are stored at
− 20 °C for at least 1 year or until the case is closed by the pros- Plasma = Serum + fibrinogen
ecutor. Different materials enable complementary information. Because of different water content and different binding
Therefore, the investigation of different materials can be used to to proteins in blood cells and plasma, the concentration of
obtain more evidence about the state, the time course, and the a drug may be different in whole blood and in plasma (≈
circumstances of the intoxication. serum). For instance, the plasma (serum)/whole blood con-
centration ratio is 1.23 for alcohol and 1.4–1.6 for the can-
Venous blood nabinoid THC. This difference can play a role in forensic
interpretation by use of legal limits.
From all materials, venous blood is the most important sam-
ple for the following reasons: (1) It is available in almost
all vital and postmortem cases. (2) It is most characteristic Heart blood
for the situation at the time of sampling in vital cases or
at the time of death in fatal cases since it is in equilibrium Heart blood is sufficiently available during autopsy. It is
with all essential organs and tissues until collection or death. used if no or not enough venous blood is available. However,
(3) Concerning interpretation, there are the most data avail- the concentrations of a drug in heart blood are sometimes
able from living and death cases for comparison. Therefore, much higher than in venous blood. There are different expla-
if ever possible, the analysis should include venous blood nations: (1) Agonal aspiration of vomited gastric content

Table 1  Human materials for Materials from living individuals Postmortem materials
toxicological analysis from
living and deceased individuals Venous blood Venous blood Brain, cerebrospinal fluid
Urine Heart blood Vitreous humor
Vomit/gastric lavage fluid Urine Hair
Saliva Gastric content Bone, bone marrow
Sweat, sebum Intestinal content
Hair Liver, bile
Meconium kidney, lung, muscle

Fig. 7  Materials collected for

toxicological analysis. a Typical
set of samples routinely retained
during a forensic autopsy. b
EDTA-blood sample collected
in a survived poisoning case

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4 Page 10 of 95 ChemTexts (2025) 11:4

with very high concentration and transport from the lung effect) and on the pH of the urine. Particularly the active
to the heart through the large blood vessels shortly before parent compounds are often not found in urine because
death. (2) Death during infusion or intravenous injection of of their higher lipophilicity. Besides, results from urine
the drug and no further distribution to the peripheral circu- represent the time period after the previous miction and
lation. (3) Postmortem distribution from the stomach and cannot describe the state at the time of collection or death.
surrounding tissues to the blood. As an example, in a case of Metabolites in urine: There are two kinds of metabo-
suffocation with a plastic bag over the head a short time after lism (biotransformation) of xenobiotics (foreign sub-
oral intake of a drug mixture, the concentration of codeine stances): Phase I metabolism includes reactions of the
was only 0.18 µg/mL in venous blood but 7.5 µg/mL in heart parent molecule such as hydroxylation, dealkylation,
blood. The lower value of 0.18 µg/mL is more realistic for deamination, N-oxidation, oxidation of hydroxy groups
the toxic effect, since the drug was not yet distributed from to carbonyl groups, or reduction of nitro groups to amino
the heart to the brain and other organs at the time of death. groups. Phase II metabolism (conjugation) is the binding
On the other hand, heart blood should preferentially be of glucuronic acid, sulfate, and other species to hydroxy
used for determination of CO-Hb because of slow loss of CO or amino groups of the parent molecule or of phase I
from peripheral blood in fresh air and false positive findings metabolites to form glucuronides or sulfates. As a rule,
in peripheral blood after longer presence of the corpse in a metabolism increases the hydrophilicity and facilitates
CO-containing atmosphere. the excretion by the kidneys. Glucuronides and sulfates
Stability of drugs in blood: Blood still has enzymatic often complicate the analysis in urine and, therefore, are
activity after sample collection or death. Particularly hydro- hydrolyzed by glucuronidase/sulfatase before extraction
lases and hydrogenases are active for longer. Therefore, e.g., or other tests.
the hydrolysis of heroin to the metabolites 6-acetylmorphine
and morphine, and of cocaine to benzoylecgonine, continues
after sampling. Furthermore, some microbiological action Vomit, gastric lavage fluid, and gastric content
on drugs is also possible, particularly in postmortem cases.
This can lead to a wrong interpretation. These problems can In clinical cases, vomit or fluid from gastric lavage (removal
be minimized by addition of 2% NaF to the sample, with the of poisons from the stomach) is sometimes available. In
disadvantage of hemolysis (degradation of erythrocytes) and forensic lethal cases, the volume of the gastric content is
that plasma or serum are not available thereafter. regularly determined and it is examined for tablets and
residues of the last meal. An aliquot is collected for toxi-
Urine cological analysis. In toxicological investigation of clinical
emergency cases, these materials are only analyzed if results
Urine is secreted from blood by the kidneys and collected from blood or urine are insufficient whereas in forensic death
in the bladder between the mictions. As a result of the func- cases the analysis of gastric content is a regular part of the
tional mechanism of the kidneys, hydrophilic substances and analytical investigation.
metabolites are strongly accumulated in comparison to blood Generally, the detection of high concentrations of a toxic
whereas lipophilic substances are retained in blood. There- substance in the stomach proves its oral intake. However,
fore, the more hydrophilic metabolites dominate mostly over small concentrations of basic substances can also be secreted
the parent substances in urine. Urine consists of more than into the stomach together with the gastric acid after par-
99% water with a pH of 4.8–7.6 depending on alimentation. enteral application. For instance, the intravenous injection
From analytical point of view, the advantages of urine are of heroin leads to a low positive result of morphine in the
the presence of only few endogenous substances, high optical gastric content. Furthermore, the concentration in gastric
transparency for optical tests, no enzymatic activity (higher content cannot be interpreted concerning the degree of the
stability of some analytes), higher concentration of metabolites intoxication, since the substance was not yet absorbed into
than in blood, and extended detection time window. Therefore, the systemic circulation. On the other hand, the concen-
urine can often be analyzed without any sample preparation. tration in comparison to blood can qualitatively be inter-
Detection of a substance or its metabolites in urine proves its preted with respect to the survival time after intake of the
passage through the body, which means uptake, consumption, substance.
or administration of the substance. An analytical advantage of the gastric content is the high
The main disadvantage is the impossibility of the inter- concentration after oral application which enables an easier
pretation of determined concentrations with respect to the detection of the poison. It is also possible to identify the
degree of intoxication since it depends, among others, unchanged substance because of the absence of metabolites.
strongly on the volume of drunken beverages (dilution

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ChemTexts (2025) 11:4 Page 11 of 95 4

Liver and bile barrier which is formed by endothelial cells of the blood
capillary walls and restricts the passage of hydrophilic com-
Liver tissue is the most frequently analyzed postmortem pounds into the brain. However, lipophilic drugs and poi-
sample material in addition to blood. Therefore, liver con- sons can easily diffuse through the membranes and enter the
centrations are described in literature for many substances. central nervous system. Some authors believe brain concen-
As a result of protein binding, these concentrations are fre- trations to be most suitable for toxicological interpretation
quently much higher (up to tenfold) as compared to blood. since poisons frequently act on the CNS [37]. It is frequently
Unlike other tissues, the liver retains essential enzyme activ- analyzed and brain concentrations of lethal poisoning cases
ity after death which can lead to a decrease of parent drug are often described.
in favor of metabolites. For instance, in a suicide with the Cerebrospinal fluid is a clear, colorless body fluid that
cholinesterase inhibitor propoxur (2-isopropoxyphenyl- surrounds the brain and spinal cord. It is equally separated
N-methylcarbamate), the concentrations of the parent com- from blood by the blood–brain barrier. It is produced at a
pound and its main metabolite 2-isopropoxyphenol were 4.3 rate 25–40 mL/h by the choroid plexus in the ventricles of
and 2.8 µg/mL in venous blood and < 0.3 and 56 µg/g in the brain and has a total volume of 100–160 mL. It has a
liver tissue [33]. Besides the more difficult interpretation, the low protein level (0.3–0.6% of that of blood) and very low
sample preparation is more laborious because of the disturb- enzyme activity. For diagnostic purposes in patients, it is
ing high content of lipids and endogenous compounds, as is collected by lumbar punction (lumbar CSF) but seldom
generally the case for tissue samples. used for toxicological analysis. In lethal cases, it is obtained
Bile (400–800 mL/day) is secreted from the liver into the during opening of the skull (cranial CSF) during autopsy.
gallbladder. In case of a poisoning, bile contains enriched Analytical advantages are its high optical transparency and
concentrations of substances with a medium molecular low analytical interference by endogenous substances. Drug
weight, and particularly glucuronides. In the past, bile was concentrations in CSF correspond in equilibrium to the free
often included in the analysis. However, the sample prepa- fraction (not bound to proteins) in plasma and, therefore, are
ration is significantly affected by the high content of sur- for most drugs much lower than in blood. The time course
factants and it is only seldom analyzed today. of concentrations is delayed as compared to blood. Use of
CSF is recommended if venous blood is not available [38].
Tissue from lung, kidney, and muscle A special use is the investigation of fatal complications in
spinal or peridural anesthesia [39]. The determination of
Samples from the lungs, the kidneys, or the muscle are only the concentration sum of glucose and lactic acid in CSF and
included in special cases. Lung tissue can contain extremely in vitreous humor for postmortem diagnosis of diabetes is
high poison concentrations since it is the entry site of vola- common practice [40, 41]. A hyperglycemic coma can be
tile toxic substances by inhalation or smoking and the first assumed if glucose + lactate in CSF is > 415 mg/dL and in
capillary system after intravenous injection. For instance, vitreous humor is > 410 mg/dL.
in case of fire victims, it can be analyzed for detection of The vitreous of the eye is a network of collagen fibrils
volatile fire accelerants in arson [34]. Furthermore, high and hyaluric acids and consists of 98–99% H ­ 2O, the vitre-
concentrations may originate from agonal aspiration of ous humor. Total volume is approximately 4 mL. It is, like
vomited gastric content. Particularly for exhumed corpses CSF, separated from the blood circulation by the blood–reti-
after extended times in an earth grave (up to several years), nal barrier, has a protein level of about 0.04%, low enzyme
residues from kidneys, muscles, or from liver are often still activity, and high optical transparency. It can be easily col-
available. lected during autopsy by punction and has the same use for
forensic analysis as CSF including detection of hyperglyce-
Bone and bone marrow mic coma.

The analysis of bone and bone marrow is important in case Hair

of skeletonized corpses if no other sample materials are left
[35, 36]. Bone marrow of skeletonized corpses has been used Hair is the most rapidly growing human tissue with a growth
for drug detection to prove a possible intoxication. Bone can rate of 0.6–1.4 cm/month (on average 1.1 cm/month). It is
be investigated for chronic exposure to toxic metals. formed by steady mitosis from matrix cells within the hair
follicle. This is surrounded by a rich blood capillary system
Brain, cerebrospinal fluid (CSF), and vitreous humor that provides the growing hair with necessary metabolic
material. Keratinization in the middle part of the root leads
Brain and spinal cord (central nervous system, CNS) are to its solid and durable nature. These very complex pro-
separated from the blood circulation by the blood–brain cesses are described in detail in a previous paper [42]. In an

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4 Page 12 of 95 ChemTexts (2025) 11:4

episode of poisoning, the toxic substance (e.g., a drug) is drugs and metabolites are preferentially excreted with the
durably incorporated from blood into the hair matrix during sweat. Apocrine sweat glands are essentially located in the
this growing and hardening process. The resulting poison- armpits and the genital region. They continuously secrete
positive zone moves steadily away from the skin surface a concentrated lipid-rich sweat which comprises proteins,
as the hair grows. From this point of view, the hair can be sugars, and ammonia.
regarded as a recorder leading to a timetable of exposure to Analytical tests are performed with eccrine sweat. For
drugs and poisons. Depending on the length of the hair, their this purpose, special tamper-resistant sweat patches were
detection is possible up to several years after intake. developed which can be worn for up to 7 days [45]. They
On the basis of the long-term storage of foreign sub- are used for, e.g., drug abstinence control and enable only
stances, hair is used for the detection of chronic and ret- qualitative results.
rospective exposure to drugs or poisons. Segmental analy- Sebum is produced by sebaceous glands, which open into
sis (e.g., in 1-cm segments) enables in addition to that an the upper part of each hair root. Sebum consists mainly of
estimation of the exposure time. However, these advantages triglycerides, wax esters, cholesterol esters, cholesterol, and
are limited by a possible external incorporation into the free fatty acids. Lipophilic drugs and poisons are preferen-
completed hair shaft, by the variation of the growth rate, tially excreted with sebum. Special Sebutapes were devel-
and by the presence of 5–20% telogen (not growing or dor- oped for collection of sebum [46]. However, a mixture of
mant) hair. Despite these limitations, hair testing for illicit sweat and sebum is always collected with sweat patches as
and medical drugs and poisons has been developed into an well as Sebutapes [47]. For detection of drugs on the skin
important branch of toxicological analysis in vital as well as surface, immunoassay-based “Drugwipes” were developed
postmortem cases. which collect the sample by wiping, for instance, the fore-
head of the person [48].
Saliva, oral fluid

The salivary glands produce in unstimulated state about Strategy and performance of systematic
0.32 mL/min and altogether 1.0–1.5 L/day of saliva. It con- toxicological analysis
sists of 98–99% ­H2O and 0.5–1.5% dissolved substances
(electrolytes, amylase) and has a pH of 6.0–6.5. The con- Indications from the case history, the site of the incident, the
centration of drugs in saliva corresponds, in the same way symptoms of a patient, or external and internal postmortem
as for CSF and vitreous humor, to the free (not protein- investigation as shown in Sect. “Indications from case his-
bound) fraction in blood and is much lower than in blood or tory, clinical symptoms, or autopsy” are not sufficient for
urine. Since it can be mixed with residues from food, drinks, proving an intoxication in the forensic context. The poison
smoke, and oral or nasal drug intake, the sample collected must unambiguously be identified and quantified by chemi-
from the oral cavity is called oral fluid. With increasing sen- cal analysis. The systematic search for the poison is very
sitivity of the analytical methods in the last few decades, oral difficult and requires a special strategy for the following
fluid has become the most promising material for road-site reasons:
and workplace drug testing [43, 44]. Unlike urine, oral fluid
can be collected under supervision by non-medical person- – There are a large number of possible toxic substances as
nel in a non-intimate environment. For this purpose, special shown in Sect. “Diversity of toxic substances and toxi-
saliva collection and on-site test systems were developed cological relevance”.
which deliver a rapid immunoassay result and collect suf- – These substances exhibit very different analytical prop-
ficient material for subsequent confirmation analysis by mass erties and there is no general structural feature that dis-
spectrometric methods. criminates highly toxic from less or nontoxic substances.
– Because of the different toxicity, the substances are pre-
Sweat and skin surface lipids (sebum) sent in very different concentrations. For example, lethal
concentrations in blood of the heart glycoside digoxin
Equally to oral fluid, sweat and skin surface lipids are ana- (> 10 ng/mL) are by 5 orders of magnitude lower than
lyzed for living individuals. There are eccrine and apocrine those of the antipyretic drug acetylsalicylic acid (> 1 mg/
sweat glands. Eccrine sweat glands are found in virtually mL) but both occur in fatal poisonings.
all skin, with the highest density in the palms of the hands, – The additional or even exclusive presence of metabolites
soles of the feet, and on the head. They produce under nor- of the parent substance essentially increases the number
mal conditions 300–800 mL/day of eccrine sweat which of possible analytes.
consists of 99% ­H2O, 1% NaCl, and traces of organic and – There are interferences by endogenous substances. Espe-
inorganic compounds, and has a pH of 5–6. Hydrophilic cially putrefaction products provide often similar analyti-

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ChemTexts (2025) 11:4 Page 13 of 95 4

cal signals as exogenic drugs. These endogenous com- dehydrogenase (ADH) method and the headspace gas chro-
pounds must be recognized as such, otherwise it could matographic method (HS-GC). Clinical laboratories confine
be an unknown poison. themselves to the ADH method. The HS-GC method includes
– There are often combined intoxications with more than regularly the determination of methanol and acetone and can
one toxic substance. be extended to volatile poisons such as diethyl ether or chlo-
roform (cf. Sect. “Toxic gases and volatile substances”). If
It is obvious that this analytical task cannot be solved by the long-term alcohol consumption of the individual needs to
parallel tests for each of the many possible poisons. Instead, be assessed, the determination of the alcohol markers ethyl
the vast majority of toxic substances must be detectable in a glucuronide (EtG) and ethyl palmitate (EtPa) in a hair sample
limited number (e.g., three or four) of screening procedures. is the method of choice [50].
For the remaining poisons which do not suit one of these
procedures, specific single or group tests must be applied. Carbon monoxide, CO‑Hb
In view of the available analytical techniques and of the
frequency of substance occurrence in poisonings, the sys- In history, town gas (coal gas) with a carbon monoxide con-
tematic toxicological analysis is conducted in four branches: centration of 9–18% was generally used for cooking in towns
until around 50 years ago when it was step by step replaced by
(A) Preliminary tests and specific single or group tests for natural gas with methane as the main component. As a result
frequently occurring poisons which cannot be tested in of this easy access and the relatively soft and painless way of
branches B, C and D dying, inhalation of carbon monoxide was the most frequent
(B) Test for toxic metals—AAS, ICP/EOS, ICP/MS suicide method in towns at that time. But carbon monoxide
(C) Tests for gases and volatile substances—Headspace GC poisonings are also not seldom at present. Besides its indus-
and GC/MS techniques trial use as an intermediate product, it is formed always if car-
(D) Tests for nonvolatile organic substances—Combined bon-containing material burns under limited access to oxygen.
chromatography/spectroscopy techniques Fire in houses and apartments, defects in chimneys of stoves
and gas heating, exhaust fumes from cars in closed garages,
In practice, not all analytical procedures must be applied or inside barbecues with charcoal are frequent reasons for
in every case, and the analytical effort can be limited accord- accidental deaths by carbon monoxide. Moreover, methods
ing to the information described in Sects. “Diversity of toxic to produce CO for suicide purposes from charcoal or formic
substances and toxicological relevance” and “Indications acid were practiced [51, 52].
from case history, clinical symptoms, or autopsy”. From eco- Carbon monoxide binds 200–300 times stronger to hemo-
nomicals point of view (specialized personnel and expensive globin than oxygen, leading to carboxyhemoglobin (CO-Hb,
equipment), it is also not possible that each laboratory can Fig. 8a). In this way, blood loses its ability to transport oxygen
have all corresponding methods at hand. Therefore, close with the consequence of internal suffocation. The degree of
cooperation between to a certain degree differently special- intoxication depends on the percentage of CO-Hb in this reversi-
ized laboratories is necessary. In particular, this concerns ble competition between CO and O ­ 2. First symptoms (headache,
expensive tests for seldom-occurring poisons of branch A. racing heart) occur at 10–20%, blackout and circulatory collapse
at 30–40%, and death between 40% and 70%.
Preliminary tests and specific single or group tests CO-Hb should be determined in heart blood (cf.
for frequently occurring poisons Sect. “Heart blood”). In principle, the concentration of CO
dissolved in blood can be determined by infrared spectros-
Alcohol copy methods [53]. However, in practice, the percentage of
CO-Hb is determined and much more suitable for interpreta-
Alcohol (ethanol) is omnipresent in the population, and sur- tion. The methods are based on the different absorption spec-
vived or fatal intoxications with alcohol as the only harm- tra of the hemoglobin species deoxy-Hb, O ­ 2-Hb, and CO-Hb
ful substance occur very often. Combination with medicines (Fig. 8b). It is usually included in the automatic determina-
or illicit drugs is common. It was shown that when alcohol tion of the hemogram in clinical laboratories whereas in
is present, relatively small overdoses of drugs may result in legal medicine separate spectrophotometric methods are
fatal poisoning in comparison to drugs alone [49]. Therefore, often used.
determination of alcohol in blood according to validated CO-Hb is much more stable against heat and alkaline pH
methods is routine in every forensic case. For better assess- than deoxy-Hb or O ­ 2-Hb. This is used in preliminary tests
ment of the actual drinking event, the alcohol concentration during the autopsy. On heating a postmortem blood sample
is also determined in urine. In forensic laboratories, two in a test tube for 5 min in a boiling water bath, the cherry-
independent methods must be used, the enzymatic alcohol red color of CO-Hb remains stable and is visible above a

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4 Page 14 of 95 ChemTexts (2025) 11:4

Fig. 8  a Structure of car-

boxyhemoglobin in equilibrium
with CO and ­O2. b Visible
absorption spectra of ­O2-Hb,
deoxy-Hb, and CO-Hb. c
Visible absorption spectra at
increasing CO-Hb concentration
in a method based on the differ-
ent stability of deoxy-Hb and
CO-Hb [55]

content of 40% whereas it turns gray-brown in the absence of endogenous oxidation processes, but this is limited to
CO-Hb. The higher stability of CO-Hb in alkaline medium a proportion of about 1.5% by the NADH-dependent
is also used in a spectrophotometric method for its determi- enzyme methemoglobin reductase.
nation (Fig. 8c). There are two mechanisms for the formation of toxic
CO-Hb is regularly determined in fire victims. In these high Met-Hb concentrations. Inorganic oxidizing sub-
cases, the cause of death is usually complex. Besides stances such as ­NaClO 3 (previously used as total her-
CO, fire gases may contain HCN (Sect. “Cyanide”), ­SO2, bicide) or ­NaNO 2 (used as pickling salt) directly oxi-
­NO 2, HCl, and organic irritants such as formaldehyde, dize hemoglobin to Met-Hb. Nitrate in well water which
depending on the burning material. Inhalation of soot, mainly originates from inorganic fertilizers has the
heat, and decreased oxygen content in air add to these ­ O 3− > 10 mg/L is particularly dangerous
same effect. N
hazards to a different extent. High CO-Hb and soot in for siblings whose methemoglobin reductase is not yet
the airways prove that the victim was still alive during developed. The second mechanism is the oxidation of
the fire. On the other hand, burning outside (victims of hemoglobin with oxygen under the catalytic action of
self-burning or of murder by burning) showed only low aromatic amino or nitro compounds. The essential step
CO-Hb values. in this complex mechanism is the reaction of O­ 2-Hb with
the arylhydroxylamine to form Met-Hb and the corre-
Methemoglobin (Met‑Hb)‑forming substances sponding nitroso aromate from which the arylhydroxy-
lamine is regenerated by the NADPH/NADP + redox
Met-Hb is formed from hemoglobin by oxidation of the system. The required arylhydroxylamine can arise from
iron ion of the hemin moiety from F ­ e 2+ to F
­ e 3+. As a both the amino and the nitro compound in the biological
result, similar to the formation of CO-Hb, hemoglobin environment. On the other hand, injection of methylene
loses is ability to bind and to transport oxygen. Small blue as the antidote operates as a catalyst for the enzy-
amounts of Met-Hb in blood are steadily formed by matic reduction of Met-Hb.

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ChemTexts (2025) 11:4 Page 15 of 95 4

Typical symptoms of a Met-Hb intoxication are cya- are necessary. Inorganic oxidants are best detected in
nosis and chocolate brown blood. Concentrations of Met- gastric content, and ion chromatography with conduc-
Hb are given as % MetHb of total hemoglobin. The ana- tivity detector may be useful as in the case of chlorate
lytical determination in hemolyzed blood is based on its [56], or nitrite [57] where mainly nitrate was found after
absorption band at 630 nm, which disappears after addi- metabolic oxidation. Sodium nitrite as a mild and eas-
tion of excess KCN [54] (Fig. 9a). Total hemoglobin is ily accessible means of suicide has become increasingly
measured from an aliquot of the same solution after oxi- popular recently as a result of commentary on the inter-
dation of all hemoglobin to Met-Hb with K ­ 3Fe(CN)6. At net [58, 59]. Aliphatic esters of nitrous acid lead to Met-
a methemoglobin level of 20–50%, patients experience Hb formation as well, as seen for amyl nitrite, which is
dizziness, fatigue, headache, and weakness; levels above abused as “poppers” for sexual stimulation [60]. By the
50% cause dysrhythmia, central nervous system depres- way, generation of a controlled Met-Hb level by injection
sion, coma, metabolic acidosis, seizure, and tachypnea. of sodium nitrite as well as inhalation of amyl nitrite has
Levels above 70% are lethal. been proved as an efficient antidote in cyanide poison-
If a lethal Met-Hb level is detected, the source must ing [61].
be found. In most cases, this becomes clear from the cir- For aromatic amino and nitro compounds as the reason
cumstances of death (Sect. “Site where the unconscious for high Met-Hb, the methods described in Sect. “Non-
person or the corpse was found”). If not, further tests volatile organic toxic substances” are usually successful.

Fig. 9  Spectrophotometric determination of Met-Hb in a p-nitroaniline the brownish colored gastric mucosa. c Identification of p-nitroaniline and
poisoning case [62]. a Typical absorbance of Met-Hb at 630 nm, which its metabolites p-nitroacetanilide (M3) and 2-hydroxy-4-nitroaniline (M4)
disappears after addition of cyanide. b p-Nitraniline residues adhering on by HPLC–DAD in blood of this case

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4 Page 16 of 95 ChemTexts (2025) 11:4

In the case of a 15-year-old boy who took a p-nitroaniline capture detector is based on the derivatization to cyanogen
overdose in order to try it as a recreational drug, the chloride Cl-CN with chloramine T in a reaction precolumn
substance was visible as a deposit on the gastric mucosa [71, 72]. Direct measurement of HCN was performed by
(Fig. 9b) and was clearly identified and quantified by HS-GC with nitrogen-phosphorus detector and acetonitrile
HPLC–DAD (Fig. 9c) and by GC–MS [62]. Acute poi- as internal standard [73]. This was improved by mass spectro-
sonings with aniline derivatives used as components metric detection (HS-GC–MS) and using K ­ 13C15N as internal
of hair coloring [63] and with nitrobenzene [64] were standard [74]. In the laboratory of the author of this paper,
reported. cyanide was determined by isotherm distillation of HCN at
room temperature into 0.1 M NaOH solution and subsequent
Cyanide measurement on a cyanide-sensitive electrode (Fig. 10).
Instead of blood, urine and tissue homogenates can be ana-
Cyanide is a classical poison which frequently occurs in lyzed, too. Gastric content can directly be measured after, e.g.,
accidental, suicidal, and criminal cases. Therefore, each 1:100 dilution with 0.1 M NaOH. A colorimetric test is used
clinical or forensic toxicological laboratory should be able as a second method for confirmation. The limits of determina-
to perform its detection and quantification. KCN is the most tion for all methods in blood are 0.01–0.05 µg/mL.
frequently used cyanide, but other alkali or alkaline-earth Cyanide concentrations in blood of unexposed individu-
cyanides or liquid HCN (boiling temperature 26 °C) are als are below 0.1 µg/mL. Values above 1 µg/mL are life-
common. It is practically used in electroplating, as a chemi- threatening. Values between 0.05 and 10 µg/mL were found
cal intermediate, or in baits for killing rats or pigeons. It in blood of fire victims, depending on the burning material.
has a cruel history of mass killings in Nazi concentration For instance, for a couple who died during a fire of an artificial
camps during the Second World War and, e.g., in the mass polyamide Christmas tree the concentrations were 8.5 and
suicide of the Peoples Temple sect in Jonestown Guyana in 10 µg/mL. The postmortem concentrations after oral intake
1978 [65]. HCN was used for execution of death penalties of KCN are usually 10–100 µg/mL.
in the USA [66], and the “German Society of Human Dye-
ing” sold KCN to their clients for up to 5000 DM (Deutsche Ethylene glycol, diethylene glycol
Mark)/g in the 1990s [67]. A biogenic source is amygdalin,
a cyanide-containing glycoside in bitter almonds. HCN is Ethylene glycol HO–CH2–CH2–OH and diethylene glycol
always formed if nitrogen-containing material burns under O(CH2–CH2–OH)2 are viscous, water-miscible liquids of
limited air access and is, therefore, regularly present in gases low volatility and sweet taste which are used as antifreeze,
of burning apartments or houses. lubricants, and solvents. They are toxic and oral doses > 1 mL/
The toxic mechanism is the binding of ­C N − to F ­ e 3+ kg are believed to be fatal. Accidental mixing up with bever-
of the cytochrome oxidase in the respiratory chain of the ages is the cause of most intoxications [75], for instance, of a
mitochondria, with the consequence of a changed redox diethylene glycol-containing smoke fluid [76], but there are
potential and the blockade of this enzyme. O ­ 2-Hb which also suicides and homicides with ethylene glycol [77, 78].
is transported by blood circulation to all cells cannot be Mass poisonings by diethylene glycol used as an inappropriate
depleted. As a result, oxygen-saturated bright-red blood is excipient for preparation of medications were reported [79].
also contained in the veins and leads to the red color of the The toxic effect is, like methanol, caused by acidic metabo-
skin and of the livor mortis in death cases. lites such as glycolic acid and oxalic acid for ethylene glycol
The lethal dose of HCN is 70–140 mg for adults and 2-ethoxyhydroxyacetic acid for diethylene glycol which
(1–2 mg/kg). After inhalation of HCN, death occurs within leads to severe metabolic acidosis, renal and hepatic failure,
seconds. However, the lethal effect may be delayed after seizures, coma, and death. Postmortem concentrations in
oral intake of NaCN or KCN since C ­ N− is not resorbed blood between 0.3 and 2.4 mg/g were reported.
from the alkaline solution and must first be transformed Typical clinical symptoms are acidosis, high osmolar
into HCN by the gastric acid. Rescue is impossible at high and anion gaps, and calcium oxalate crystals in urine or
doses. But at smaller doses, antidote therapy with thiosul- kidney [77, 80]. Despite the high concentrations, deter-
fate, hydroxocobalamin, and Met-Hb-forming substances mination of these compounds and their metabolites in
­(NaNO2, amyl nitrite) can be successful [68]. human samples needs special methods because of their
Various methods are used for determination of cyanide high hydrophilicity and low volatility. Direct measure-
[69]. For spectrophotometric detection, HCN is separated ment from blood was possible by thermal desorption gas
from the acidified human sample by isotherm diffusion into chromatography [81]. The simultaneous GC–MS measure-
NaOH solution and subsequently transformed into a colored ment of ethylene glycol, 1,2-propylene glycol, and glycolic
derivate (pyridine-barbiturate reaction) [70]. The headspace acid was performed after derivatization of the carboxylic
gas chromatographic determination (HS-GC) with electron groups with isobutyl chloroformate and of the aliphatic

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ChemTexts (2025) 11:4 Page 17 of 95 4

Fig. 10  Determination of
cyanide in blood by a cyanide-
sensitive electrode. a Isothermal
distillation of HCN from acidi-
fied blood to 0.1 M NaOH at
room temperature. b Measure-
ment setup for the cyanide-sen-
sitive electrode with ionometer
and magnetic stirrer. c Cyanide-
sensitive electrode with A­ g2S
membrane from below. d Test
tube with electrode, magnetic
stirring bar, and cyanide-con-
taining NaOH solution

OH groups with phenylboronic acid [82]. GC–MS [83] and 86] and reviews [87, 88]. Immunologic analysis as the essential
positive ESI-LC–MS with measurement of the N ­ a+ adduct principle of a large variety of advanced spot testings was also
of ethylene glycol [84] were also described. presented in a previous lecture of ChemTexts [89]. The principle
In the laboratory of the author of this paper, a simple is based on the highly sensitive and selective, quasi-irreversi-
method for determination of ethylene glycol was applied ble reaction of the analyte (antigen) with specifically tailored
which could easily be integrated into the HS-GC measure- antibodies.
ment of alcohol. Ethylene glycol is cleaved with periodate
into two molecules of formaldehyde which are reduced to
Antibody + Antigen (Analyte) → Immunocomplex (2)
methanol with sodium borohydride. Antibodies are proteins with a Y-shaped structure and
NaIO4 NaBH4 consist of four chains, two light (M ≈ 20,000) and two heavy
HO-CH2 -CH2 -OH ⟶ 2 H2 C = O ⟶ 2 H3 C-OH (1) (M ≈ 50,000–70,000) chains which are linked together by
disulfide bonds (Fig. 11a). Besides a constant region, each
In this procedure, 1 mL H ­ 2O and 3 mL 4% H ­ ClO4 are
chain has a variable region which enables the specific binding
added to 1 mL blood or serum. After homogenization and
of the antigen. Under natural conditions, antibodies are formed
centrifugation, 0.2 mL of the supernatant it transferred into
only against antigens (peptides, proteins etc.) with a molecular
a headspace vial, and 0.2 mL of 10% N ­ aIO4 solution are
mass > 3000. But the antigens carry usually a smaller determi-
added. After 5 min, about 2 mg solid N ­ aBH4 is mixed with
nant group (epitope) by which the antibodies recognize and
this solution, the vial is closed, and the contents are ana-
distinguish them from body’s own tolerated molecules. This
lyzed for methanol according to the routine HS-GC alcohol
enables the preparation of antibodies with high affinity for
method with tert-butanol as internal standard. Propylene
smaller molecules: In an animal experiment, the target analyte
glycol yields an equimolar mixture of methanol and ethanol.
is chemically bound to albumin (Fig. 11b). After reinjection, the
immune system of the animal recognizes the modified albumin
Immunoassays for drugs and poisons
as foreign with the analyte moiety as the determinant group and
forms specific antibodies. These have a high affinity also to the
Immunoassays have gained increasing importance in various
non-bound, free analyte and can be isolated and multiplied by
bioanalytical fields such as clinical diagnostics, environmental
biochemical techniques to form the analyte-specific reagent.
monitoring, and food testing. The principles, methodologies,
Such antibodies often have a cross-affinity for metabo-
and applications were comprehensively described in books [85,
lites of the analyte and to other substances with the same

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4 Page 18 of 95 ChemTexts (2025) 11:4

Fig. 11  Reagents used in

immunoassays. a Structure
of drug-specific antibodies. b
Morphine differently bound to
albumin to form opiate-specific
antibodies. The cross-reactivity
to individual opiates other than
morphine depends on the site of
binding. Binding at the 3-OH
group leads to a high cross-
sensitivity against codeine and
morphine-3-glucuronide, where
the N-CH3 group can be “seen”
by the antibodies, whereas bind-
ing to the nitrogen atom leads to
higher cross-sensitivity against
normorphine or nalorphine with
an N–H or N-allyl group and
the 3-OH group “visible” to
the antibody. c Immobilized or
labeled antibodies and or target
drugs used in immunoassays

or similar structural features and, therefore, exhibit a group glucose-6-phosphate and ­NAD + which is transformed
specificity for, e.g., opiates, amphetamines, or benzodiaz- to NADH with its characteristic UV absorption band at
epines. The degree of cross-sensitivity of the antibodies to 340 nm. The increase of the absorbance at this wavelength
other compounds depends on the kind of binding to albumin within a definite time is used for determination of the enzy-
during their preparation, i.e., which part of the bound mol- matic reaction rate. After addition of antibodies and labelled
ecule the antibody “sees” during its formation (Fig. 11b). analyte to the sample, analyte and the enzyme-labelled ana-
The direct assessment of the analyte–antibody reaction is lyte compete for the antibodies. It is crucial for this method
not possible. For this purpose, the following auxiliary rea- that the enzyme is sterically blocked in the labelled ana-
gents are necessary depending on the respective test assay lyte—antibody complex. The remaining rate of NADH for-
(Fig. 11c): the immobilized analyte or the immobilized mation increases with the analyte concentration as shown
antibody, bound to the wall of a test vessel or to a test strip, by the calibration curve in Fig. 12. This method can be
and the labelled analyte or the labelled antibody for visual calibrated and can be performed in all automated clinical
or instrumental determination. Practically used labels which laboratory systems.
are chemically bound to the analyte or to the antibody are A prerequisite of EMIT is the optical transparency of
125
I (radioimmunoassay, not used anymore), colloidal gold the sample at 340 nm. For this reason, its direct application
(visual test plates and strips), an enzyme in combination with is mainly limited to urine. A suitable sample preparation
a suitable substrate (enzyme immunoassay) or fluorescein also allows the use of other materials [90]. Because of the
(fluorescence polarization immunoassay). There are a large group specificity, the predominant presence of metabolites
variety of implementations of immunoassays [86]. In forensic in urine, and the general problems in quantitative interpreta-
and clinical toxicology, enzymes are the most common labels tion of urine results (Sect. “Urine”), EMIT is mainly used as
employed in immunoassay methods. The enzyme-catalyzed a pretest in toxicological screening for methadone, opiates
reaction enables amplification of the signal and in this way (target substance morphine), cocaine (benzoylecgonine),
increases the sensitivity of the method. amphetamines (methamphetamine), ecstasy (MDMA), can-
An example of a homogenous competitive enzyme immu- nabinoids (THC-COOH), and benzodiazepines (oxazepam).
noassay is the enzyme multiplied immunoassay technique Test kits for medical drugs and drug classes are available,
(EMIT) with the analyte labelled with the enzyme glucose- e.g., tricyclic antidepressants. For interpretation, cutoffs
6-phosphate dehydrogenase (Fig. 12). The substrates are were defined which should as far as possible exclude false

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ChemTexts (2025) 11:4 Page 19 of 95 4

Fig. 12  Principle of the enzyme

multiplied immunoassay
technique (EMIT). D = drug to
be tested; AB = drug-specific
antibody; E-D = enzyme-labeled
drug, glucose-6-phosphate
dehydrogenase chemically
linked to the drug; S = substrate
of the enzyme, glucose-6-phos-
phate + ­NAD+; P = product of
the enzymatic reaction, NADH
with specific absorption at
340 nm. This is a kinetic test
in which the increase of the
absorbance of NADH at 340 nm
under constant conditions is
used for drug determination

negative results. Positive samples must be confirmed, spec- colored product. The reaction is stopped after a certain time
ified, and quantified by mass spectrometric techniques in by addition of sulfuric acid and the color turns yellow with
order to gain judicial validity. an absorption maximum at 450 nm. For a positive sample,
Besides application to drug-driving cases or workplace the lower enzyme concentration in the well leads to a less
drug testing, urine testing with EMIT or another immu- intense color. The absorbance at 450 nm vs. analyte concen-
noassay (fluorescence polarization immunoassay [FPIA] tration calibration curve is inverse.
[91], cloned enzyme donor immunoassay [CEDIA] [92]) is The providers of ELISA test kits supply automated pro-
regularly performed as part of the systematic toxicological cedures by employing robotic workstations equipped with
analysis in emergency and death cases. washing and dispensing modules, automated mechanical
Another broadly applied method is the enzyme-linked handling and analysis, and microplate readers. The con-
immunosorbent assay (ELISA) [93]. Owing to extremely ventional 96-well formats were transformed into higher-
high sensitivity, specificity, precision, and throughput it is throughput 384- and 1536-well plates.
used for a plethora of analytes. It serves as a pretest for drug Besides the application for large series pretests, immuno-
testing in large sample series of blood [94], oral fluid [95], assays were also developed for special poisons, which are
and hair [96]. Optical transparence of the sample is not nec- difficult or impossible to detect by other methods and partly
essary. Among the different kinds of ELISA available [93], could also be misused as warfare agents [97, 98]. Examples
the competitive technique is usually preferred for molecules are poisons with peptide or protein structure:
with the lower molecular size of most drugs and poisons. The toxins of the mushroom death cap (Amanita phal-
The principle is shown in Fig. 13. loides), α-amanitin and β-amanitin, are cyclic octapeptides
The test is performed in “micro wells” (small tubes) of with a lethal dose of about 0.1 mg/kg body weight. They can
a 96-well microplate coated with analyte-specific antibod- be determined in urine by an ELISA method [99]. Besides
ies. The sample with the analyte and a defined amount of the relatively frequent accidental poisonings with this mush-
the enzyme-modified analyte (enzyme: horseradish per- room, murder cases are known. In one case, the perpetrator
oxidase) are pipetted into the well. The enzyme-modified prepared a mushroom juice and injected it subcutaneously
analyte competes with the analyte of the sample material into the 26-year-old anesthetized victim [100]. The autopsy
for the available binding sites of the antibodies. In the next revealed acute liver necrosis and the poison was detected by
step, the unbound material is washed out and 3,3′,5,5′-tetra- radioimmunoassay in the bile.
methylbenzidine (TMB) + ­H2O2 is added as the substrate Insulin (molecular mass 5800) is an endogenous hormone
of the enzyme. In case of a negative sample, high enzyme for regulation of glucose metabolism and a medication used
activity remains in the well and TMB is oxidized to a blue by people with diabetes. Overdose leads to hypoglycemia

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4 Page 20 of 95 ChemTexts (2025) 11:4

Fig. 13  Reagents and work-

ing principle of enzyme-linked
immunosorbent assay (ELISA).
For explanations, see text

and can be lethal. Murder cases were repeatedly described Special methods for further poisons
[101–103]. Immunoassays are used for analytical determi-
nation [104]. For differentiation between endogenous and There are many further poisons which occurred in survived
exogeneous origin, the C-peptide (connecting peptide) is or lethal poisonings but are not detectable by the more
important in addition to insulin [105]. C-peptide, a compo- universal techniques described in Sects. “Toxic metals” to
nent of proinsulin, is cleaved off for insulin formation and is “Nonvolatile organic toxic substances”. Some salient exam-
excreted in a ratio of 1:1 together with insulin. However, it ples shall be mentioned.
is not contained in therapeutically injected insulin prepara- Hydrofluoric acid H2F2 and fluorides are frequently used
tions. For the comprehensive clarification of an insulin over- in industry. Fluorides also play a role in dental prophylaxis.
dose in postmortem cases, mass spectrometric techniques, Accidental and suicidal poisonings [114, 115] as well as
if available, are more suitable than immunoassays [106]. a mass poisoning with 263 victims [116] were reported.
An ELISA kit was developed for the extremely poisonous Hypocalcemia with inhibition of blood coagulation, subse-
ricin, a polypeptide from the African wonder tree (Ricinus quent hyperkalemia, and blocking of enzymes are some of
communis) with a lethal oral dose of 0.3–20 mg [107]. Previ- the toxic mechanisms [117, 118]. It is an important aspect
ously, a radioimmunoassay was used [108]. of fluoride toxicity that concentrated hydrofluoric acid exists
Botulinum toxin, a protein with a molecular mass of about almost entirely in non-dissociated state and, as a small neu-
149,000, is thought to be the most toxic substance known. tral molecule, penetrates lipid membranes very fast. After
The gold standard for its detection is still an animal experi- extensive accidental dermal exposure, it diffuses rapidly
ment, the mouse bioassay [109]. Analytical methods includ- through the skin and, besides local necrosis, reaches sys-
ing immunoassays were developed for its determination in temic circulation within less than 5 min. There, under neu-
food and animal feces [110, 111]. However, there seems to tral pH conditions, the toxic effects of fluoride ions may be
be no suitable method for detection in blood or tissue sam- fatal within a few hours [119, 120].
ples thus far. Despite the extreme toxicity and the broad A fluoride-sensitive electrode is preferentially applied for
application as Botox in medicine, no criminal poisoning was detection and quantification of fluoride in blood, urine, and
found in the literature, but serious adverse reactions after tissue extracts [121–123]. ­F− concentrations between 2.6
cosmetic injection were reported [112, 113]. and 56 µg/mL were measured in postmortem blood of fatal
fluoride poisonings.
Hydrogen sulfide H2S is an extremely toxic gas and causes
cellular hypoxia by inhibiting mitochondrial cytochrome
oxidase, which may rapidly lead to multiorgan dysfunction

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ChemTexts (2025) 11:4 Page 21 of 95 4

[124]. Breathing of air with a hydrogen sulfide concentra- elements (Me) are transformed into their exited states Me*
tion > 0.1% is lethal within minutes. Hydrogen sulfide occurs and emit light of characteristic wavelengths.
naturally in crude oil, natural gas, volcanic gases, and hot
springs and is produced by bacterial breakdown of organic
Me∗ → Me + h ⋅ 𝜈 (3)
materials and human and animal waste (sewage). Numer- The UV/VIS emission is focused into the entrance of a UV/
ous accidental death cases by ­H2S poisoning were reported, VIS spectrometer. Up-to-date ICP-OES spectrometers have
mainly by sudden inhalation of sewer gases (typical example an echelle grating monochromator matched to a CCD (charge
in [125]). An outbreak of suicides with ­H2S in Japan (208 coupled device) detector which measures simultaneously the
deaths within 3 months) was provoked by communication of full spectrum of this emission between 167 and 785 nm on
the method on the internet [126]. The poison was liberated 70,000 two-dimensional arranged pixels with a wavelength
in deadly amounts simply by mixing a bath additive (lime resolution around 0.01 nm. As an example, the computer-oper-
sulfur, containing calcium polysulfide) and a toilet deter- ated Varian Vista-PRO CCD Simultaneous ICP-OES instru-
gent. For determination of ­H2S in blood, it is liberated with ment with a built-in wavelength library of 32,000 lines enables
0.5 M ­H3PO4 and trapped in 0.1 M NaOH for potentiometric the simultaneous determination for up to 70 elements within
measurement with a sulfide-selective electrode [127, 128]. 35 s [134, 135]. The elements measurable by this method and
the detection limit ranges related to the mineralized analyte
Toxic metals solution are shown in Fig. 14. Not measurable are C, N, O, Tc,
Cs, Pm, the halogens, and noble gases. The detection limits
Analytical methods for most elements (0.1–10 µg/L) and the wide linear dynamic
range of four to six orders of magnitude are acceptable for all
Besides typical metals like mercury, lead, or thallium, the forensic problems. An application to postmortem material is
term “toxic metals” includes also some elements which do not described in [136].
have typical metallic properties such as arsenic and selenium. Inductively coupled plasma–mass spectrometry (ICP-
Toxic metals are not toxic in elemental state but in the form MS): An introduction to the function principle, the analyti-
of soluble salts or oxides by interaction of their cations with cal aspects, and the broad applications of ICP-MS is given in
vital biological systems on the cellular or molecular level. numerous reviews and brochures from the instrument manu-
From an analytical point of view, they can be detected and facturers [137–143]. In this method, the cations of the analyte
quantitatively determined today by comprehensive screen- formed in the plasma are analyzed. The ionization occurs by
ing procedures using inductively coupled plasma–optical ­ r+. Ar has a first ionization potential of
electron transfer to A
emission spectroscopy (ICP-OES) and inductively coupled approximately 15.8 eV. The first ionization potential of most
plasma–mass spectrometry (ICP-MS). These methods have elements is lower (4–12 eV). Therefore, they can easily be
replaced atomic absorption spectrometry, electrochemical ionized by ­Ar+.
techniques, and other specific methods for certain metals
mainly because of the possibility of multielement analyses Me + Ar+ → Me+ + Ar (4)
which enable one to determine simultaneously a large number A small portion of the ions is directed into the high vac-
of elements with high sensitivity and specificity. uum of the mass spectrometer through a suitable interface,
Before analysis, the biological material (blood, urine, tis- is separated there according to their mass/charge ratio (m/e),
sues) is mineralized in a microwave oven with nitric acid/ detected, and recorded as a mass spectrum of the ion abun-
H2O2 (4:1) [129]. The plasma is generated in specially dance as a function of m/e. Special techniques are applied
designed torches by ionization of an argon gas stream with to remove interferences by plasma gas, sample solvent, or
a radiofrequency electromagnetic field of a copper coil (e.g., polyatomic species. Quadrupole mass spectrometers (sin-
40 MHz, 700–1500 W) and consists of A ­ r+ ions and elec- gle or triple quadrupole) are mainly used, but time-of-flight
trons. The plasma has a temperature of up to 10,000 K. The instruments (ICP-TOF-MS) with a higher mass resolution
mineralized sample is transported into the instrument as a were also developed [144].
liquid stream. Inside the instrument, the liquid is converted The elements which are measurable by ICP-MS are
into an aerosol through nebulization. This sample aerosol shown in Fig. 14. Only seven elements cannot be measured:
is then transported to the extremely hot plasma where it is H and He are below the mass range of the mass spectrom-
desolvated, vaporized, atomized, and excited and/or ionized. eter, Ar, N, and O are present at high level from the plasma
Inductively coupled plasma–optical emission spectros- and air, and F and Ne are not ionized in an argon plasma.
copy (ICP-OES): The principles and practical details of ICP- Most elements have more than one isotope, which leads to
OES are reviewed in several papers [130–133]. Under the different peaks in the mass spectrum with intensity ratios
extreme conditions of the plasma, a portion of the sample reflecting their natural abundance. With detection limits

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4 Page 22 of 95 ChemTexts (2025) 11:4

Fig. 14  Elements detectable by

inductively coupled plasma–
optical emission spectroscopy
(ICP-OES) and inductively
coupled plasma–mass spec-
trometry (ICP-MS). Modified
figure [131]. Not measurable
by ICP-OES are C, N, O, Tc,
Cs, Pm, the halogens, and noble
gases, and by ICP-MS are H,
He, Ne, Ar, N, O, and F. For
the remaining elements in non-
colored fields no applications
are described

between < 1 ng/L and 10 µg/L, the method has a higher sen- Co, Cu, Fe, Ge, Hg, Li, Mn, Ni, Pb, Pt, Sb, Se, Sn, Tl, U, V,
sitivity for most elements than ICP-OES. W, and Zn. These data considerably support the appropri-
The computer-operated instruments enable the simultane- ate assessment of results in corresponding poisoning cases.
ous determination of up to 85 elements within a measure- Among the toxic metals, arsenic, mercury, and thallium have
ment time of around 20 s. Depending on the equipment for a higher incidence of acute poisonings and shall be dealt
sample supply, up to 1200 samples per day can be measured. with more in detail in the following sections.
On the basis of a calibration with multielement standards, Poisonings with toxic metals have become seldom in the
the efficient software delivers qualitative and quantitative last few decades and, therefore, partly lost the attention and
information about the sample from the full mass spectrum experience of the scientific community with the risk of being
of a sample within a very short time. overlooked in serious cases or misinterpreted as a bacte-
By use of ICP-OES or ICP-MS, the concentrations of rial infection because of the initial gastrointestinal symp-
many elements in blood, urine, or hair were measured for toms. In principle, a screening for toxic metals should be
the unexposed population of different countries in numerous performed as a regular part of the systematic toxicological
larger studies, e.g., for Germany [145], France [146], Poland analysis. However, not every forensic toxicological labora-
[147], Italy [148], Russia [526], Australia [149], Cape Verde tory can afford the expensive instruments, operating costs,
[152], or Brazil [151]. The importance of ICP-MS for clini- and specially trained personnel. Therefore, in practice, the
cal and forensic toxicology was reviewed by Goullé [146]. samples are sent to laboratories specialized in ICP-MS and
Toxic and lethal concentrations in blood, urine, and tissue ICP-OES if indications point to toxic metals or if poisoning
samples together with the used analytical methods, metabo- is suspected and no other poison is found. This is a very
lism, and poisoning symptoms were reviewed by Baselt in unsatisfactory situation and a large number of undetected
separate monographs for each element in his comprehensive cases must be assumed as shown for some examples in the
reference book Disposition of Toxic Drugs and Chemicals next sections.
in Man [152] for Ag, Al, As, Au, Ba, Be, Bi, Cd, Cs, Cr,

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ChemTexts (2025) 11:4 Page 23 of 95 4

Arsenic entered the scientific literature. In newer case reports, from

87 victims, 55 survived under medical treatment and 32 died.
Arsenic has a long history in both intentional and unin- Forty-two cases were suicides or suicide attempts, 27 were
tentional poisoning for humans. It was called as the “king accidents (mainly confusion with food or drinks), and 15
of poisons” and the “poison of kings” as it was highly were criminal administrations. The ingested or administrated
potent and efficient after administering with the intent to arsenic compounds were ­As2O3 (n = 43), ­NaAsO2 (n = 3),
succeed the throne or power. The Borgias, Pope Alexander ­NaAsO3 (n = 6), methylated arsenic pesticides (n = 9), ele-
IV, his son Cesare, and Cesare’s half-sister, Lucrezia, were mentary As (n = 2), ­AsCl3 (n = 1), ­As2S3 (n = 1), and ­As4S4
a family of poisoners from the late fifteenth century. The (n = 1). In 18 cases, mainly criminal administrations, the
odorless and tasteless arsenic trioxide ­(As2O3) appeared substance was unknown. Although elementary arsenic and
to be an ideal poison. Arsenic poisoning was difficult to the arsenic sulfides are believed to be nontoxic because of
detect as the symptoms initially mimicked food poison- their low solubility, one death occurred after intake of 30 g
ing. A well-known Italian arsenic poisoner of the mid- arsenic powder [163], possibly by superficial ­As2O3.
seventeenth century was Giulia Tofana who was executed The mechanism of the poisoning is the binding of As(III)
together with her daughter in Rome in 1659 for involve- to the SH groups of proteins which leads to the functional
ment in the murder of several hundred victims. In another loss of more than 200 enzymes and other biomolecules. Fur-
infamous arsenic case, Marie-Madeleine de Brinvilliers thermore, an exchange of phosphate for arsenate in adeno-
poisoned several family members in Paris at about the sine triphosphate is discussed.
same time. The ingested dose of ­A s 2 O 3 in lethal cases was
The secret poisoning with A ­ s2O3 became more difficult between 1 and 60 g and the concentrations in blood were
when James Marsh in 1836 discovered a way to detect the 0.26–10.2 mg/L, depending on the survival time. In sev-
poison in biological material [15]. Today, the Marsh test is eral cases, opacities in the x-ray of the stomach were the
exercised in the practicum of every chemistry student. It is first indication of a heavy element intoxication as shown
based on the reduction of the As(III) compound to form the in one example after intake of 54 g ­A s 2O 3 in Fig. 15a
volatile ­AsH3 by Zn and ­H2SO4 added to the sample. In the [164].
subsequent decomposition of ­AsH3 in the hydrogen flame, It was shown that arsenic poisoning can also be detected
an arsenic mirror on a cold ceramic surface is deposited. In after cremation by analysis of the ash [165, 166]. The poi-
this way 0.02 mg As could be detected. soning proceeds in two phases. The symptoms of the first,
The most abundant arsenic compound is ­As2O3, 30,000 to gastrointestinal phase begin normally within the first hour
50,000 t/year of which is produced as a byproduct of metal after intake and are nausea, vomiting, severe and sustained
smelting worldwide. The compound is applied in metallurgy, diarrhea with vomiting, heavy loss of water and electro-
the glass industry, the electronics industry, and taxidermy. lytes, metabolic acidosis, hypotension and tachycardia,
The chemistry of arsenic, its occurrence in the earth, and unconsciousness, dilated pupils, pallid skin, and death after
release from industry into the environment as well as the 3 h to 6 days. The situation is easily mistaken for cholera
history of arsenic compounds in human and veterinary medi- or botulism. If this phase is survived, a broad spectrum
cine and as herbicides and insecticides were covered in two of impairments of internal organs and particularly of the
previous lectures in ChemTexts [153, 154]. Arsenic pesti- nervous system (polyneuropathy) take place in the second
cides were banned in 1974. phase.
Despite the restricted access to arsenic compounds and The toxicity of arsenic differs strongly between the
their improved analytical detection, arsenic poisonings ingested compounds. In animal experiments, As(III) com-
continued to occur. Its infamous reputation as an efficient pounds were found to be 60 times more toxic than As(V)
and secretly administrable poison promotes the choice of compounds, and the ­LD50 was 10–15 mg/kg for ­As2O3 and
this substance. The symptoms, time course, and analyti- 700–1800 mg/kg for ­CH3AsO3HNa [161, 167].
cal findings of survived and lethal arsenic poisonings were Toxicity of arsenic compounds:
thoroughly described in toxicological textbooks [155–157],
Inorganic Inorganic
review articles [158–161], and numerous case reports. In a
As(III) − compounds > As(V) − compounds
criminal mass poisoning of participants of a festival in Japan
As2 O3 , NaAsO2 Na3 AsO4
1998 by arsenic-laced curry, four persons died and 67 were (5)
Organic
severely ill [162].
> Arsenic compounds
The case reports in the scientific literature were almost ( )
CH3 AsO3 HNa, CH3 2 AsO2 Na
always written by the treating physician. But there are
many criminal poisonings with arsenic which were broadly
reported in daily newspapers, e.g., as court reports, but never

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4 Page 24 of 95 ChemTexts (2025) 11:4

Fig. 15  Detection of metallic poisons by x-ray. a ­As2O3 in the stom- the legs after 4 years. b Elementary mercury deposits in the lung of
ach of a 43-year-old man after suicidal intake of 54 g (from [164], a 34-year-old man after homicidal injection of 40 mL mercury (from
© Elsevier with permission). The poison was removed by surgery [175], used under creative commons CC-BY license). The patient sur-
(gastrotomy) and the patient survived and was discharged from hos- vived after surgical excision of the metal, continuous blood purifica-
pital after 55 days and showed chronic slight distal polyneuropathy of tion, plasma exchange, alveolar lavage, and chelation

Therefore, and for determination of the applied poison for a long time in the form of mercury globules as visible
in forensic cases, the binding state of arsenic is determined by x-ray. The victim of the homicidal first case (injection
by speciation [168, 169]. Since ICP-MS and ICP-OES do of 40 g Hg) showed initial pyrexia, chill, fatigue, body
not differentiate between different arsenic compounds, an aches, and pain of the dorsal aspect of right foot and
additional extraction and chromatographic separation is nec- survived after surgical excision of the metal, continu-
essary for this purpose. ous blood purification, plasma exchange, alveolar lavage,
Arsine AsH3 is an extremely toxic gas with a garlic-like and chelation. In the suicidal second case of injected
odor. It is widely used in the semiconductor industry. Unlike 15 g Hg, sufficient mercury slowly entered the nervous
other arsenic compounds, inhalation of arsine leads to mas- system and the patient finally died from mercury toxic
sive hemolysis and renal failure. The poisonings are gener- encephalopathy and chemical pneumonia about 9 months
ally accidental [169, 170]. Arsenic concentrations in blood after injection.
were 0.1–0.6 mg/L in lethal cases. Acute vapor exposure of metallic mercury can cause
pneumonia, respiratory distress syndrome, progressive
pulmonary fibrosis, and death. In one case, a 4-month-
Mercury old child died a short time after the father had evaporated
two spoons of mercury on a red-hot stove plate in the
Mercury has a broad practical application and belongs to kitchen [176]. In another similar case, a 14-month-old
the classic poisons [171]. Acute poisoning can occur by child died and the mother had to be treated in hospital
elementary mercury, mercury salts, and organic mercury [177]. In a case of suspected homicide, the possibility of
compounds such as methylmercury. These groups differ criminal poisoning with mercury-manipulated cigarettes
substantially in their toxicity [172, 173]. was systematically investigated with the result that the
Elemental mercury: The liquid metallic mercury is evaporable amount in one cigarette is not sufficient to
poorly absorbed orally and as much as 204 g have been do any harm [178]. In a later case of attempted murder,
taken with little adverse effects [174]. After intravenous the victim smoked 14 cigarettes with grains of mercury
injection of considerable amounts in two cases [175], over 16 h and noticed flu-like symptoms but no further
the mercury entered the pulmonary circulation along physical abnormalities [179]. In serum, 0.99 µg/L Hg
the affected veins, passed through the pulmonary cap- was determined.
illary bed, and finally distributed throughout the body
(Fig. 15b). It has a stable nature in blood, and can remain

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ChemTexts (2025) 11:4 Page 25 of 95 4

Chronic metallic mercury inhalation is known to dangerous and can easily penetrate the skin. In a case of
induce symptoms from the central nervous system includ- a chemistry professor, about 0.5 mL of the substance was
ing tremors, delusions, parkinsonism, memory loss, and absorbed though the gloves and the skin during an experi-
neurocognitive disorders [180]. This “mercurialism” has ment [190]. The first neurologic symptoms were observed
a long history and the sources of mercury are manyfold. after a latency period of 154 days with 4.0 µg/mL Hg in
Because of the relatively high vapor pressure of Hg, it blood. Despite comprehensive medical treatment, the patient
occurred always in mercury-contaminated environments became unresponsive to all visual, verbal, and light-touch
such as gold extraction with mercury in small-scale gold stimuli 176 days after exposure and died on day 298. In the
mining, electrochemical production of NaOH in mercury severely degenerated brain, 3.1 µg/g mercury were deter-
cell plants, mercury gilding, preparation of amalgam fill- mined. The long latency between exposure and symptoms
ings in dental practice, or regular mercury use in chemi- makes these substances insidious poisons. However, the
cal laboratories (e.g., polarography). Another less known very long half-life in human blood and tissues gives a good
example is the large-scale production of tin–mercury chance of its detection even after several months provided
mirrors (lamination of glass panes with a layer of tin that an analysis is performed.
amalgam) which were replaced by silver mirrors only at
the end of the nineteenth century [181]. In the Bavarian Thallium
town Fürth, 60–70 tons of mercury per year was used for
this production and led to persistent contamination of Different from the long history of arsenic and mercury,
houses and chronic exposure of inhabitants [182]. thallium was discovered only in 1861. It is used in glass,
Mercury salts HgCl2, HgSO4: Oral intake of mercuric metal, and electronics industries and the world production
chloride (sublimate, ­HgCl2) can produce a range of toxic is only about 10 t/year [191]. The toxic properties of thal-
effects including corrosive injury, severe gastrointestinal lium were noted in 1863 by one of its discoverers, Claude-
disturbances, acute renal failure, hypovolemic shock, cir- Auguste Lamy, who developed general lassitude and pains
culatory collapse, and eventual death [183–185]. The initial of the lower limbs, which he attributed to the vapors of thal-
symptoms are like those of arsenic. The lethal dose is about lium [192]. He fed dogs, ducks, and hens with thallium-
1 g. ­Hg2+ is known to bind to sulfhydryl groups, producing containing milk and all animals died within 68 h. The use
damage to cellular proteins and enzymes. The half-life in of thallium as a rodent poison originated in about 1920,
untreated cases is between 30 and 100 days. Since ­Hg2+ does when Bayer AG in Germany introduced it as a proprietary
not permeate the blood–brain barrier, brain dysfunction is rat poison (Zelio paste and grain, 2.5% T ­ l2SO4). The reputa-
less evident than with other forms of mercury. tion of thallium as an efficient rat poison led to its frequent
Postmortem concentrations in blood were 0.8–22 µg/mL misuse in suicides and criminal poisonings. The symptoms
depending on the survival time. Toxicological analysis of and the progression of the poisoning were well known at
samples by cold vapor atomic absorption spectrometry from this time and were comprehensively described [191]. The
a corpse, exhumed 6 years after homicidal death, revealed use as a rodent poison was banned in 1970 in Europe, North
1.2 µg/g in liver, 1.0 µg/g in heart muscle, and 0.23 µg/g in America, and other countries, and the concession of the last
hair [186]. thallium pesticide in Germany expired in 1992.
Organic mercury compounds, CH3HgCl, (CH3) 2Hg: Newer case reports and reviews about survived and
Alkylmercury compounds are extremely toxic and are lethal thallium poisonings were quoted in a previous paper
infamous mainly because of Minimata disease from eating [193]. They often come from China but also from the Czech
contaminated fish [187] and the mass poisoning in Iraq by Republic, Germany, India, Iraq, Italy, Japan, Russia, Slova-
eating bread which was baked from wheat treated with mer- kia, Syria, and the USA. Two cases of murder and attempted
curial fungicide [188]. Typical was a delayed onset of the murder in the last 2 years in Germany emphasize the actual-
symptoms from 10 to 150 days (average 3 weeks). Of the ity of this poison [193, 194]. Instead of rat poisons, purchase
various clinical features encountered, neurological symp- of ­Tl2SO4 via the internet has become the poison source
toms and signs were predominant and included muscular which is difficult to control in the globalized world. The
weakness, numbness, unsteady gait, paresthesia, dysarthria, insidious course of the intoxication (see below) makes it
mental disturbances, and, in severe cases, blindness, partial a dangerous tool for malicious murder. For this reason, it
deafness, stupor, coma, and death. was also used in political murder by secret services, e.g., in
Self-injection of methylmercury in a 23-year-old man led the assassination of the Cameroon opposition leader Félix-
to severe neurological symptoms, progressive multiorgan Roland Moumié by the French “La Main Rouge” in 1960
failure, and death 10 months later [189]. Analysis of blood [195] and the attempted murder of the escape-helper Wolf-
on hospital admission several days later showed 2.9 µg/mL gang Welsch by the GDR Stasi in 1981 [196].
Hg and 1.53 µg/mL ­CH3Hg+. Dimethylmercury is even more

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4 Page 26 of 95 ChemTexts (2025) 11:4

The toxic mechanism of thallium is based on the fact

that the monovalent ion ­Tl+ has the same ion radius as ­K+
and replaces the potassium ions in most biological pro-
cesses [197]. It has a 10 times higher affinity for the N ­ a +/
+
K -ATPase, crosses the blood–brain barrier, and efficiently
enters and accumulates in the cells. There, it inhibits a num-
ber of enzymes by reactions with thiol groups and interferes
with vital metabolic processes. Reaction with membrane-
bound enzymes and receptors changes transport processes
and structure of membranes, disturbs the function of the
mitochondria, and inhibits cellular respiration. It attacks
particularly the peripheral and central nervous system. The
damage of peripheral fibers was shown by nerve biopsy
[198] and electron microscopy [199].
Symptoms of thallium poisoning are diverse and nonspe-
cific. The key symptoms which are described in almost every
case report are abdominal pain, vomiting, and nausea within
the first few hours, later persistent obstipation and oliguria,
neuralgic stinging pains after 3–5 days with hyperesthesia
of the legs, which progressively increase and extend to the
abdomen and sternum, tuft-wise loss of hair about 2 weeks
after the first complaints, and Mee’s lines (white lines on the
finger and foot nails) after 1 month (Fig. 16).
The intoxication can also be associated with neuropsy- Fig. 16  Dermal symptoms of thallium poisoning. a Diffuse alope-
chological symptoms, psychosis, and signs of personality cia of a young boy after ingestion of an unknown amount of T
­ l2SO4
(from [202], used under creative commons CC-BY license). b Mee’s
changes such as depression, apathy, anxiety, confusion, lines at the fingernails of a young boy 7 weeks after ingestion of
delirium, and hallucinations. Mental symptoms such as rest- cookies containing an unknown amount of thallium (from [203], used
lessness and wailing can convey the impression of hysteria. under creative commons CC-BY license)
The sensorimotor polyneuropathy leads to muscle weak-
ness of legs and arms, gait disorder, and paralysis. The most
severe impairments in many thallium intoxications are visual strength until he died, unable to walk and blind [201].
problems such as paralysis of the peroneal muscles, nystag- Despite typical indications, a diagnosis of poisoning was
mus, color vision constraints, atrophy of the optic nerve, not made and all medical treatment failed. Suspicion arose
and blindness. only some time after death with the detection of thallium
The diversity of the symptoms and the limited experi- in tissues of the exhumed corpse. Thallium mass poison-
ence of physicians with this rather seldom intoxication in ings were reported in Iraq [202] and Syria [203].
recent years often complicate making the correct diag- The average lethal dose of soluble thallium salts is
nosis; Guillain–Barré syndrome (autoimmune-induced about 1 g. In blood of severe survived poisonings, con-
weakness and paralysis of muscles), porphyria, botulism, centrations between 1 and 8 μg/mL were measured after
diabetic peripheral neuropathy, or Crohn’s disease were 1–21 days, and in postmortem samples 0.5–11 μg/mL. The
assumed instead. Often, it ends with a psychosomatic half-life increases with the dose and was reported to be
explanation. In one criminal case, within 7 weeks, the between 2–4 days and up to 30 days. This large variation
victim was successively examined in six hospitals with of the half-life is caused by accumulation in tissues and the
a wide variety of diagnostic methods, but a conclusive essential contribution of the enterohepatic circulation after
explanation of the complaints was not found until the biliary excretion combined with obstipation as a typical
perpetrator herself initiated a test for toxic metals [193]. symptom of thallium. The determination of thallium by
The delayed diagnosis of thallium poisoning is rather the the methods described in Sect. “Analytical methods” is no
rule and not an exception. The mean interval from expo- problem, even for unexposed persons (< 0.04–0.65 ng/mL
sure to diagnosis of 86 cases described in 13 reports was in Germany), if the analysis is performed.
23.6 days (range 2–150 days) [200]. In one case, the wife
of a 45-year-old miner mixed repeatedly within 3 years
altogether 5 g thallium in his meals and saw him gradually
pine away with unbearable pains and steadily decreasing

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ChemTexts (2025) 11:4 Page 27 of 95 4

Toxic gases and volatile substances is temporarily interrupted for a preset time. This enables the
pressurized gas in the vial to expand onto the column, result-
Headspace gas chromatographic techniques ing in a flow of a definite amount of the mixed headspace gas
from the vial to the column.
The identification and quantitative determination of gases Since every forensic toxicological laboratory uses a gas
and volatile substances in human material is generally per- chromatographic method with flame ionization detector
formed by headspace gas chromatography (HS-GC) [204]. (HS-GC-FID) for routine determination of alcohol, a method
The broadly applicable technique is based on the formation of for determination of the most important volatile poisons is
a temperature-dependent distribution equilibrium of volatile easily installed on this instrument and includes frequently
substances between the liquid or solid sample and the gas occurring solvents, for instance, methanol, propanol, higher
phase in a vial according to Henry’s law (Fig. 17). This ena- alcohols, acetone, butanone, chloroform, diethyl ether, ethyl
bles one to determine the concentration of the volatile analyte acetate, dioxan, tetrahydrofuran, acetonitrile, and essential
via its presence and concentration in the gas phase. aliphatic and aromatic hydrocarbons. Since for identifi-
The different automatized techniques to transfer the head- cation only the retention time is used, a second run on a
space gas from the vial into the injection port of the gas different GC column must be performed for confirmation.
chromatograph and the corresponding theoretical relation- The accuracy of identification is much improved if a mass
ships were described in detail by Tipler [205]. Besides the spectrometric detector is applied (GC–MS). The software of
automated gas syringe injection and the valve loop injection, GC–MS instruments includes automatic searching in large
the pressure-balanced sampling provides the most straight- MS libraries, e.g., Wiley Registry®/NIST Mass Spectral
forward means of determining the amount of headspace Library 2023 with over three million mass spectra from
vapor injected since the sample vapor from the headspace about 950,000 compounds [206], which enable identifica-
vial flows directly into the GC column (Fig. 17). After equi- tion of even exotic volatile substances.
libration, the vial is pressurized with the carrier gas to the Special 10- or 20-mL headspace glass vials with gas-
gas inlet pressure of the column. Then, the carrier gas flow tight PTFE-coated septa, which do not absorb lipophilic

Fig. 17  Headspace pressure-balanced sampling in HS-GC. After interrupted for a preset time by closing valves 1 and 2 and the pres-
equilibration between sample and gas phase, the hollow needle is surized gas in the vial expands onto the column for chromatographic
injected and the vial is pressurized with the carrier gas to the gas analysis
inlet pressure of the column. Then, the carrier gas flow is temporarily

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4 Page 28 of 95 ChemTexts (2025) 11:4

substances, and aluminum screw or crimp caps were devel- of consecutive measurements. Since the decline of the peak
oped. Subtle techniques for collection of samples during area from measurement to measurement occurs according to
autopsy without loss of the volatile or gaseous analytes first-order kinetics, in principle two measurements are suffi-
were used [207–209]. This includes aspiration with gas- cient for determination of the total peak area by extrapolation
tight syringes directly from the trachea, lung tissue, or stom- of all measurements. Since this total peak area depends only
ach, or collection of gas liberated from the lung placed in on the total analyte amount in the sample, the MHE result is
a water bath (Fig. 18). Blood and tissue samples are tightly independent of the matrix. Therefore, the calibration can be
enclosed into pre-weighed headspace vials immediately dur- performed with the standard in absence of any matrix or, e.g.,
ing autopsy. in aqueous solution.
The routine quantification in HS-GC for a constant matrix, The cyclic performance of MHE with the pressure-bal-
e.g., ethanol determination in blood, is performed by cali- anced sampling technique, shown in Fig. 19a, proceeds by
bration with an internal standard which cannot occur in the changing the position of the needle and closing of valves of
sample, e.g., tert-butanol. However, since the determination of the autoinjector in four steps: 1. equilibration, carrier gas
other volatiles is rather seldom, various matrixes are analyzed flows directly to the column, 2. pressurisation of the vial
for the same substance, and the same matrix (e.g., liver tissue) to the precolumn pressure, 3. transfer of a definite part of
differs from case to case, the standard addition method is more the headspace to the column by interrupting the carrier gas
suitable. This is also a general procedure for other methods. supply, and 4. venting the vial through the upper opening of
After a first measurement, a definite amount of the analyte is the needle. As an example, the chromatograms of six MHE
added to either the gas or to the sample phase in the vial, and cycles of a postmortem heart blood sample (propane sniffing
after equilibration the measurement is repeated. The increase death case, 490 µg/mL) are shown in Fig. 19b [210].
of the analyte peak area corresponds to the added concentra-
tion. By repeated additions, a calibration curve of the analyte
in the original sample can be obtained. The concentration Suffocation in gas atmosphere
after a single standard addition is calculated by Eq. (6).
Inhalation of gases can act on the human organism by suf-
Abefore addition
Csample = × Cadded (6) focation (displacement of oxygen) and by toxic impairment
Aafter addition − Abefore addition of life processes. Typical examples of toxic gases are CO
C = concentrations, A = peak area. (Sect. “Carbon monoxide, CO-Hb”), ­SO2, or HCl. Examples
Another very efficient method of headspace quantifica- of pure suffocating effect are the noble gases and nitrogen. A
tion is the multiple headspace extraction (MHE). This can be mixed effect exists for carbon dioxide and hydrocarbons with
applied if in the equilibrated sample vial an essential part of increasing toxic action from methane to butanes.
the analyte is in the gas phase (high distribution constant KG/S Carbon dioxide CO2: The concentration of ­CO2 in ambi-
of the analyte between the gas phase and the sample at the vial ent air is about 0.04%. Exhaled air contains about 4%.
temperature, high volume ratio VG/VS of gas and sample phase Inhaled air with 8–10% ­CO2 leads to deepened breathing,
in the vial). Under these conditions, the analyte is step by step breathlessness, tachycardia, headache, and finally uncon-
completely extracted from the sample by a sufficient number sciousness [211]. Concentrations above 20% are lethal. On

Fig. 18  Collection of a gas

sample before autopsy in a case
of accidental suffocation in an
argon atmosphere [209]. a Aspi-
ration of the gas sample from
the bronchia through a probe
after installation of a trocar. b
Transfer of the gas from the
syringe into the headspace vial
with avoidance of mixing with
surrounding air

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ChemTexts (2025) 11:4 Page 29 of 95 4

Fig. 19  Multiple headspace

extraction (MHE) for gas chro-
matographic determination of
gases and volatile substances. a
Steps of an MHE cycle. b GC-
FID chromatograms (6 MHE
cycles) of propane in the heart
blood sample of a lethal inhala-
tion case (sniffing). Resulting
propane concentration 490 µg/
mL

inhalation of very high concentration, the person can sud- Helium: Natural gas with a content of about 0.2% is the
denly collapse and die within 5–10 min. The mechanism is essential source of helium. It is not toxic and freely obtain-
the development of hypercapnia and respiratory acidosis. able via the internet as balloon gas. However, in the last two
Severe acidosis results in a depression of the respiration and decades, asphyxiation in helium atmosphere has become an
the circulation. ­CO2 sources in accidental or suicidal fatali- increasingly popular method of suicide [218]. In digital and
ties were volcanic gases [212], dry ice [213], fermentation printed publications, the readers can find all the necessary
[214], fire extinguishers [215], or mixing baking soda with instructions. With a plastic bag secured over the head by
citric acid [216]. In a murder-suicide, a man killed his wife, a rope, a rubber band, or adhesive tape fixed around the
his two sons, and finally himself with ­CO2 from a 500 g sin- neck, the flow of helium (density 0.178 g/L compared to
gle-use cylinder using synthetic inhalation face masks [217]. air 1.23 g/L) through a tube into the bag easily removes
Postmortem identification of carbon dioxide intoxication the air. Hypoxia is a fast process. It is estimated that loss
is difficult, and scene investigation was the most important of consciousness due to oxygen deprivation can occur in
factor in determining the cause of death in most cases. Blood 5–10 s and within 60 s cerebral damage due to hypoxia can
analysis for ­CO2 has only a limited diagnostic value, as ­CO2 be irreversible.
rapidly accumulates after death. In one case, an elevated A peculiar aspect of helium inhalation (as well as
­CO2 content in lung gasses has been described after vacuum of argon, nitrogen, or methane, see below) is the lack of
degassing of the lungs in an argon atmosphere and GC–MS the breathing reflex, that the victims do not feel the urge
[214]. to breath. In fact, the breathing reflex is not triggered by

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4 Page 30 of 95 ChemTexts (2025) 11:4

oxygen deficiency, but by carbon dioxide excess, which is tract as compared to unexposed cases could merely support
not present in the case of helium suffocation since C ­ O2 can evidence from the circumstances. Recently, suffocation with
be freely exhaled. There is no relevant stimulation of the nitrogen was applied to enforce a death penalty in Alabama
respiratory center and the feeling of suffocation as a warn- [227].
ing signal as well as defense reactions are missing. This Methane: Besides the danger of explosion, methane led
together with the easy availability is the main reason that also to death of coal miners by asphyxia [228]. The ­CH4
helium is often used in euthanasia procedures. Helium was concentrations in cardiac blood of 21 victims determined
also described in a homicide by asphyxiation of three chil- by GC–MS were 6.8–26.8 µL/g. For interpretation, animal
dren after sedation with doxylamine [219]. For determina- experiments at different methane concentrations in air were
tion by GC–MS, the usual carrier gas helium was replaced performed. A suicide by breathing from a tube linked to
by nitrogen or hydrogen [219–222] or by a special gas inlet the domestic methane gas system [229] and a homicide of
of the MS [218]. In one case, the concentrations in gas were a husband by his wife after sedation with lorazepam [230]
20% from the trachea, 12% from the lung, and 1.5% from were described.
the stomach [218], far above the natural concentration in air
of 0.0005%. Narcotic action of volatile substances and solvent abuse
Argon: Only each one accidental and one suicidal asphyx-
iation death case were described for argon [209, 222]. The Medical use and mechanism of action: The medical
accident of a 31-year-old engineer occurred during repair use of volatile anesthetics such as nitrous oxide (­ N2O),
work in the reaction vessel of a bulb factory which was filled diethyl ether, and chloroform has been known since the
with argon (Fig. 20). The analytical detection of argon in the middle of the nineteenth century. Ether and chloroform
body of the diseased was complicated by 70-min resuscita- were replaced by mainly fluorinated substances with low
tion with artificial respiration and by the natural content of boiling point such as halothane, isofluorane, desfluorane,
0.93 vol% Ar in air. Nevertheless, increased Ar concentra- and sevofluorane. However, there are many other volatile
tions were determined in the trachea, as well as in heart substances with a quite different structure and a narcotic
blood as compared to nonexposed bodies. and (at lower concentrations) intoxicating effect. The
Nitrogen: Accidental or also increasingly suicidal cases anesthetic mechanism is not yet completely known [231].
of suffocation in N­ 2 atmosphere were reported [223–226]. The only common property of all narcotic acting vola-
­N2 sources were liquid nitrogen, nitrogen steel cylinders, or tiles is their high lipophilicity. Therefore, it was assumed
scuba nitrogen tanks. Because of its high concentration in that they penetrate the nervous cell membranes, lead to an
air, the analysis of N ­ 2 in postmortem cases still represents increased membrane fluidity (expansion), and indirectly
a major challenge in forensic toxicology. Increased N ­ 2 con- disturb the imbedded ion channels and receptors [232].
centration or N­ 2/O2 concentration ratios in the respiratory Newer research also suggests a direct action on diverse
receptors [233].
Volatile substance abuse: The phenomenon of deliber-
ate inhalation of the vapor of volatile substances in order
to become intoxicated is known as glue sniffing, inhalant
abuse, solvent abuse, or volatile substance abuse [234,
235]. It had a particularly high prevalence in European and
American countries in the 1980s and 1990s. For instance,
in the UK in 1985, 6.1% of 11- to 19-year-olds had tried
volatile substance abuse and a further 0.7% were current
abusers [234]. A large variety of solvent containing domes-
tic and sanitary products are abused such as fuel gases, pet-
rol, paints and paint thinners, paint strippers, spot removers
and dry cleaners, adhesives and glues, nail varnish and
nail varnish remover, deodorants, antiperspirants, and hair
lacquers.
Fig. 20  A 31-year-old engineer was found dead in this vessel (height There are different practices of inhalation depending on
1.8 m, diameter 0.8 m) in a bulb factory some minutes after he had the abused product. For instance, contact adhesives are usu-
entered it for maintenance work. It was filled with argon from the ally poured into plastic bags or paper bags. The top is then
previous production step. Influx of compressed air through the black gathered together and placed over the mouth and the vapor
tube was not efficient to replace the heavier argon. Suffocation in the
argon atmosphere was determined as the cause of death (from [209], is repeatedly in- and exhaled. The inhalation leads to eupho-
© Elsevier with permission) ria, disinhibition, and a feeling of invulnerability. At higher

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ChemTexts (2025) 11:4 Page 31 of 95 4

doses, bizarre and frightening hallucinations may occur. The preferably involved substance was chloroform which has
Continued inhalation can cause a severe solvent intoxication a long history in criminal abuse [241]. A cloth, e.g., a hand-
with ataxia, impaired speech, eye movement disorder, som- kerchief, soaked with the toxic liquid is forcibly held over
nolence, increasing unconsciousness, and coma. Repeated mouth and nose of the victim and leads to unconsciousness
solvent abuse leads to addiction and chronic impairments. within a few seconds. Because of the high cardiotoxicity of
The major risk associated with volatile substance abuse is chloroform, sudden death may occur by cardiac arrest. In
that of sudden death. In Great Britain, 1396 deaths from a robbery case in which the dust cloth was found still over
volatile substance abuse were reported between 1971 and the mouth and nose of the dead victim, MHE-HS-GC was
1993 [234]. Besides aspiration of vomit and asphyxia associ- used for the analysis [242]. The high chloroform concen-
ated with the use of a plastic bag, cardiac arrhythmia leading trations in blood (200–280 µg/mL), brain (770 µg/g), liver
to cardiac or cardiorespiratory arrest is presumed to cause (800 µg/g), lung (190 µg/g), and kidney (250 µg/g) indicated
most deaths. a prolonged inhalation before death. The higher blood con-
Figure 21 shows a case of lethal chloroethane inhalation centration in the left ventricle (280 µg/mL) in comparison to
through a military gas mask from an ampulla, which origi- the right ventricle (200 µg/mL) showed that the poison was
nally was intended for local anesthesia. The concentrations still inhaled when the victim died.
in blood and lung tissue determined by HS-MHE-GC were
37 µg/mL and 43 µg/g [210]. Much higher concentrations Headspace solid phase microextraction HS‑SPME
were measured in heart blood in a sniffing case with propane
(490 µg/mL) and in an industrial accident with acetylene Solid phase microextraction (SPME) is a miniaturized and
(490 µg/mL) [210]. solvent-free sample preparation technique for chromato-
A frequently abused sniffing gas is nitrous oxide ­(N2O, graphic–spectrometric analysis by which the analytes are
laughing gas) [236]. It is easily available in ampoules for extracted from a gaseous or liquid sample by absorption in, or
whipped dairy cream dispensers. The reported effects are adsorption on, a thin polymer coating fixed to the solid surface
lowered consciousness, dizziness, deformation of vision of a fiber, inside an injection needle or inside a capillary [243].
and sound, better known as psychedelic effects, accompa- The headspace extractions on fibers (HS-SPME) or needles
nied by euphoric feelings which explain the label “laughing (solid phase dynamic extraction, HS-SPDE) combined with
gas”. The lethal course of the recreational use of ­N2O was GC methods (preferentially GC/MS) are the most advanta-
frequently caused by suffocation and not by toxic effects geous versions of SPME because of very pure extracts and
[237]. Chronic impairments, caused by long-term excessive the availability of automatic samplers. The principle is shown
use, can be neurological deficits including degeneration of in Fig. 22. Surprisingly, substances with boiling points well
the spinal cord. ­N2O is extensively used as an anesthetic above 250 °C which are usually not found in headspace, such
gas in surgery and inadvertent fatal hypoxemia resulting as methadone, tricyclic antidepressants, or phenothiazines,
from technical errors in this application are described can be extracted by HS-SPME from aqueous samples. Besides
[238]. volatility, the hydrophobicity of the analyte is an important
Criminal use of the anesthetic effect of volatile substances prerequisite for this. A possible explanation is that these “sem-
occurred in sexual assault, robbery, and murder [239–241]. ivolatile” hydrophobic substances are accumulated at the sur-
face of the aqueous phase and are transported by tiny droplets
formed above the heated sample to the fiber. The applicability
and sensitivity of SPME in combination with GC–MS was
essentially extended by in-sample or on-fiber derivatization.
Many specific and very sensitive HS-SPME–GC–MS
methods were developed for determination of illegal and
medical drugs, pesticides, or alcohol markers from human
samples. It proved to be, for example, a suitable technique
for the fast detection of γ-hydroxybutyric acid (GHB) in
blood or urine which is to an increasing extent abused for
recreational purposes and as a “knockout substance” in drug-
facilitated crimes. Since this substance is very hydrophilic, it
is transformed into the more volatile γ-butyrolactone (GBL)
for HS-SPME by addition of an acid to the sample [244].
Fig. 21  Sniffing death by inhalation of chloroethane from an ampulla However, HS-SPME proved not to be a broadly applicable
through a military gas mask. The C ­ 2H5Cl concentrations in blood and
lung tissue determined by HS-MHE-GC were 37 µg/mL and 43 µg/g sample preparation technique for systematic toxicological
[210] analysis, and the majority of the “semivolatile” substances

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4 Page 32 of 95 ChemTexts (2025) 11:4

Fig. 22  Headspace solid phase

microextraction (HS-SPME).
The coated fused silica fiber
is fixed on a stainless steel rod
and is withdrawn in the syringe
needle for injection through
the septum and is exposed for
absorption and desorption

are equally detected by the methods described in the next For identification of loose tablets or capsules, e.g., found
section. beside the corpse or undecomposed in the gastric content,
the “Yellow List Identa” app enables fast identification of
Nonvolatile organic toxic substances the medication [246]. Moreover, the fast identification of
medical and illicit drugs in the form of tablets, powders, or
As was shown in Sect. “Diversity of toxic substances and liquids can be performed by a portable FTIR (Fourier trans-
toxicological relevance”, the majority of toxic compounds form infrared) spectrometer equipped with a diamond ATR
are nonvolatile organic substances. They are usually solids (attenuated total reflection) sample interface in combination
or high-boiling liquids, have a molecular mass < 1000, and with a database of hazardous substances and illegal drugs
do not considerably evaporate at room temperature. The [247]. This technique is regularly used in police laboratories
molecules have a chain-shaped and/or cyclic carbon skel- with a high occurrence of seized drug samples, but in toxi-
eton with the possibility of nitrogen, oxygen, and/or sulfur cological laboratories, these samples are usually analyzed
included in this skeleton and hydrogen as well as various by the same methods as described below for blood or urine.
heteroatom-containing functional groups and substituents For systematic toxicological analysis from human sam-
bound to it. The rules of chemical bonding between these ples, the ab initio structural identification is completely inap-
atoms enable a myriad of different structures which only propriate considering the complexity of samples from which
partly exist in reality. the unknown toxic substance (if there is any) must first be
Today, the structure of any organic compound can be clar- isolated in pure state and in sufficient amount. Instead, ana-
ified by combined application of up-to-date spectroscopic lytical procedures were developed which include as many
methods such as MS, NMR, UV, and IR spectrometry. How- as possible toxic substances and consist of a suitable sam-
ever, despite all progress, this ab initio structural elucidation ple preparation followed by chromatographic separation of
is still a time-consuming and very expensive approach. In potentially present poisons and identification of the chro-
forensic toxicology, it is used for the first identification of matographic signals by mass and/or UV spectroscopy and
seized unknown new psychoactive substances (NPS), when library search in large spectroscopic databases of toxic com-
the pure compound is available in sufficient amount [245]. pounds. However, these techniques were only enabled by the

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ChemTexts (2025) 11:4 Page 33 of 95 4

rapid development of analytical instruments and computer substances in 1856 by the German chemist and pharmacist
technology in the last few decades and are based on a long Friedrich Julius Otto (1809–1870) [248]. For identification
and laborious development. of the isolated alkaloids, besides odor and taste, color tests
or precipitation tests with F­ eCl3, ­AuCl3, ­PtCl4, curcuma
History of organic poison detection and Stas–Otto paper, iodine tincture, ­HIO3, chlorine water, conc. ­HNO3,
separation procedure chromosulfuric acid, and others allowed one to distinguish
between, e.g., atropine, brucine, colchicine, coniine, mor-
Historically, the proof of intoxication by organic poisons phine, nicotine, strychnine, and veratrine.
relied for a long time on the circumstances of death, a com- The Otto method was based on solubility differences in
bination of signs and symptoms in life, and internal and ether or chloroform at acidic and alkaline pH. Concerning
external appearances after death. At that time, organic poi- the acid–base behavior, organic substances can be classi-
sons originated exclusively from plants, and merely a thor- fied as:
ough inspection of the gastric content for plant residues such
as fruits, leaves, or stems could give some clues. This situ- – Basic substances with –NH2, –NRH, –NR2, =N–H, =N–R
ation changed when the Belgian chemist Jean Servais Stas or heterocyclic nitrogen, forming cations by protonation
(1813–1891) isolated and identified nicotine in the famous at acidic pH
murder case of the count de Bocarmé in 1850 [16]. The – Acidic substances with –COOH, phenolic –OH, –SH,
murderer had doused the corpse of the victim with vinegar –CO–NH–CO–, forming anions by deprotonation at alka-
in order to cover the nicotine smell, and, after autopsy, alco- line pH
hol was added to the gastric content and the organ samples – Amphoteric substances with both basic nitrogen and a
before sending them to the laboratory of Stas. This combina- –COOH group are always in ionic state (cationic, zwit-
tion of alcohol and acetic acid led to protein precipitation, terionic, or anionic, depending on pH)
and, when Stas distilled the remaining fluid with KOH, he – Neutral substances without any of these groups are
recognized the smell of “mouse urine” which he knew to always in nonionic state independent of pH
be typical for nicotine. He identified the alkaloid with spe-
cific color reactions known at this time. On the basis of this Examples are shown in Fig. 23. Relatively nonpolar
evidence, the murderer was executed by guillotine in Mons and with water immiscible solvents like ether or chloro-
in 1851. form extract the organic substances from aqueous solution
The extraction procedure for basic alkaloids developed only in the lipophilic nonionic state, i.e., basic substances
by Stas was extended to the isolation of neutral and acidic in non-protonated state at alkaline pH, acidic substances in

Fig. 23  Examples of basic, neu-

tral, and acidic substances. The
different acid–base properties
are important for separation by
liquid–liquid extraction

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4 Page 34 of 95 ChemTexts (2025) 11:4

non-deprotonated state at acidic pH, and neutral substances liquid phase was evaporated and the residue dissolved
always independent of pH. Besides acid–base properties, in 50 mL water. This solution was then separated by
the solubility in water as well as in organic solvents depends altogether five liquid–liquid extraction steps in separa-
also on molecular size, polarity, and hydrophilicity of the tion funnels into the five fractions of different acid–base
substance. Most natural constituents of the human sample properties. Aliquots of these extracts were then spotted
material such as proteins and carbohydrates remain undis- together with selected standard substances on altogether
solved, and fats are removed by re-extraction into aqueous 11 TLC plates which were run in six different mobile
phase at appropriate pH. phases and developed by observation under UV light
When the number of possible poisons steadily grew, and spraying with nine different reagents. Identification
mainly due to synthetic drugs, the Stas–Otto method was occurred by identical R f values and color of the spots.
further optimized, and ensuing separation of the fractions The TLC plates were manually prepared until commer-
by paper- and thin-layer chromatography (TLC) was intro- cial plates on aluminum foil became available. The TLC
duced. For identification, physicochemical methods such as conditions were chosen in reference to a running project
UV or IR spectra were used [249, 250]. Essential contribu- of the Deutsche Forschungsgemeinschaft (DFG) and
tions to this were also done by the English toxicologist Alan the International Association of Forensic Toxicologists
Stewart Curry (1925–2007) [251]. (TIAFT) [252]. For semiquantitative estimation of the
The procedure used until 1990 in the Institute of Legal concentrations, the weight of the extracts and the size
Medicine of the Charité Berlin is shown in Figs. 24 and and intensity of the TLC spots in comparison to the spots
25. Fifty mL blood, 30 mL gastric content, and a mixed of a series of standard concentrations were applied, or
tissue sample of 100 g liver and 100 g kidney, were the undeveloped spots were extracted and measured by
levigated in a mortar with anhydrous N ­ a 2 SO 4 and for UV/VIS spectrometry.
12 h incubated with 200–300 mL tartaric acid in etha- This very labor-intensive procedure required more than
nol. After filtration and addition of 20 g (­ NH 4)2SO4, the 1 week of a technical assistant’s time per case and huge

Fig. 24  Modified Stas–Otto extraction scheme for postmortem samples as it was performed in the author’s laboratory in the year 1987

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ChemTexts (2025) 11:4 Page 35 of 95 4

Fig. 25  Postmortem sample

preparation in the author’s labo-
ratory in 1987. a Homogeniza-
tion of a mixed liver-kidney
sample with a kitchen homog-
enizer. b Liquid–liquid extrac-
tion with a laboratory shaker. c
Thin-layer chromatography. d
Thin-layer plate after spraying
with Dragendorff reagent

amounts of solvents and reagents were consumed which at two wavelengths were used as the search parameters
were a health hazard for the analyst. And, it fulfilled less for identification [255–257]. Later, more subtle computer
and less the requirements with respect to the increas- methods were developed for calculation of spectra simi-
ing number of possible poisons as well as to the neces- larity (see below).
sary sensitivity and accuracy. Therefore, it was a relief HPLC–DAD has become a kind of workhorse in foren-
when combined chromatographic-spectrometric methods sic and clinical laboratories. Its advantages are high
became available. robustness, easy handling, lower investment and running
costs, exact spectral reproducibility, long-term reproduc-
High performance liquid chromatography with photodiode ibility of concentration–absorbance relationships, low
array detector (HPLC–DAD) sensitivity to matrix interference by components such
as fatty acids, cholesterol, or carbohydrates, high detec-
The invention of high pressure liquid chromatography, tor sensitivity, and, as shown further below, high UV
later renamed high performance liquid chromatography, spectral specificity. The steps of a systematic toxicologi-
in the 1960s and its broad availability since the 1980s led cal analysis with this technique from sample preparation
to its increasing application also in forensic toxicology to the qualitative and quantitative results are shown in
[253]. UV or UV/VIS detectors were preferentially used Fig. 26 and shall be explained in detail below.
besides electrochemical (ECD) and fluorescence detec- For efficient use of this technique an HPLC–DAD
tors (FLD). With the primarily used fixed wavelength database of more than 3300 toxicologically relevant
detectors (e.g., 254 nm with high pressure mercury lamp) substances was compiled by the author of this paper and
or variable wavelength detectors, only the retention time his colleagues [258]. A data set of a compound consists
under constant conditions of the improved chromato- of a substance code, the international non-proprietary
graphic separation could be used for substance identifica- substance name or the IUPAC name, the CAS number,
tion [254]. The development of the computer-controlled the structural formula, the main effect or use, the UV
diode array detector (DAD) enabled the continuous spectrum between 195 and 380 nm (Shimadzu version)
measurement of the UV/VIS spectrum over the whole or 195–400 nm (Agilent version), maxima, minima, and
chromatogram and, therewith, its additional use for elu- shoulders of the spectrum, the relative retention time
ent identification. In first applications, maxima, minima, (RRT), and the specific peak area between 220 and
and shoulders in the UV spectrum and absorbance ratios 230 nm. Furthermore, the library is additionally divided

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4 Page 36 of 95 ChemTexts (2025) 11:4

Fig. 26  Steps of systematic

toxicological analysis by
HPLC–DAD

into 12 sublibraries LIB01 to LIB12 according to use or of clinical or forensic cases venous blood (full blood, serum,
effect of the substances in order to specify library search. or plasma) is the most important sample material since the
A typical example of a date set as printed in the manual qualitative and (semi)quantitative results allow immediate
of the library is shown in Fig. 27. Detailed instructions interpretation with respect to the severity of poisoning and the
for practical use are given in the manual of the UV spec- prognosis based on comprehensive reference data available in
tra library. A summarizing example of application will literature. In most cases the parent drug predominates in blood
be given in Sect. “Example of application” and the number of metabolites is limited. Therefore, as a rule,
chromatograms are well resolved even in case of multidrug
Sample preparation for HPLC–DAD Methods were developed poisonings.
for blood, urine, gastric content, and tissues, as well as tablets, There are three principal methods of sample prepara-
powders, syringe contents, etc. In a toxicological screening tion for toxicological screening: liquid–liquid extraction

Fig. 27  Data set of a substance

in the UV spectra library of
toxic compounds [258]

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ChemTexts (2025) 11:4 Page 37 of 95 4

Fig. 28  Sample preparation for

systematic toxicological analy-
sis by HPLC–DAD [258]

(LLE), solid phase extraction (SPE), and online extrac- since they have no essential UV absorption above 195 nm.
tion, from which LLE is widely preferred in HPLC–DAD Furthermore, the operation of the HPLC technique is not
analysis. A simple and very fast procedure for blood spoiled by injection of large excess of these matrix con-
(serum, plasma) samples which is regularly used in the stituents. Cleaner extracts were obtained for basic drugs by
author’s laboratory is shown in Fig. 28 and involves extraction with hexane/isoamyl alcohol (99:1 v/v) and sub-
acidic and basic extraction with ­C H 2Cl2, as well as pro- sequent re-extraction into aqueous HCl [263]. For gastric
tein precipitation with acetonitrile [259–261]. ­C H 2Cl 2 content with high concentrations after oral intake, direct
as a solvent of medium polarity is a reasonable compro- injection into HPLC after, e.g., 1:30 dilution with 0.01 M
mise between extract purity by exclusion of polar matrix HCl was described [264]. An overview of microextraction
constituents and sufficient extraction yield for a broad techniques in analytical toxicology including liquid–liq-
variety of nonpolar or only moderately polar substances. uid extraction was given by Flanagan et al. [265]. It also
Further advantages are the low boiling point (40 °C, includes procedures using methyl t-butyl ether, butyl ace-
easily removable by a nitrogen stream), immiscibility tate, and methyl ethyl ketone as extraction solvents and the
with water, and high density (1.33 g/mL). It forms the salting-out effect by ammonium chloride, ammonium sul-
lower layer after centrifugation which is partly protected fate, sodium chloride, sodium citrate, sodium sulfate, and
against evaporation losses by the upper sample layer but potassium carbonate for encouraging the partition of rela-
must be separated by a syringe. Many drugs are extracted tively water-soluble analytes into an immiscible solvent.
from 0.5 mL blood by only 0.4 mL of the solvent with For solid phase extraction (SPE), columns filled with
yields between 60% and 100% [262]. diatomaceous earth or chemically modified silica are used
Substances with higher hydrophilicity such as paraceta- [266]. A detailed description of these techniques will be
mol or salicylic acid are extracted only with low yield and given in Sect. “Gas chromatography–mass spectrometry”.
reproducibility by C ­ H2Cl2 but are determined with high The principle of SPE using diatomaceous earth is like
accuracy from the supernatant of the protein precipita- the liquid–liquid extraction. The aqueous sample (blood,
tion which is additionally analyzed. Since in most cases urine, tissue homogenate) is completely adsorbed on the
drug–protein binding is destroyed by the interaction with hydrophilic surface of the diatomaceous earth, a porous
acetonitrile, the yield of this sample preparation is often material in the extraction column which acts as support
nearly quantitative. The procedures can be improved for spe- for the aqueous phase. It provides a large surface area for
cial substances or substance groups by use of other solvents partitioning into the elution solvent, which flows through
and/or other pH. the immobilized sample under gravity. The eluate is evap-
It is an advantage of diode array detection that choles- orated to dryness and dissolved in 100 µL of the mobile
terol, fatty acids, lipids, and carbohydrates do not interfere phase for HPLC–DAD measurement. Advantages are

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4 Page 38 of 95 ChemTexts (2025) 11:4

superior recoveries in comparison to conventional LLE,

avoidance of centrifugation, aspiration, and filtration steps,
and the prevention of emulsion formation.
The online processing of sample preparation for
HPLC–DAD was implemented in the Remedi instrument
in the 1990s, which fully automatically analyzed plasma or
serum samples for more than 500 drugs [267]. The manufac-
turer Biorad provided all consumables, and the instrument
delivered semiquantitative results within 20 min. Although
it proved to be useful in clinical emergency cases [268], it
remained a “black box” for the analyst. The instrument was
not broadly accepted, mainly because of the high costs, and
the principle was not further developed.
Column-switching systems, in which a pre-column for
online extraction is coupled to the analytical HPLC, have been
used successfully for the analysis of drugs and metabolites in
biological media and especially in plasma. An example of a Fig. 29  Flow diagram of online solid phase extraction for HPLC–
typical flow diagram is shown in Fig. 29 [269]. DAD with a ten-port valve and two equal extraction columns EC1
Restricted access materials (RAM) proved to be particu- and EC2 [269]. In the presented position, extraction and clean-up
occurs on EC1 whereas the previous sample is eluted from EC2 in
larly suitable for this purpose and were reviewed by Souve-
back-flush direction and separated on the analytical column AC. After
rain et al. [270]. These materials consist of silica particles the valve is switched, EC2 extracts the next sample and EC1 is eluted
with, for instance, a diameter of 25 µm and a pore diam- for separation on AC
eter of 6 nm. The particles are modified on the outside by
alkyldiol silica and within the pores by usual RP materials.
The outer layer prevents adsorption and denaturation of pro- are best fulfilled by mixtures of acetonitrile with acidic phos-
teins and excludes macromolecules (e.g., M > 15,000) from phate buffer. RP8 or RP18 columns (particle size 5 µm) are
adsorption on the internal RP surface, whereas the target mainly used as the stationary phase. Since toxic substances
compounds of lower molecular weight can be extracted. encompass the full range from very hydrophilic to lipophilic
RAM-RP materials for cation and anion exchange were and from highly polar to nonpolar structures, it is difficult to
developed. As alternatives to the continuously used pre- find conditions for elution of all possible poisons in a single
columns, single-use sorbent columns, which are changed chromatographic run with optimal distribution over the run
before each new run in robotic sample processors [271], and time. A gradient elution with acetonitrile in phosphate buffer
the automatic packing of the online microcolumn from a sus- increasing from, e.g., 10% to 70% on an end-capped RP8ec
pension reservoir before each sample [272] were described. column proved to be successful.
In the author’s laboratory, a database of UV spectra and
Separation by high performance liquid chromatography The relative retention times of more than 3300 toxic substances
components of a typical HPLC–DAD device are shown in was compiled between 1992 and 2007 under the follow-
Fig. 30. In the solvent delivery system (reservoir for the com- ing chromatographic conditions: RP8ec, isocratic elution
ponents of the mobile phase, vacuum degassing, binary pump) with phosphate buffer/acetonitrile (pH 2.3) at three volume
a controlled flow rate and composition of the mobile phase is ratios 63:37 (A), 37:63 (B), and 20:80 (C), wavelength range
generated. A defined volume of the sample is injected into this 195–380 nm [258–262]. The retention times were related to
flow by the autosampler. The separation of possibly present a reference compound for each A, B, and C. This is the only
toxic components of the sample occurs on the HPLC column at commercially available HPLC–DAD database of toxic com-
controlled temperature in the column compartment. Finally, in pounds worldwide. The application in systematic toxicologi-
the photodiode array detector, UV spectra of the effluent from cal analysis must be performed under the same HPLC–DAD
the column are measured, e.g., every 1 s, and stored to form the conditions. The substance identification in the search analy-
three-dimensional HPLC–DAD chromatogram. sis is based on both the relative retention time RRT​and the
The mobile phase used for HPLC–DAD must be opti- UV spectrum. The relative retention time is used to com-
cally transparent above 195 nm to exploit the full wavelength pensate experimental differences, e.g., in column length or
range of the absorption spectra. Furthermore, all measure- flow rate, and is calculated for substance X from the absolute
ments (of samples as well as of reference substances) must retention time of X RTX, of the reference standard RTStand,
be performed at a constant pH since the UV spectra of acidic and the death time t0 (time of unretained peak) as follows:
or basic substances mostly depend on pH. These conditions

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ChemTexts (2025) 11:4 Page 39 of 95 4

Fig. 30  HPLC–DAD device in overview. a Components of an Agilent HPLC–DAD instrument. b Computer control of all parameters

(7) challenged improved technologies on all parts of instrumen-

( )/ ( )
RRTx = RTx − t0 RTStand − t0
tation. Smaller peaks and shorter run time (< 5 min) require
Considering the large number of possible poisons, the a higher sampling rate of the DAD to have sufficient points
contribution of RRT for substance identification is relatively per peak. This can decrease the quality of the UV spectrum.
small, and the main contribution is accomplished by the UV
spectrum which is continuously measured by the photodiode Photodiode array detector The photodiode array detector
array detector (DAD). is a single-beam spectrophotometer with a flow cell. As an
If, different from the database, a gradient with the same example, the light beam of the Agilent detector is shown in
components (acetonitrile/phosphate buffer pH 2.3) or Fig. 31a. Detectors from other manufacturers differ in some
another column is used, the UV spectra of the database can details but deliver spectra of equal quality. For covering the
still be used since they do not or only marginally depend whole UV and visible range, a tungsten and a deuterium
on the buffer/acetonitrile ratio. Of course, RRT is unusable lamp are arranged in line as light sources. Since only a very
then. small portion of the toxicologically relevant substances pro-
With the intention to increase chromatographic resolu- vides light absorption above 400 nm, the wavelength range
tion and to shorten measurement time, ultra performance of the deuterium lamp is generally sufficient for toxicologi-
liquid chromatography (UPLC) methods were developed cal investigations. The light is directed onto the flow cell by
using particle sizes < 2 µm [273–275]. This is also important an achromatic lens system, passes a slit, and is dispersed by
for LC–MS/MS applications (Sect. “Liquid chromatogra- a holographic grid according to wavelength. The dispersed
phy–mass spectrometry”). The smaller particles dramati- spectrum is recorded wavelength by wavelength by the pho-
cally increase column efficiency (mass sensitivity, analyti- todiode array, a silicon chip with 512 or even 1024 parallel
cal resolution, and speed). A typical 2.1 × 100 mm RP8 or photodiodes (Fig. 31b).
RP18 column of 1.7 µm particle size with a flow rate of The primary spectra obtained by reading out the
0.4 mL/min requires pressures > 1000 bar (> 100 MPa). This diodes every 10–40 ms are averaged over, e.g., 0.5 s and

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4 Page 40 of 95 ChemTexts (2025) 11:4

Fig. 31  a Light beam of a

photodiode array detector. b
Photodiode array chip

transformed into final spectra by correction for the dark sig- On the first view, UV spectra look not very specific as
nal of the diodes and calculation of the absorbance accord- they have only one or two maxima and minima, sometimes
ing to Lambert–Beer’s law by use of the light intensity of the even not that. However, the substance identification is not
mobile phase which is measured at the start of the chroma- based on maxima and minima alone but compares wave-
togram. The wavelength accuracy is usually < 0.5 nm, and length by wavelength the whole shape of the spectrum with
the wavelength resolution 0.7–1.2 nm/diode. The digital that of the reference compounds. In case of, e.g., a spectrum
spectrum obtained from the chromatographic eluent every ranging from 200 to 380 nm, this are at least 181 wave-
0.5 s is a list of the absorbance for each nanometer over the length–absorbance pairs. Mathematical approaches for this
measured wavelength range and is stored on the computer. spectral matching were reviewed by Sievert and Drouen
At the end of a chromatographic run, a file is stored on the [276].
computer as a three-dimensional graph of absorbance vs. A preferred model for the assessment of spectral simi-
wavelength and time (Fig. 32). For qualitative and quantita- larity uses the description of the spectrum as a vector in
tive evaluation of the peaks, sections are extracted from this n-dimensional space, where n is the number of absorb-
file in the absorbance–time plane at constant wavelength ance–wavelength pairs. The principle is shown in Fig. 33
(chromatogram) or in the absorbance–wavelength plane at using only three wavelength–absorbance pairs in three-
constant time (UV spectrum). dimensional space. The direction of the vector in space is
determined by the specific shape of the spectrum and is
UV spectrum and substance identification Different from independent of the concentration, and only the length of the
IR, NMR, or mass spectra, the UV/VIS absorption bands vector is proportional to the concentration.
cannot directly be attributed to the structure of a molecule. The similarity index (SI) between two spectra is defined
It is not possible to calculate the exact UV/VIS spectrum in as the cosine of the angle θ between their two corresponding
solution from the structural formula. Therefore, the use of vectors and is numerically calculated by Eq. (8).
UV spectra for peak identification is generally based on the ∑
comparison with reference compounds. Absorption of light 𝜆i [Abs1 (𝜆i )Abs2 (𝜆i )]
SI 1,2 = cos𝜃1,2 � (8)
in the UV/VIS wavelength range arises from the interaction
�∑
∑ 2 2
𝜆i Abs1 (𝜆1 ) 𝜆i Abs2 (𝜆i )
with the electrons of the molecule. In the wavelength range
of 200–800 nm, the conjugated system of π-electrons and
SI1,2 = Similarity index of spectra 1 and 2.
free electron pairs of heteroatoms is essentially responsible
θ1,2 = Angle between vectorized spectra 1 and 2.
for light absorption. The corresponding part of the molecule
Abs1(λi) and Abs2(λi) = Absorbance of spectra 1 and 2 at
is referred to as the chromophore. Saturated substances such
wavelength i.
as alkanes or aliphatic alcohols have no light absorption
i = Every wavelength in the compared range, e.g.,
above 200 nm.
200–380 nm.

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ChemTexts (2025) 11:4 Page 41 of 95 4

Fig. 32  Three-dimensional chromatogram obtained by HPLC–DAD of a blood extract in an emergency case. a Three-dimensional view. b Con-
tour plot. c Chromatogram at a given wavelength or averaged over a given wavelength range. d UV spectrum at a given time-point

Fig. 33  Calculation of the simi-

larity index SI between spec-
tra 1 and 2 using the description
of the spectra as vectors in the
n-dimensional space, for better
understanding exemplified for
three absorbance–wavelength
pairs

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4 Page 42 of 95 ChemTexts (2025) 11:4

Different concentrations lead to the same SI. In case of camazepam would wrongly be considered identical with that
two identical spectra, both vectors point in exactly the same of temazepam.
direction and the SI has the maximum value of 1.0000. In the To evaluate the specificity of UV spectra for substance
computerized post-run library search, the SI values between identification, the similarity indices of all possible pairs
an unknown spectrum and thousands of library spectra are among 2682 substances (about 7.2 × ­106 indices) were cal-
calculated by this elaborate equation within seconds and culated and entered into a similarity matrix [261]. By use
arranged in decreasing order. of the criterion of SI > 0.9990 for identity, 1619 (60.4%) of
UV spectra of organic compounds measured in the same the 2682 substances showed unique spectra. For 399 com-
solvent at constant pH are highly reproducible. However, pounds (14.9%) there was one other and for 175 compounds
in practical analysis, the spectra may be altered to a small (6.5%) there were two further substances each with an indis-
extent by noise, matrix effects, or fluctuations of experi- tinguishable spectrum (Fig. 34d, ochre bars). However, there
mental conditions. This leads only to a small decrease of SI, were also spectra much less specific. For example, 28 sub-
since near its maximum of 1, the cosine function is relatively stances were found to have 15 further candidates each with
insensitive to small changes of θ. Therefore, the threshold an identical spectrum.
value 0.9990 was experimentally determined for spectrum If the less specific relative retention time (RRT​) was used
identity [261]. As an example, the spectrum of temazepam as an additional criterion, the percentage of unambiguously
is compared with the spectra of three other benzodiazepines identified substances increased from 60.6% to 84.2%, the
in Fig. 34a–c. It can be seen that, despite minor structural number of substances with two hits (positive search results)
difference, all three spectra differ from that of temazepam. decreased from 14.2% to 10.6% and of those with three posi-
When the threshold of 0.9990 is used, only the spectrum of tive hits from 7.0% to 2.6% (Fig. 34d, blue bars). There were

Fig. 34  UV spectra similarity in HPLC–DAD. a–c Comparison of from temazepam. d Frequencies of identical spectra from 1993 com-
temazepam with camazepam, flurazepam, and lormetazepam. The pounds by using the threshold of the similarity index ≥ 0.9990 for
spectra of flurazepam and lormetazepam are regarded as different identity

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ChemTexts (2025) 11:4 Page 43 of 95 4

no compounds with more than six hits. Comparison of the

discrimination power in that study confirmed HPLC–DAD
to be a highly selective method for substance identification
with a discrimination power similar to that described for
GC–MS with much smaller substance numbers [277, 278].
In practical analysis, the overlapping of chromatographic
peaks can seriously change the spectrum. Therefore, the
peak purity is automatically checked and displayed before
library search. The “pure peak” criterion is the uniformity of
the spectrum over the whole peak length. In case of overlap-
ping peaks, the undisturbed spectra of both components can
often be extracted at the leading or the tailing of the peak.
Otherwise, a change of the chromatographic conditions (e.g.,
of the buffer/acetonitrile ratio) may lead to a separation of
both overlapping components.

Metabolites in HPLC–DAD Metabolite peaks occur in most

HPLC–DAD chromatograms from blood samples of poi-
soning cases and can often be identified by UV spectrum
and shift of the retention time in relation to the parent com-
pound. Concerning the spectrum, there are two possibilities
as shown in Fig. 35.

– The UV spectra of the metabolites are very similar or

even identical to those of the drug, if the reaction does
not occur at the chromophore. This is the case, e.g., for
nortriptyline formed by demethylation from amitriptyline
(Fig. 35a) and is found in the majority of metabolization
reactions.
– The UV spectra are different from those of the drug if
the reaction occurs immediately at the chromophore. The
metabolization is bound to a change of the light-absorb-
Fig. 35  UV spectra of metabolites. a The UV spectra of the metabo-
ing conjugated electron system. Examples are the forma- lites are very similar or even identical to those of the drug, if the reac-
tion of pentobarbital from thiopental (Fig. 35b), aromatic tion does not occur at the chromophore, example demethylation of
hydroxylation, oxidation of aromatic methyl groups to amitriptyline. b The UV spectra of the metabolites are different from
carboxylic groups, or reduction of aromatic nitro groups those of the drug if the reaction occurs at the chromophore, exam-
ple hydrolysis of the C=S group of thiopental. Chromophore = con-
to amino groups. jugated system of π-electrons and free electron pairs at heteroatoms

The shift of the retention time on RP columns can also

help to allocate a peak to a certain metabolite [279]. A
general task of the metabolization of xenobiotics is to The allocation of a peak to a metabolite is an additional
favor the renal excretion by increasing the hydrophilicity. confirmation of the drug identification. Furthermore, it
In view of the retention mechanism on reversed phases excludes that the peak originates from another poison.
this leads to a shortening of RRT​. This is valid, e.g., for
hydroxylation, N- or O-demethylation, reduction of nitro Quantification with HPLC–DAD In a general unknown
to amino groups, and formation of glucuronides and sul- investigation, one or more substances may be detected
fates of the parent compound. The more seldom decrease and, particularly in emergency cases, results, including
of the hydrophilicity (increase of RRT​) occurs particularly a statement about the concentrations, are often required
in acidic mobile phases by a desamination, if the proto- within a short time. However, calibration and validation
nated ammonium group (–NH3+ or –NRH2+ or –NR2H+) of the quantification cannot be carried out for all possible
is replaced by the less hydrophilic hydroxy group. compounds in such a way as is performed, for instance, in
therapeutic drug monitoring where large series of samples
containing the same analyte are analyzed. On the other

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4 Page 44 of 95 ChemTexts (2025) 11:4

hand, in clinical intoxications, a certain deviation from the acidic and basic ­CH2Cl2 extraction from blood (Fig. 28). In
real value can be tolerated considering the large biological most cases, between 60% and 80% were found. The use of
variability of concentration–symptom relationships. specific peak areas is particularly favorable for the protein
In comparison to mass spectroscopic methods, precipitation for instance with acetonitrile since the recovery
HPLC–DAD has the advantage of excellent reproducibil- of this sample preparation is very close to 1 for many drugs.
ity of the peak areas measured under constant chromato- Although this is only a rough approximation, it proved
graphic conditions since there are no essential fluctuations to be very helpful and sufficient for a decision about the
in the absorbance measurement. A further advantage is the therapy under the time pressure of emergency cases. Nev-
proportionality between concentration and UV absorbance ertheless, only preliminary results can be obtained in this
(validity of the Lambert–Beer’s law) over a large concen- way, and it is necessary to be aware of the limitations of this
tration range. There are two possibilities to obtain semi- procedure.
quantitative results from HPLC–DAD peak areas under
these conditions in a relatively short time: the method of
calibrated standard addition and the use of specific peak Example of application
areas for a defined wavelength range.
The principle of the calibrated standard addition was HPLC–DAD was regularly used in the author’s laboratory
explained in Sect. “Headspace gas chromatographic techniques”. for systematic toxicological analysis in death cases as well
A prerequisite of this method is that the reference substance is as in clinical emergency cases with suspected poisoning or
available in the laboratory. It is an advantage of the method of unknown reason for death or illness. The application shall
calibrated standard addition that the specific effects of the sam- be demonstrated by the example of a 72-year-old woman
ple under investigation on the yield of the sample preparation who was found in her apartment in unconscious state and
are taken into account. brought to the rescue center of the hospital. The blood sam-
During the profiling of the HPLC–DAD database [258], ple was liquid–liquid extracted according to the procedures
the peak areas (averaged over the defined wavelength range of Fig. 28. HPLC–DAD was performed for the basic and
of 220–230 nm) obtained after injection of 1 µg of all sub- the acidic extract (Fig. 36). Library search of all essential
stances present in the database were measured and listed peaks and semiquantitative estimation of the concentrations
in addition to the UV spectra. These specific peak areas from the specific peak areas (Eq. 9) led to the detection of
A1µg (mAU·min/µg) are well reproducible in HPLC–DAD caffeine (1.6 µg/mL), the hypnotic zopiclone (2.9 µg/mL),
and can be used as a one-point calibration for the rough desmethylzopiclone (0.7 µg/mL), and traces of three further
estimation of the concentration by Eq. (9). zopiclone metabolites with similar UV spectrum, diazepam
(0.17 µg/mL), its main metabolite nordazepam (0.30 µg/
Asample × Factor of sample preparation
Csample (𝜇g∕mL) = mL), and traces of the diazepam metabolites temazepam and
A1𝜇g × Recovery of sample preparation oxazepam. Desmethylzopiclone was hidden below the caf-
(9) feine peak in Fig. 36 and became visible only after repeated
C = Concentration. chromatography with decreased acetonitrile/phosphate
A = Peak area. buffer ratio (1:4 instead of 1:2 v/v) and with a better separa-
Factor of sample preparation = portion of the extract of tion of caffeine and zopiclone. Furthermore, a small amount
1 mL or 1 g sample which is finally injected into HPLC. of hydrocortisone was detected, which is always present in
For instance, in the dichloromethane extraction (Fig. 28) blood as an endogenous substance. Diazepam was confirmed
0.5 mL blood is liquid–liquid extracted, only 50% of the by a positive immunoassay test for benzodiazepines.
organic phase is separated, and only 50 % of the dissolved The result was interpreted as a high overdosage of the
residue after evaporation is injected into the HPLC sys- sleeping drug zopiclone (comatose-lethal range above
tem), the injected amount is 12.5 % of the extract of 1 mL 0.6–1.8 µg/mL [280, 281]) which fully explained the uncon-
(0.125 mL). In this case, the factor of sample preparation scious state of the patient. The toxic effect was augmented
is 1/0.125 = 8. by diazepam (therapeutic range 0.2–2.0 µg/mL [280, 281])
Recovery of sample preparation = Extraction yield. and its main metabolite nordazepam. The high nordazepam/
A prerequisite of the use of tabulated specific peak areas diazepam ratio was explained by a chronic intake of diaz-
A1µg is the exact agreement between the spectrum measured epam or one occurring several hours prior. The caffeine
in the application and the library spectrum. The most serious concentration was in the range of usual coffee or tea con-
errors in using Eq. (9) arise from not knowing the recovery sumption. The result and interpretation were conveyed to
of the sample preparation (extraction yield). This was deter- the clinic about 2 h after the sample reached the laboratory.
mined for about 100 frequently occurring drugs with the

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ChemTexts (2025) 11:4 Page 45 of 95 4

Fig. 36  HPLC–DAD chromatogram and UV spectra obtained for the result was interpreted as a high overdosage of the sleeping drug zopi-
blood sample of a 72-year-old woman who was taken to the rescue clone (comatose-lethal range above 0.6–1.8 µg/mL)
center in comatose state after suicide attempt with medicines. The

Gas chromatography–mass spectrometry (GC–MS) for substance identification, which were measured under
identical GC conditions. However, in view of the large
Historical role of gas chromatography in toxicological number of possible toxic substances, reference standards
screening Gas chromatography (GC) on packed and cap- were not always at hand. In order to get some clue from the
illary columns was already previously used for toxicologi- retention time also in the absence of reference substances,
cal screening [282, 283]. Because of the higher chroma- the retention indexes IK and IL were introduced, in which
tographic resolution, capillary columns were preferred. A the retention time is normalized using the homologous
prerequisite for the use of this technique is that the analyte n-alkane series as the retention references according to
is evaporated in the injection port of the gas chromatograph Eq. (10) [286].
without decomposition. This is not possible for many non-
log(tR(S) ) − log(tR(n) )
volatile organic poisons. Therefore, sophisticated meth- IK (S) or IL (S) = 100n + 100 (10)
ods for derivatization of the instable groups such as –OH, log(tR(n+1) ) − log(tR(n) )
–COOH, –NH2, –NHR, or –SH, often a combination of
IK(S) = Kováts retention index for compound S, valid for
acylation and alkylation or a trimethylsilylation, were devel-
isothermal GC conditions
oped which coincidently lowered the evaporation tempera-
IL(S) = Linear retention index for compound S, valid for
ture of the analytes [284, 285].
isothermal GC conditions, valid for linear increase of the
The detection occurred mainly by flame ionization
temperature during the chromatographic run
detector (FID). Particularly for the prevailing nitrogen-
tR(S) = Retention time of the substance S
containing analytes, the nitrogen–phosphorus detector
tR(n), tR(n+1) = Retention times of two adjacent alkanes
was applied with much higher sensitivity for these com-
before and after the compound S
pounds [283]. With these detectors, only the retention time
in comparison to the reference compounds could be used

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4 Page 46 of 95 ChemTexts (2025) 11:4

The Kováts retention index IK was defined for isother- the solvent vapor passes. Then, a temperature ramp (e.g.,
mal GC condition while the linear retention index IL was linear with 20°/min to 300 °C) is applied and the analytes
defined for ramped temperature condition (linear increase of subsequently start moving along the capillary in order of
the temperature during the chromatographic run). Retention their boiling points, are further separated by the interaction
indices of several 100,000 compounds, categorized by capil- with the coating, and go one by one directly from the out-
lary column class, were acquired in general-purpose mass let of the capillary into the 70-eV electron impact (EI) ion
spectra libraries of NIST [287] and Wiley [288] as well as source of the mass spectrometer. The ions formed there are
in the specific toxicological mass spectra library reported by separated by the quadrupole and measured by a secondary
Maurer et al. [289]. electron multiplier. In the scan modus of the instrument, an
The practical use of this index in toxicological casework EI mass spectrum (plot of the ion abundance as a function of
appeared to be tedious. Instead, an in-house list of retention the mass-to-charge ratio m/z) of the column outlet is stored
times under the specific GC conditions of the laboratory was in preselected intervals, e.g., every 0.5 s, on the computer.
generally preferred. Nevertheless, the reliability of substance The result of the GC–MS measurement is a file which con-
identification based on the retention time alone remained tains all mass spectra over the chromatographic run time, e.g.,
low, and additional evidence was always required. Therefore, 2400 spectra in 20 min. The total ion chromatogram (TIC) is
the availability of the computerized combination of capillary the plot of the abundance sum of all ions of each spectrum as a
gas chromatography and mass spectrometric detection was function of the time and can be shown on the computer screen
huge progress. (Fig. 38a, upper window). Every peak indicates a compound
in the sample, and the corresponding mass spectrum can be
visualized in the lower part of the screen by shift of the cursor
Toxicological screening by GC–MS A GC–MS instrument (lower window, peak at 17.38 min).
in overview and a view into the column oven are shown in Besides the molecular ion peak (m/z = 361), the spectrum
Fig. 37. The main components are the gas chromatograph contains isotope peaks which result from the presence of
with the injection port and the column oven, the autosa- natural isotopes, e.g., 2H, 13C, 15N, or 17O, and many frag-
mpler, the quadrupole mass spectrometer (mass selective ment ion peaks. The molecular ion formed by the impact of
detector, MSD), and the computer with the necessary soft- the analyte molecules with 70-eV electrons carries exces-
ware for automatic control of the measurement steps and sive energy which leads to cleavage of bonds and rearrange-
for post-run analysis. A frequently used quartz capillary col- ments. These fragmentations proceed in a way that pref-
umn is HP-5, length 25 m, internal diameter 0.22 mm, inter- erentially cleaves the weakest bonds and the products are
nal coating 0.33 µm copolymer 95% dimethylsiloxane/5% as stable as possible. Therefore, the 70-eV electron impact
polydiphenylsiloxane with 1 mL/min helium as carrier gas. ionization mass spectra are very reproducible and specific
In a screening procedure, the solution of the derivatized to the chemical structure. Sometimes, they are called a fin-
sample, e.g., in methanol, is evaporated at high temperature gerprint of the substance.
(e.g., 300 °C) in the injection port. Since the temperature of The EI fragmentation mechanisms were elucidated
the column oven is still low (e.g., 130 °C), the analytes of the for almost all substance classes. A pioneer in this field
sample at first condense on the front of the capillary whereas was Fred W. McLafferty (1923–2021) [290]. Using the

Fig. 37  Gas chromatography–mass spectromety (GC–MS). a GC–MS instrument in the author’s laboratory in 2001. b View into the column
oven of the gas chromatograph

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ChemTexts (2025) 11:4 Page 47 of 95 4

Fig. 38  Post-run analysis of a GC–MS measurement. a The upper library search and the results are displayed in a new window b with
window shows the total ion chromatogram (TIC). The mass spectrum a list of hits in decreasing order of spectral agreement and the library
of a peak can be visualized in the lower part of the screen by shift spectra for comparison. In the present case, the acetylated derivative
of the cursor. Double-click on this window initiates the automatic of a metabolite of the antidepressant opipramol

fragmentation rules known from this research, one can to the molecular mass. However, this is seldom performed
directly explore structural units and the presence of heter- in practice. Instead, the spectrum of the unknown peak is
oatoms (S, Cl, Br) from the fragment spectra in addition searched in databases of mass spectra. Huge and steadily

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4 Page 48 of 95 ChemTexts (2025) 11:4

growing libraries of 70-eV mass spectra are meanwhile com- These MS spectra libraries are linked to the GC–MS soft-
mercially available for general use and for special fields of ware and allow the fast library search of an unknown peak.
chemical products. In 2023, the Wiley Registry® of Mass In the search procedure, the unknown mass spectrum is com-
Spectral Data contains 873,300 GC–EI-MS spectra [288], pared with all spectra of the library, the degree of agree-
and the NIST/EPA/NIH Mass Spectral Library includes ment is calculated, and the results are displayed in order
394,000 mass spectra [287]. of decreasing agreement. The practical approach is shown
For toxicological analysis, the Maurer, Meyer, Pfleger, in Fig. 38. A double-click on the spectrum area in Fig. 38a
Weber GC–MS library of drugs, poisons, and their metabo- starts a library search in a new window (Fig. 38b), and a
lites proved to be most useful worldwide and the 2023 edi- table shows the first 10 hits with the percentage of agree-
tion contains 10,948 mass spectra and GC retention indices ment. A visual comparison of the spectra is possible and
of toxicologically relevant compounds and their metabo- recommended. In the present case, the acetylated derivative
lites [289]. It is important from an analytical point of view of N-desethylopipramol, a metabolite of the antidepressant
that this library includes the spectra of several derivatives opipramol, has the best agreement of 97%. In newer versions
of temperature-sensitive substances, e.g., methyl-, acetyl-, of the library, the structural formula and the substance class
or trifluoroacetyl-substituted derivatization products, since are also displayed.
such derivatizations are often included in sample preparation
procedures of toxicological screenings.

Fig. 39  Solid phase extraction

(SPE) of blood, serum, or urine
samples for GC–MS. a Steps
of SPE. b Automatic perfor-
mance of SPE in the author’s
laboratory in 2002. c Insertion
of a new SPE cartridge into the
device

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ChemTexts (2025) 11:4 Page 49 of 95 4

Sample preparation for GC–MS The sample preparation and the primarily lipophilic but in part also polar toxic sub-
is crucial for the successful analysis. On the one hand, as stances. Extraction and re-extraction procedures were rec-
many as possible poisons must be included, and, on the ommended to get cleaner extracts for GC–MS [295].
other hand, matrix constituents must be excluded as far as In solid phase extraction, a defined volume, e.g., 1 mL,
possible to avoid capillary plugging and high chromato- of blood, serum, or urine is poured onto a cartridge filled
graphic and mass spectrometric background. In this context, with the solid adsorbent (Fig. 39a) [296, 297]. The ana-
urine turned out to be more suitable than blood for GC–MS lytes are retained by adsorption whereas the liquid sample
screening, mainly because of the low content of disturb- passes through the cartridge. Residues of the sample and
ing lipids. However, a high content of metabolites must be disturbing matrix constituents are removed in ensuing wash-
taken into account. The sample preparation steps are acidic ing steps. Then, the sorbent is dried by an air stream, and
or enzymatic hydrolysis of phase II metabolites (glucuron- the analytes are desorbed with a suitable solvent. The sor-
ides and sulfates), liquid–liquid extraction (LLE) or solid bents used for SPE are silica particles modified by surface-
phase extraction (SPE), and acetylation and/or methylation bound organic groups. Depending on the kind of matrix
[291, 292]. and analytes, there are cartridges of different size with
For liquid–liquid extraction, diethyl ether, methyl t-butyl nonpolar (–C8H17 or –C18H37), aromatic (–C6H5), cyano-
ether, 1-chlorobutane, ethyl acetate, n-hexane, n-heptane, propyl, dihydroxy-propoxypropyl, or aminopropyl groups
dichloromethane, chloroform, and mixtures between these but also groups with strong or weak cation exchange prop-
solvents or with a small percentage of an alcohol were used, erties or of anion exchange properties commercially avail-
which are not miscible with water and have a low boiling able. Mixed mode cartridges (­ C18H37 + cation exchange
temperatures for removal after extraction [293, 294]. A –CH2–CH2–C6H4–SO3−) are preferentially used for basic
medium polarity of the solvent is important for the discrimi- drug analysis. The absorption from the sample occurs at
nation between the mainly hydrophilic matrix constituents pH 6 whereas, after three washing steps, the elution occurs

Fig. 40  GC–MS screening of the postmortem urine sample of a administered during the medical treatment and metoprolol belonged
69-year-old man who survived 13 h after a suicidal self-burning in to his chronic medication. No indications of an influence of toxic sub-
an intensive care unit. Ketamine, fentanyl, and midazolam were stances on his behavior were found

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4 Page 50 of 95 ChemTexts (2025) 11:4

at basic pH by, e.g., ­CH2Cl2/isopropanol/conc. ­NH3 (80:20:2 some disadvantages. Besides the disturbances of the mass
v/v). All SPE steps can be automatically performed by a spectra by matrix masses, the relatively low sensitivity in
computerized device as shown in Fig. 39b. Fully auto- scan mode and the low reproducibility of the absolute chro-
mated methods using SPE online to GC–MS in a bench-top matographic peak areas caused by fluctuations of the many
GC–MS system equipped with a laboratory robot for sample instrumental parameters also make even the estimation of
preparation from blood, urine, and plasma with trimethylsi- the concentration in the absence of the reference substance
lylation were also developed [298]. impossible.

Example of application The complexity of a urine screen- Specific, sensitive, and accurate analysis by selected ion
ing by GC–MS shall be shown with an example. A 68-year- monitoring (GC–MS‑SIM) The computerized operation of
old man attempted suicide by self-burning with petrol in his the mass spectrometer enables one to confine the measure-
garden and died 13 h later in a hospital. A possible influence ment to the characteristic m/z of a limited number of ana-
of toxic substances on his behavior should be cleared. For lytes in the selected ion monitoring (SIM) mode. In this
GC–MS analysis, 1 mL urine was hydrolyzed with glucuro- way, all other disturbing or irrelevant m/z are neglected, the
nidase/arylsulfatase and extracted by automated SPE. The specific registration time of the relevant m/z is increased,
extract was acetylated with acetanhydride/2-dimethylami- and the sensitivity is enhanced by more than two orders of
nopyridine. The measurement of the total ion chromato- magnitude. Therefore, GC–MS-SIM can also be applied to
gram (TIC) was performed under the following conditions: blood and tissue samples. In order to utilize these advan-
1 µL injection; temperature program: 2 min at 70 °C, 15°/ tages, only a selected number of m/z, for instance eight, can
min to 300 °C, 10 min at 300 °C; m/z range 40–600. The be monitored at the same time. For this reason, in order to
TIC shows more than 70 peaks (Fig. 40). measure more analytes, the run time of the chromatogram is
Library search of the mass spectra identified the follow- divided into several time windows with different m/z.
ing substances (Ac = acetyl): ketamine-Ac (highest peak, Furthermore, deuterated standards in which three to
anesthetic), underivatized ketamine, a complex ketamine nine H atoms are replaced by D atoms are used as inter-
metabolite, metoprolol-Ac2 (beta-blocker), midazolam (ben- nal standards. These are commercially available and are
zodiazepine, sedative and anesthetic), 3-hydroxymidazolam- ideal internal standards for GC–MS as they have the same
Ac, and fentanyl (opioid analgesic). Furthermore, glycerol behavior in sample preparation (e.g., extraction yield) and
stearate-Ac2, two matrix-specific impurities, and one peak can be clearly distinguished and separately measured in the
resulting from capillary coating bleeding were seen. The SIM mode. As an example, the mass spectra of morphine-
other peaks showed no reasonable search result. Concen- TFA2 and ­D3-morphine-TFA2 (TFA = trifluoroacetyl) are
trations cannot be obtained from this qualitative screening compared in Fig. 42. On the basis of these spectra, the
and would not be very informative, as for urine in general. masses 364 and 477, and 367 and 480 are chosen for the
According to the patient chart, all drugs were applied in morphine time window. It turned out, that the deuterated
the hospital or part of his chronic medication. Analysis in drugs have generally a somewhat shorter retention time
blood by HPLC–DAD Sect. “High performance liquid chro- than the nondeuterated, obviously because of a lower boil-
matography with photodiode array detector (HPLC–DAD)” ing point.
confirmed the result for ketamine (18.2 µg/mL) and mida- As an example, the analysis of the blood sample of a
zolam (0.23 µg/mL), whereas metoprolol and fentanyl were 39-year-old man for drugs of abuse and their essential
not found in blood. metabolites by GC–MS-SIM in 12 time windows is shown in
The comparison of the sample spectrum and the library Fig. 43. Only methadone (0.60 µg/mL), its metabolite EDDP
spectrum for ketamine-Ac is shown in Fig. 41a. All masses (0.084 µg/mL), and traces of cocaine (0.002 µg/mL) and its
of the library spectrum are also seen in the sample spec- metabolite benzoylecgonine (0.016 µg/mL) were detected.
trum in about the same abundance ratio (99% agreement). Injection of a methadone overdose, promoted by the pre-
However, there are many additional masses in the sample existing tuberculosis, was determined as the case of death.
spectrum which obviously originate from the background GC–MS-SIM can only be used for testing of a relatively
despite SPE as an extraction technique with clean-up effect. small number of predetermined substances and is, for this
This is even more the case for the spectrum of the tiny peak reason, not suitable for a general toxicological screening.
of midazolam at 17.11 min and an agreement of only 41% However, it can successfully be applied for the sensitive con-
(Figs. 40 and 41b). Therefore, additional evidence from firmation and quantitation in case of suspected poisons or
exact agreement of retention time or other methods is often doubtful results of the TIC screening.
required also for this method.
Although GC–MS for a long time was considered to be
the gold standard of systematic toxicological analysis, it has

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ChemTexts (2025) 11:4 Page 51 of 95 4

Fig. 41  Search results in the Pfleger–Maurer–Weber mass spectra 99% agreement with library spectrum. b Tiny peak at 17.11 min, hit 1
library for the postmortem urine sample, total ion mass spectrum see midazolam, only 41% agreement, many matrix masses at lower m/z
Fig. 40. a Most intense peak at 15.1 min, identified as ketamine-Ac,

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4 Page 52 of 95 ChemTexts (2025) 11:4

Fig. 42  Commercial certified

deuterated standards for mass
spectrometry and EI mass
spectra of the trifluoroacetyl
derivatives of morphine and
morphine-D3. For GC–MS-
SIM, at least two specific
masses must be chosen for each
substance

Fig. 43  Drug analysis from blood of a methadone fatality by GC– mL) were positive besides traces of cocaine and benzoylecgonine.
MS-SIM in 12 time windows using deuterated internal standards. For The deuterated standards have a slightly shorter retention time lead-
quantification, each mass is separately monitored. In this case, only ing to a clear peak separation in case of MTDN-D9 and MTDN
methadone 0.60 µg/mL) and its main metabolite EDDP (0.084 µg/

Liquid chromatography–mass spectrometry (LC–MS) tages of liquid chromatography for application to thermo-
labile substances without derivatization, to high molecu-
General features of LC–MS LC–MS combines the advan- lar weight substances, and to ionic organic substances as

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ChemTexts (2025) 11:4 Page 53 of 95 4

described in Sect. “High performance liquid chromatogra- the electrical repellent force between the equally charged ions
phy with photodiode array detector (HPLC–DAD)” with reaches a critical point at which the ions at the surface are
the high sensitivity and specificity of mass spectrometry. ejected into the gaseous phase. They are sampled by a skim-
Because of the lower injected sample amounts in compari- mer cone and accelerated into the mass analyzer.
son to HPLC–DAD, LC columns with higher plate num- Mobile phase: The mobile phase should be completely
ber can be used. Disadvantages in comparison to GC–MS evaporable. Therefore, mixtures of water with methanol
are the higher burden of matrix constituents and, most or acetonitrile and formic acid, acetic acid, and trifluoro-
importantly, that the large excess of mobile phase must be acetic acid (acidic pH), ammonia (basic pH), or ammonium
removed. For these reasons, LC–MS has some particulari- formate or ammonium acetate (neutral buffer) are used. In
ties in comparison to GC–MS and HPLC–DAD. positive ESI, mainly protonated molecular ions ­MH+ are
LC–MS ion sources: Several coupling methods between formed by addition of protons to free electron pairs, besides
LC and MS have been developed to solve the aforemen- adducts with other cations from the matrix such as M–Na+
tioned problem [299, 300]. From these, two techniques of or M–NH4+. Negative ESI leads to deprotonated anions
atmospheric pressure ionization (API)—the electrospray ion (M – ­H+)− and requires dissociable –COOH, –OH, –SH,
(ESI) source and the atmospheric pressures chemical ioniza- or –CO–NH– groups.
tion (APCI) source—as well as a combination of both have In an APCI source, the nebulization of the mobile phase
gained the highest practical importance. occurs in a hot (typically 250–400 °C) vaporizer chamber.
In the ESI source, the mobile phase from the LC column is The spray droplets rapidly evaporate. The gas-phase solvent
sprayed through a capillary tip, which is maintained at a high molecules are ionized by the discharge from a corona nee-
positive or negative voltage (e.g., 2.5–6.0 kV) relative to the dle followed by a charge transfer (protonation or deprotona-
wall of the surrounding chamber (Fig. 44). A mist of highly tion) to the analyte molecules. In principle, the same ions are
charged droplets is generated. The application of a nebulizing formed as in ESI; however, the APCI ionization is comple-
gas (e.g., nitrogen) enhances a higher sample flow rate. With mentary to API-ES since less polar analytes are also ionized.
the aid of an elevated temperature and stream of nitrogen dry- Matrix effects in LC–MS: The efficiency of ion formation of
ing gas, the charged droplets are continuously reduced in size the analytes can be affected by other components of the sample
by evaporation of the solvent. With decreasing droplet radius, if they are not sufficiently removed in the sample preparation.

Fig. 44  Electrospray ionization

(ESI) of an aqueous analyte
solution in LC–MS. a Scheme
of the ion source for posi-
tive ionization. b The analyte
is sprayed through a highly
positive charged needle and the
charged droplets are stepwise
transformed into ions ­MH+ by
solvent evaporation and increas-
ing repellent forces at the drop-
let surface. For negative ESI,
the spray needle is negatively
charged and anions are formed
by deprotonation of the analyte

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4 Page 54 of 95 ChemTexts (2025) 11:4

An ion suppression (decreased ionization yield) is seen more dissociation (CID) is performed to increase the specificity,
frequently, but an ion enhancement is also possible. APCI is less and tandem mass spectrometry is applied to increase the
prone to ion suppression than ESI. As the mechanism, coelut- sensitivity.
ing matrix components may compete for ionization or inhibit For collision induced dissociation, the molecular ions are
efficient ionization in other ways. For example, basicity or high accelerated in an electrical field to a defined energy and then
surface activity of the impurities may decrease the ion concen- collide with argon atoms or nitrogen molecules. In single-
tration at the droplet surface in the ESI source. Matrix effects stage MS, an in-source CID can be conducted in the medium
disturb mainly quantitative determinations but also decrease the pressure range (1–3 torr) between the ion transfer capillary
sensitivity for qualitative detection. Consequently, the testing for of the ion source and the entrance of mass analyzer by accel-
matrix effects is always an important part of LC–MS method erating the ions with an orifice voltage (cone voltage, or
validation. declustering potential) of 10–60 V [309]. The resulting CID
Sample preparation for LC–MS: A wide range of proce- fragment spectra were arranged in a positive and a negative
dures are used for LC–MS sample preparation. This concerns ESI library for general unknown screening [310]. Although
liquid–liquid extraction, solid phase extraction, and protein pre- in-source CID in single-stage MS has the advantage of
cipitation as described in previous sections. The sample amount including all formed ions, it has not found large application
is usually 1 mL urine, whole blood, serum, or plasma, or 1 g because of the elevated noise and additional chemical back-
tissue, although 100 µL or 100 mg or even less is quite sufficient ground of the spectra [311].
in most cases. Simple protein precipitation with acetonitrile has In triple-quadrupole mass spectrometers (Fig. 45), the
the advantage of inclusion of very polar and hydrophilic analytes first and third quadrupoles Q1 and Q3 act as mass filters and
but the disadvantage of more impure extracts. Automatic online the second, the collision cell Q2, causes fragmentation of the
extraction for LC–MS/MS was described for blood after pro- analyte through interaction with the collision gas (Ar or N­ 2).
tein precipitation with methanol/zinc sulfate [301] and for urine The operation of the quadrupoles allows for different scan
[302, 303]. Dried blood or urine spots of 10 or 20 µL were also types from which the product ion scan and the selected reac-
successfully submitted to toxicological screening [304–306]. tion monitoring (SRM) are commonly used. In the product
Dilution of urine with mobile phase (1:10 to 1:50, “dilute and ion scan, the first quadrupole Q1 is set to select a predeter-
shoot”) after addition of deuterated standards can also deliver mined ion of a known mass, which is fragmented in Q2. The
sufficient results [307, 308]. third quadrupole Q3 is then set to scan the entire m/z range,
leading to the fragment ion spectrum of the original ion.
Liquid chromatography with triple‑quadrupole mass spec‑ In the SRM mode, the molecular ion of a predetermined
trometry, LC–QQQ or LC–MS/MS Single-stage LC–MS analyte (precursor ion) is selected in the first quadrupole
techniques have not found greater application in systematic from the total of ions, fragmented by CID in the second
toxicological analysis for two reasons. At first because of the quadrupole (collision cell), and a fragment ion of high inten-
high background noise which enabled only a low sensitiv- sity (product ion) is selected from all fragments by the quad-
ity, and secondly, because the specificity of the exclusively rupole Q3 and detected. Since two stages of mass selection
detected molecular ions ­MH+ or (M − ­H+)− is not sufficient. are utilized, interferences are widely excluded which enables
To overcome these drawbacks, fragmentation of the proto- a high sensitivity and specificity. However, for unambiguous
nated or deprotonated molecular ions by collision induced identification of an analyte, at least two SRMs are required.

Fig. 45  Operation modes of a

triple-quadrupole mass spec-
trometer

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ChemTexts (2025) 11:4 Page 55 of 95 4

In multiple reaction monitoring (MRM), Q1 and Q3 are set 15–20 analytes can be measured at the same time. In order to
to more than one precursor ion/product ion pair. include a higher number of analytes, the chromatogram can
Different from in-source CID, the collision energy in be divided into several time segments which measure the ana-
Q2 can be chosen specifically for each precursor ion. This lytes eluted at that time as described above for GC–MS-SIM.
allows one to optimize the fragmentation to obtain inform- This possibility is optimized in a “dynamic MRM” mode, in
ative spectra. In low energy CID (10–40 eV), the kinetic which the SRMs of each analyte are only measured while they
energy is converted into vibrational energy which results in are eluting from the chromatograph [315]. Applications of
bond breakage. The required collision energy for fragmen- quantitative screening for 300 pesticides in a 15-min chroma-
tation increases generally with the molecular mass since in tographic run, and for 143 new psychoactive substances (432
large molecules the energy excess can be better dissipated. SRMs) from blood with limits of detection of 0.01–3.1 ng/
The fragmentation of the even-electron molecular ions mL are described [316].
­MH+ or (M – ­H+)− obtained by ESI or APCI differs clearly MRM assays proved to be particularly useful for the specific
from that of the odd-electron radical cations ­M.+ generated analysis of a lager but limited number of target compounds in
by 70-eV EI ionization. General fragmentation rules for a human samples and are widely used in forensic and clinical
larger number of substance classes were described by Nies- laboratories. However, they allow only testing for predeter-
sen [312–314]. According to that, precursor ions and prod- mined substances whereas analytes not included a priori can-
ucts (ions + neutral molecules) always have an even electron not be detected. Several strategies were described to overcome
number. In unimolecular reactions, preferably weak bonds this drawback with further developed LC–MS/MS techniques
are cleaved with formation of preferably stable products. [317–322]. From them, combinations with time-of-flight mass
Frequently formed neutral products are ­H2O, CO, ­CO2, spectrometry (TOF-MS) have gained particular importance for
­CH2O, ­C2H4, and ­CH3NH2. As an example, the mass spec- systematic toxicological analysis.
trum and the fragmentation scheme of methylenedioxyam-
phetamine (MDA) is shown in Fig. 46. Beside the collision Hybrid quadrupole time‑of‑flight mass spectrometry (LC–
energy, the CID spectra depend on pressure of the colli- QTOF‑MS) The use of a time-of-flight mass spectrometer
sion gas (Ar, ­N2), and the instrumental configuration of the instead of the quadrupole Q3 in the triple-quadrupole MS
instrument. Unfortunately, there is not yet a standardization provides particularly favorable preconditions for a real gen-
of the conditions. eral unknown analysis. The working principle of TOF mass
The MRM mode measures several analytes (up to 50 SRM) spectrometers enables comprehensive recording of all data.
one after the other in each MS cycle time of, e.g., 500 ms. Therefore, there is no a priori limitation or prediction of the
In current instruments, the duration for each SRM (dwell substances included in the search procedure. The hybrid LC–
time) should be > 5 ms, but longer dwell times are desirable QTOF-MS device used in the author’s laboratory [323–325]
for better signal/noise and sensitivity. With two SRM/ana- and the scheme of the QTOF-MS are shown in Fig. 47.
lytes and including internal standards for quantitation, about

Fig. 46  Collusion induced mass spectrum and fragmentation scheme of methylenedioxyamphetamine (MDA) [314]. Typical for CID is the loss
of stable neutral molecules, in this case ­NH3, ­CH2O, and C
­ 2H5NH2, from cations with an even number of electrons

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4 Page 56 of 95 ChemTexts (2025) 11:4

Figure 48 shows the functional principle of the time-of- 149.138887 Da. If, for instance, from a sample the monoi-
flight mass spectrometer. The ions from the collision cell sotopic mass 149.0841 Da was determined (after subtrac-
are collected in the ion pulser. From there, ion packages are tion of H­ +), the accuracy of < 2 ppm means a possible
“shot” with a sudden potential pulse of 10 kV every 0.1 ms error < 0.0003 Da. From the five molecular formulas with
into the highly evacuated separation tube, are turned back in the nominal mass 149, only C ­ 9H11NO is within this range
the reflector by a specially arranged electrical field, and get of error (Table 2).
to the detector. Since all ions receive the same kinetic energy This result is confirmed by the accurate mass difference
E = e·V = mv2/2, the square of the ion velocity v2 is indirectly to the isotope peaks (M + ­H+) + 1 and (M + ­H+) + 2 (spacing
proportional to the ion mass m. Since all ions have the same match) and by the abundance ratio to these isotope peaks
flight pass l, it follows from v = l/t that the square of the (abundance match). All such calculations are automatically
time-of-flight t is directly proportional to the ion mass m/z performed by the MS software.
[Eq. (11)]. The knowledge of the molecular formula is a large help
for substance identification but not sufficient because of
m∕z = const. ⋅ t2 (11) the huge number of isomers. For instance, the software
ChemSpider [326] found 2438 isomeric compounds with
The multichannel plate detector is read with a frequency the molecular formula ­C9H11NO, besides cathinone several
of 1 GHz ­(109 point/s) and delivers 10,000 primary mass frequently used chemicals such as 4-dimethylaminobenza-
spectra/s with 100,000 points/spectrum. In total 2000 to ldehyde, N,N-dimethylbenzamide, or N-acetyl-o-toluidine.
10,000 primary spectra are added up to an adjustable acqui- Therefore, in TOF-MS search procedures, the list of pos-
sition rate of 1–5 spectra/s. The TOF-MS is called an “accu- sible candidates in exact monoisotopic molecular mass
rate mass spectrometer” with a mass resolution of ≈15,000 libraries was reduced to toxicologically relevant com-
and a mass accuracy < 2 ppm. pounds and their metabolites [327], and retention time
This high accuracy enables one to determine the atomic and metabolite pattern were included as additional search
composition of monoisotopic molecular ions ­MH+ consist- parameters [328, 329]. The library of Polettini et al. con-
ing of 1H, 12C, 14N, and 16O. Compounds with the same tained 50,500 substances selected from PubChem [327].
nominal mass (isobars), e.g., 149 Da, have different exact However, the most successful step for peak identification
masses because of the different mass defect of the isotopes. is the additional measurement of accurate mass fragment
The exact mass range of all monoisotopic formulas with spectra in LC–QTOF-MS coupling.
the nominal mass 149 is in the range of 148.996038 to

Fig. 47  a LC–QTOF-MS device

in the author’s laboratory. b
Scheme of the hybrid quadupole
time-of-flight mass spectrom-
eter (QTOF-MS)

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ChemTexts (2025) 11:4 Page 57 of 95 4

Fig. 48  Time-of-flight mass

spectrometer (TOF-MS). a
Ion path in the flight tube. The
ion mass is proportional to the
square of the time of flight. b
Setup of the detector. The total
amplification of multichannel
plate, scintillator, and photo-
multiplier tube is 2 × ­106

Table 2  Possible molecular Molecular formula Possible substance (example) Monoisotopic mass, Da Difference to
formulas for a nominal analyte mass,
monoisotopic mass of 149. Da
In case of a measured mass
of 149.0841, only ­C9H11NO C8H7NO2 5,6-Dihydroxyindole 149.047676 − 0.03643
is within the range of mass
C7H7N3O 2-Amino-5-hydroxy-1H-benzimidazole 149.058909 − 0.0252
error < 2 ppm
C9H11NO Cathinone 149.084060 0.00004
C8H11N3 3-Ethoxyaniline 149.095293 0.01119
C10H15N Methamphetamine 149.120443 0.03634

The fully computer-controlled instrument is equipped ion chromatogram from a blood sample extract is shown in
with comprehensive, easy-to-use software and can be oper- Fig. 49a and for four measurement cycles in Fig. 49b. In a
ated in three modes: measurement cycle of 1.1 s, at first the total MS spectrum
is measured for 0.33 s, three of the most abundant precur-
1. MS mode: The quadrupole is fully open, no collisions sor ions are selected, and their optimal collision energies
are performed, all ions from the ESI source are continu- are calculated according to Eq. (12):
ously measured by the TOF.
2. MS/MS mode: The quadrupole selects molecular ions
Collision energy = 4.00 + 0.06 ⋅ m∕ z (12)
­MH+ which are fragmented in the collision cell, and the
fragment spectrum is measured by the TOF. Then, the three precursor ions are subsequently selected
3. Auto-MS/MS mode (data dependent acquisition): Alter- by the quadrupole for each 0.25 s and the three fragment
nating MS and MS/MS mode with a cycle time of, e.g., spectra are recorded. To enable the assessment of other
1.1 s. precursor ions, these three ions are excluded for, e.g., 6 s.
An abundance threshold (e.g., 1000 counts) is set for the
From these, the auto-MS/MS mode fulfils the prerequi- precursor ions to include only essential components in the
sites for a real systematic toxicological analysis. The total measurement of CID spectra. The total ion mass spectrum

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4 Page 58 of 95 ChemTexts (2025) 11:4

Fig. 49  Analysis of a blood sample after protein precipitation by LC– m/z = 349.2032, 237.1021, and 495.1797, automatically selected for
QTOF-MS in auto-MS–MS mode. a Total ion chromatogram. b Part MS–MS measurement d CID fragment spectrum of [M1 + ­H]+. e
of the total ion chromatogram between 9.83 and 9.87 min shown with CID fragment spectrum of [M2 + ­H]+. f CID fragment spectrum of
increased time resolution and indication of the MS and the three MS– [M3 + ­H]+. The collision energies given in d, e, and f were automati-
MS acquisitions. c Mass spectrum in MS mode at 9.845 min with cally calculated as (4 + 0.06 × m/z) eV (from [323], © Elsevier with
indication of the three ions [M1 + ­H]+, [M2 + ­H]+, and [M3 + ­H]+ at permission)

and the three fragment spectra acquired in the measurement The post-run evaluation of the huge amount of data from
cycle are shown in Fig. 49c–f. In this way, a three-dimen- such a chromatographic run (> 1 GB) is done by several
sional chromatogram is obtained which contains all essential easy-to-use software tools. Details are described in a pre-
molecular ions and their fragment spectra. vious paper by the author [323]. First, the background is
removed by subtraction of constant or slowly changing

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ChemTexts (2025) 11:4 Page 59 of 95 4

signals. Then, all masses with the same elution profile are from a blood sample injected after protein precipitation. An
grouped by molecular feature extraction to a so-called set example with 6256 compounds and the components belong-
of “compounds”. Such “compounds” consists, for instance, ing to the “compound” paracetamol are shown in Fig. 50.
of the protonated molecule [M + ­H]+, cluster ions with For identification of these “compounds”, a search in
sodium, potassium, or ammonium, dimers of this ion spe- theoretical mass spectrometric databases using the accurate
cies, the corresponding isotope peaks, and the CID spectra mass of the molecule and of fragments is performed [330].
of [M + ­H]+ in the retention time range of the peak. Several Large MS/MS databases are available such as METLIN MS/
thousands such “compounds” are usually in a chromatogram MS spectrum search [331, 332]. The almost unambiguous

Fig. 50  Post-run analysis of an LC–QTOF-MS file. a Grouping of all and the abundance ratio between the isotope peaks are used in addi-
masses with the same elution profile to 6256 “compounds”. b Total tion to the accurate mass. d CID fragment spectrum of the precur-
ion chromatogram at 1.805 min with typical masses belonging to the sor m/z = 152.0691, collision energy 13.1 eV (from [323], © Elsevier
“compound” paracetamol. c Isotope peaks of the peak m/z = 152.0691 with permission)
mainly caused by 13C. For peak identification, the m/z mass difference

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4 Page 60 of 95 ChemTexts (2025) 11:4

identification is possible by accurate mass spectra library spectrum), and molecular formula generation score (gen-
search. A commercially available database of accurate mass eration of the molecular formula from all MS data of the
data and molecular formulas of more than 7500 toxicologi- sample spectrum) are calculated and summed up to a total
cally relevant substances and accurate mass spectra of more score of identity.
than 2500 of these substances for positive and negative ESI
at three collision energies (10, 20, and 40 eV) was com- Identification of metabolites and semiquantitative deter‑
piled in the author’s laboratory [323] which has steadily mination of concentrations by LC–QTOF‑MS The chroma-
been extended by other authors in cooperation with Agi- tograms obtained from urine samples but also from blood
lent. As an example, the three spectra of the synthetic can- samples of poisoning cases are frequently dominated by
nabinoid JWH 073 are shown in Fig. 51. The inclusion of metabolite peaks. Unfortunately, metabolites were rarely
all three spectra from different collision energies increases available as reference substances, and are in most cases not
the accuracy of search results which is mainly based on the in the spectra libraries. To fill this gap, a metabolite tool
presence of fragment accurate masses and less on fragment “Identify Metabolites” was developed to search for possi-
abundances. ble metabolites of a parent substance or vice versa to search
Several scores such as the database score (agreement of for a possible parent substance if a peak originates from a
accurate molecular mass), the reverse search score (pres- metabolite [324, 333]. This possibility is based on the fact
ence of library masses in the sample spectrum), the for- that for each kind of metabolization a specific mass differ-
ward search score (presence of sample masses in the library ence between parent compound and metabolite exists. For

Fig. 51  Accurate mass spectra

of the synthetic cannabinoid
JWH 073 at CID energies of
10, 20, and 40 eV as stored in
the library. Increasing colli-
sion energy leads to advanced
fragmentation. Cleavage 1 leads
to the fragments m/z = 200
and 127, and cleavage 2 to
m/z = 155. In the library search,
all three spectra are included

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ChemTexts (2025) 11:4 Page 61 of 95 4

instance, the molecular ion mass of every metabolite formed 2100 substances in the presence of 33 deuterated standards
by cleavage of a methyl group (demethylation, loss of C ­ H2) were determined under standardized conditions. During the
is 14.0157 Da lower than that of the parent compound. Some application to a real case sample which was analyzed under
frequently occurring metabolization reactions are listed in the same conditions, the software selects the internal stand-
Table 3. These reactions can also proceed two- or three-fold ards with retention times closest to the analyte and calculates
or in combination. automatically the concentration based on the stored peak
In the metabolite search procedure in a chromatographic area ratios.
file, the tool calculates the molecular formulas of all possi-
ble metabolites to an identified peak and looks for agreeing Example of application The LC–QTOF-MS procedure
“compounds”. The found peaks are shown in an extracted shall be demonstrated using the example of a 54-year-old
ion chromatogram. As an example, for the synthetic opi- multimorbid woman, who died in hospital after unsuccess-
oid tramadol in the chromatogram of a postmortem blood ful resuscitation attempts. A lethal intoxication had to be
sample, seven metabolites are shown in Fig. 52. These were excluded. Protein precipitation was chosen as the sample
formed by mono-, di-, or tri-demethylation, hydroxylation, preparation: 100 µL venous blood, 400 µL acetonitrile, and
or N-oxidation. In this way, unknown peaks in the chromato- a mixture of internal standards were vortexed for 1 min.
gram are identified, and the parent compound is confirmed. After centrifugation, the supernatant was separated, evapo-
As shown in the previous sections, at least the approxi- rated in a nitrogen stream to dryness, and the residue dis-
mate concentration of the identified substances is needed solved in 80 µL mobile phase (acetonitrile/0.1% HCOOH in
for forensic interpretation. The exact quantification with ­H2O, 35:65 v/v). For analysis, 5 µL was injected.
LC–QTOF-MS usually requires a second measurement with The total ion chromatogram of the 20-min run was shown
standard addition or a calibration using deuterated internal in Fig. 49a and the result of the software tool “Find com-
standards. A method to obtain semiquantitative results for pounds” with 6256 results in Fig. 50a. The search procedure
frequently occurring substances in the first measurement was identified 24 drugs or drug metabolites by MS spectrum
developed [334]. For this purpose, the peak areas of about (Fig. 53) and proposed a further 266 substances only by
molecular formula. The results of the quantification, in the
Table 3  Change of the molecular formula and the accurate monoisotope
present case by HPLC–DAD, were codeine 1.1 µg/mL, tram-
molecular mass in frequently occurring metabolization reactions adol 8.4 µg/mL, carbamazepine 9.6 µg/mL, and meproma-
zine 0.3 µg/mL. It was concluded that the high concentration
Metabolization Added or Mass difference, Da
removed group of tramadol in combination with the other drugs could at
least have facilitated the death of the woman.
Hydroxylation, epoxidation, +O + 15.9949 For an experienced and specialized analyst, the time for
N-oxidation, sulfoxidation
post-run analysis of one chromatogram, including automatic
Dehydrogenation − ­H2 − 2.0156
molecular feature extraction, database and library identifi-
Demethylation − ­CH2 − 14.0156
cation, manual exclusion of false positives, and generation
2 × Demethylation − 2 × ­CH2 − 28.0312
of the report, is altogether 10–30 min, depending on the
Deethylation − ­C2H4 − 28.0312
complexity of the sample. Unambiguous identification by
Oxidative desamination − ­NH3 + O − 1.0316
library search was possible for typical basic drugs down to
Glucuronidation + ­C6H8O6 + 176.0321
0.5–2 ng/mL and for benzodiazepines down to 2–20 ng/mL.
Sulfation + ­SO3 + 79.9568

Fig. 52  Metabolites detected

by LC–QTOF-MS in the blood
sample of a tramadol poison-
ing case with the tool “Find
metabolites”. Besides the shown
7 demethylation, hydroxyla-
tion, and N-oxidation products
12 further metabolites from
combinations of these reactions,
hydroxylation at different posi-
tions, and glucuronidation were
found with lower concentrations

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4 Page 62 of 95 ChemTexts (2025) 11:4

Fig. 53  Result of the peak identification of the LC–QTOF-MS file The high concentration of tramadol in combination with the other
from the postmortem blood sample of a multimorbid patient. The drugs could have facilitated the death of the woman From [323],
quantification by HPLC–DAD lad to codeine 1.1 µg/mL, tramadol ©Elsevier with permission
8.4 µg/mL, carbamazepine 9.6 µg/mL, and mepromazine 0.3 µg/mL.

It is a big advantage of this method that the mass informa- Illicit drug abuse and fatalities Systematic analytical search
tion of every substance in the injected sample is stored in for illicit drugs plays a major role in clinical and forensic
the chromatographic run file. In case a poison is suspected toxicology laboratories because of their addictive proper-
later, the file can be specifically searched for this compound ties and endangerment of health, life, and social conditions
even after years without a new measurement. of the population. They are banned by international and
national narcotic acts, and special paragraphs in road traffic
Poisonings with nonvolatile organic compounds acts were issued because of impaired fitness to drive under
their influence. Despite huge efforts of politics, justice, and
The methods described in the previous sections are regularly social authorities, illicit drug criminality remains an endur-
applied to solve suspected poisoning cases or for the diag- ing problem. The number of illicit drug death cases per year
nosis in cases with unclear reason for death or illness. The is to a certain degree a reflection of this problem. As shown
following sections give an overview and typical examples in Fig. 54 for Germany, it developed from the 1970s in two
of intoxications with the main groups of nonvolatile organic increasing waves to a level between 1000 and 2000. In all
poisons. A comprehensive and detailed description of this these cases, consumption of drugs was analytically proved
widely ranging matter is beyond the scope of this paper. as the reason for death.
Heroin, illegally prepared from the natural opiate
morphine by di-acetylation, is a frequent poison in drug

Fig. 54  Number of illicit

drug death cases in Germany
between 1973 and 2022

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ChemTexts (2025) 11:4 Page 63 of 95 4

Fig. 55  Metabolism of heroin

death cases. Because of its higher lipophilicity in com- reason, the interpretation of a heroin death from analyti-
parison to morphine, it penetrates the blood–brain barrier cal results is complicated and needs broader evidence
much faster after injection and leads to a much higher from the circumstances. A clue about chronic use and
euphoria and stronger addiction than morphine. Besides adaptation can be obtained from hair analysis.
the euphoric effect on the limbic system, it suppresses Although heroin itself is usually the death-causing
protective reflexes such as pain, coughing, and breathing. poison, there are cases with lethal effect of adulterants.
The respiratory depressive effect is the reason for death This occurred in a series of fatalities after injection of
after acute heroin overdose. The victim does not feel the heroin with a high content of the local anesthetics lido-
urge to breath and softly passes away from hypoxia, often caine or tetracaine [335]. In such cases, the bolus injec-
with the injection needle still in the vein. tion leads to ventricular fibrillation and death by heart
A strong adaptation is typical for heroin, as for opi- failure; unlike heroin death, it occurs with severe cramps.
oids in general. The intravenous dose of, e.g., 10 mg Cocaine is worldwide one of the most frequently
in a beginner can rise to more than 100 mg within 1 abused drugs with a very strong addiction potential. The
or 2 weeks to have the same effect. Since this adapta- stimulating drug is preferentially snorted or smoked
tion is lost after abstinence, e.g., lack of the drug in jail, but also injected. Death cases by cocaine poisoning are
resumption of heroin use with the pre-abstinence dosage relative seldom in view of its high prevalence and occur
is often fatal. A further uncertainty is the unknown and often after injection, similar to that described above for
very variable heroin content in illicit drug preparations. lidocaine with seizures and heart failure. Accidental
After injection, heroin (t 1/2 ≈ 6 min) is metabolically death by oral cocaine overdosage occurred repeatedly
hydrolyzed to 6-acetylmorphine (6-AM, t 1/2 ≈ 40 min) for “body packers” who illegally transported the drug in
and morphine (t 1/2 ≈ 3–4 h, Fig. 55). Since this enzy- a large number of swallowed small packets and one or
matic hydrolysis is continued after death, neither heroin more of these packets leaked [336, 337]. Cocaine more
nor 6-AM is found in postmortem blood. Therefore, for often is detected in living individuals in the context of
distinguishing illegal heroin from legal morphine, 6-AM drug driving, criminal activities, and social misconduct.
has to be detected in urine or CSF which has no enzyme Recklessness and hubris under the influence of cocaine
activity. Postmortem morphine concentrations of heroin are often the reason for fatal accidents.
fatalities vary greatly between 0.05 and more than 3 µg/ Besides the drug itself, the metabolites benzoylecgo-
mL, depending on injected dose, survival time, degree of nine, ecgonine methyl ester, and norcocaine are measured
adaptation, and residues of previous injections. For this in the analysis (Fig. 56). Furthermore, cocaethylene as a

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4 Page 64 of 95 ChemTexts (2025) 11:4

Fig. 56  Metabolism of cocaine.

Half-lives in blood are 0.7–1.5 h
for cocaine, ≈ 4.5 h for ben-
zoylecgonine, and ≈ 3.1 h for
ecgonine methyl ester

combined metabolite and marker of simultaneous ethanol consumed for enhancement of mental and physical per-
and cocaine use and anhydroecgonine methyl ester as a formance as well as for recreational purposes. Similar
pyrolytic marker of cocaine smoking (crack marker) are to most other stimulants, they have an anorectic affect.
included. They are illegally traded as salts (amphetamine sulfate
The postmortem cocaine concentration in blood var- and methamphetamine hydrochloride) and mainly taken
ies greatly between 0.9 and 21 µg/mL and depends on orally, but also snorted or injected. The more volatile
dosage, route of administration, survival time, and sam- methamphetamine hydrochloride is also frequently
ple storage conditions. Despite their different effects, smoked. Chronic intake with increasing dosages can lead
cocaine and heroin are frequently used together. to total exhaustion with paranoid hallucinatory condi-
The complexity of the toxic exposure of a cocaine–her- tions and attacks of vandalism. Tachyphylaxis as a spe-
oin co-consumer is shown in Fig. 57 by the LC–QTOF- cial form of drug tolerance may occur. The usual dosage
MS chromatogram of the systematic toxicological is 5–30 mg, and the lethal doses of 0.75 to 4 g were
analysis in hair [338]. In addition to cocaine and its described, with extreme agitation, seizures, unconscious-
metabolites, both E,Z-isomers of cinnamoylcocaine as ness, heart and respiratory arrest, and death. The analysis
concomitant alkaloids of the coca plant, the adulterants is confined to the drug itself with amphetamine as the
paracetamol, lidocaine, and tetramisole, heroin and its main metabolite of methamphetamine. In postmortem
metabolites together with the concomitant Papaver som- blood, concentrations of 0.5 to 40 µg/mL were deter-
niferum alkaloids papaverine and noscapine as well as mined. Both illicit substances can occur as metabolites
the tobacco alkaloid nicotine and its metabolite cotinine of medical drugs. This can lead to a wrong interpretation
were detected. The neuroleptic drug risperidone and its in forensic cases [339].
metabolite 9-hydroxyrisperidone point to a psychiatric Methylenedioxymethamphetamine (MDMA) and its
treatment. analogue methylenedioxyethamphetamine (MDE) are also
Amphetamine and methamphetamine (speed, crys- illegally synthesized and have been increasingly used
tal meth) are stimulants with a high addiction potential since the 1990s as illegal party or rave drugs “Ecstasy”.
which are synthesized in clandestine laboratories and are On the one hand, they are known for an “oceanic feeling

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ChemTexts (2025) 11:4 Page 65 of 95 4

Fig. 57  Systematic toxicological analysis of the hair sample of a mg), 12 = norcocaine, 13 = heroin, 14 = acetylcodeine,15 = cin-
drug fatality. 1 = paracetamol, 2 = anhydroecgonine methyl ester, namoylcocaine, isomer 1, 16 = cocaethylene, 17 = 9-hydroxyrisperi-
3 = phenethylamine, 4 = cotinine, 5 = morphine (0.16 ng/mg), 6 = nic- done, 18 = cinnamoylcocaine, isomer 2, 19 = papaverine, 20 = risp-
otine, 7 = benzoylecgonine (22 ng/mg), 8 = tetramisole, 9 = 6-acetyl- eridone, 21 = lidocaine, 22 = noscapine, and 23 = hydroxyzine (from
morphine (2.2 ng/mg), 10 = 3-hydroxylidocaine, 11 = cocaine (27 ng/ [338], © Elsevier with permission)

of togetherness”, warmth, faith and being in love, and on Δ 9 -Tetrahydrocannabinol (THC) is the active com-
the other hand for strong physical stimulation allowing ponent of cannabis (marihuana and hashish) and is the
many hours of dancing, followed by insomnia, avolition, worldwide most frequently used drug. The structure and
and depression. The usual oral dose is 100 mg. High the metabolism are shown in Fig. 58. It is much less toxic
doses together with strong physical activity lead to tach- than the drugs described above and cannabis fatalities are
ycardia (> 160/min), hyperthermia caused by impaired practically nonexistent. The illicit status was based on its
regulation of the body temperature up to 43 °C, and addictive properties and the impairment of the physical
extreme dehydration with a loss of up to 5 L of water. In and mental performance (e.g., driving ability) under its
fatal ecstasy cases, rhabdomyolysis, acute kidney failure, action. At present, liberalization of cannabis is gaining
disseminated intravascular coagulation (DIC), and brain ground in many countries. Nevertheless, the cannabi-
bleeding were determined as the causes of death. The noids THC, cannabidiol (CBD), and cannabinol (CBN)
analytical determination of MDMA and MDE together as well as the THC metabolites 11-hydroxy-THC (THC-
with the N-demethylation metabolite methylenedioxyam- OH) and the final oxidation product 11-nor-9-carboxy-
phetamine (MDA) is generally unproblematic. The rec- Δ 9 -tetrahydrocannabinol (THC-COOH) are regularly
reational blood concentration is < 0.1 µg/mL. In death tested in drug-driving cases and cases with criminal and
cases, concentrations of 0.3–7.3 µg/mL MDMA were social background. Since August 2024, a THC concen-
measured. tration above 3.5 ng/mL in blood proves recent cannabis
consumption and contradicts fitness to drive in Germany.

Fig. 58  Structure and metabo-

lism of cannabinoids. Δ9-
Tetrahydrocannabinol (THC)
and cannabidiol (CBD) are
the main natural cannabinoids
of hemp and marihuana. The
inactive cannabinol (CBN) is
formed by oxidation during
smoking or preparation of
hashish

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4 Page 66 of 95 ChemTexts (2025) 11:4

Lysergic acid diethylamide (LSD) is a very potent men-

tal, visual, and auditory hallucinogen [340]. The usual
dose is only 50–200 µg, and effective plasma concentra-
tions are in the order of < 1 to 5 ng/mL. The usual mental
effects are delusions, visual hallucinations, distortion of
the sense of time and identity, impaired depth and time
perception, artificial sense of euphoria or certainty, dis-
torted perception of the size and shape of objects, move-
ments, color, sounds, touch and the user’s own body
image, severe, terrifying thoughts and feelings, fear of
losing control, fear of death, and panic attacks. Although
these effects are highly contradictory to traffic safety, LSD
is not included in programs for regular road site testing,
mainly for analytical reasons. LC–MS/MS methods were
described for plasma [341] as well as for oral fluid [342].
Fig. 59  Structure and blood concentrations of synthetic opioids in
Case reports about accidents and bizarre actions under fatal cases [354]
LSD are rare in the scientific literature, but there may be a
large number in view of the difficult detectability. Exam-
ples of self-inflicted injuries were reviewed by Le Daré is only an activity-specific pretest, further specification is
et al. [343] and include a case of manual self-amputation necessary in case of a positive result.
of both testes [344]. Death cases by NPS intoxications (opioids, cathinones,
Designer drugs and new psychoactive substances (NPS) synthetic cannabinoids, and other substances) were reviewed
are structurally derived from drugs of abuse as described by Ferrari Júnior [354]. The structures and postmortem con-
above in order to circumvent the legal control but also to centrations of some illicit synthetic opioids, mainly derived
preserve or enhance the pharmacological effects [345]. They from fentanyl, are shown in Fig. 59. An aerosol of carfentanil
were traded mainly via the internet as “Research Chemi- and remifentanil was reportedly used by the Russian secu-
cals”, “Bath Salts”, or “Legal Highs”. In the scientific litera- rity forces in the hostage drama at the Moscow Dubrowka
ture, the umbrella term new psychoactive substances (NPS) Theater in 2002 against the Chechen hostage-takers and led
was introduced and includes essentially the four groups: to the death of 125 of the 979 hostages [355, 356].
synthetic stimulants, synthetic cannabinoids, synthetic hal- The United Nations Single Convention on Narcotic Drugs
lucinogens, and synthetic depressants (synthetic opioids and and established national drug laws exclusively ban single
benzodiazepines) [346]. NPS are often by orders of magni- substances and are not suitable to keep pace with the steadily
tude more efficient than the conventional drugs, with cor- changing NPS market. Manufacturers continually develop
respondingly much lower doses and concentrations in blood new chemicals to replace others that have been banned. In
or urine to be detected. This and the steady appearance of this respect, NPS chemical structures constantly change and
new substances on the market posed a huge challenge to develop in an attempt to stay ahead of the national and inter-
analytical methods as well as to legislation. national banning laws. Therefore, simultaneously regulatory
Although screening procedures for a broad spectrum and legislative groups interested in banning drugs have been
of NPS in blood or urine [347, 348], oral fluid [349], and introduced worldwide to counteract the trade and use of
hair [350, 351] by LC–MS/MS were described, the abuse emerging NPSs more effectively [357, 358]. For instance,
of these drugs in the broad population remains essentially in Germany, the New Psychoactive Substances Act (Neue-
undetected. Activity-based bioassays could be a new way psychoaktive-Stoffe-Gesetz, NpSG [359]) prohibits at pre-
of group testing for NPS as was shown for synthetic can- sent seven substance groups with (1) compounds derived
nabinoids [352]. This assay is based on the mechanism of from 2-phenethylamine, (2) cannabimimetics derived from
cannabinoid receptor CB1 or CB2 activation in combination indole, pyrazole, and 4-quinolone and from 3-sulfonylamido
with the principle of the functional complementation of a benzoic acid, (3) benzodiazepines, (4) compounds derived
split Nanoluc luciferase, a highly stable ATP-independent from N-(2-aminocyclohexyl)amide, (5) compounds derived
bioluminescent protein. Binding of any cannabinoid at the from tryptamine, (6) compounds derived from arylcyclohex-
receptor leads to restoration of the luciferase and to meas- ylamine, and (7) compounds derived from benzimidazole.
urable bioluminescence. In the same way, a test for opioid The structural formulas with possible variations and substit-
activity by activation of the μ-opioid receptor was applied uents are clearly defined in an appendix; however, its appli-
successfully to 107 authentic postmortem blood samples for cation appears to be complicated and requires considerable
the detection of synthetic opioids [353]. However, since this chemical expertise.

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ChemTexts (2025) 11:4 Page 67 of 95 4

Fig. 60  Compounds derived

from 2-phenethylamine banned
by the German New Psychoac-
tive Substances Act [359]. The
possible combinations of ring
systems and substituents leads
to a huge number of banned
compounds

As an example, the structural description of the concentration of cyclobarbital (5-cyclohexenyl-5-ethyl-

2-phenethylamine group is shown in Fig. 60. Variation of barbituric acid) together with three other medicines was
structure and substituent pattern in these groups results in detected in the mysterious death of the prime minister of
a huge number of proactively banned compounds. Since the German state Schleswig–Holstein Uwe Barschel (1944–
important intermediates for industry and research are also 1987) [361, 362]. The interpretation of the concentrations
among them, additional bureaucracy and difficulties are neg- in blood and urine concerning suicide or murder led to an
ative aspects of this legislation. In a critical review about the endless controversy among experts in this case.
efficiency, van Amsterdam et al. conclude that this generic At present, the therapeutic application of barbiturate is
drug legislation has not affected the availability of NPSs on essentially obsolete. However, the prescription of pentobarbi-
the scene, likely because legal sales of NPS were replaced tal for assisted suicide is legal practice of right-to-die organi-
by illegal sales via street dealers and e-commerce, and that zations such as Exit International or Dignitas in Switzerland
NPSs are arguably an overestimated phenomenon [358]. [363, 364]. A lethal dose of the drug is orally taken, or, if
the person willing to die is not able to take it by themselves,
Suicide, murder, drug‑facilitated crimes, and accidental poi‑ by active switching on a pre-installed infusion. Thiopental,
soning by medical drugs The majority of medical drugs in which C=O in position 2 of pentobarbital is replaced by
have toxic effects on overdose, and the concentrations in C=S, is infused for anesthesia as the first step of executions in
blood are categorized in the literature into therapeutic, combination with the muscle relaxant pancuronium and KCl
toxic, and comatose-lethal ranges [280, 281]. Figure 61 on death row in the USA [365] and in China [366].
shows examples of drugs which occurred frequently in Other sleeping drugs occurring in lethal poisonings
lethal poisonings. Barbital (Veronal) was the first barbi- were methaqualone [367], diphenhydramine [368], and
turate developed as a synthetic sleeping drug in 1903 by later benzodiazepines (e.g., nitrazepam [369]) and the
Emil Fischer and Josef von Mering. This was followed by so-called z-drugs (zolpidem [370], zopiclone [371], and
a larger number of other 5,5-disubstituted barbiturates from zaleplon [372]). A high suicide rate is seen in cases of
which, among others, phenobarbital was used as an antie- mental illness such as schizophrenia, bipolar disorder, and
pileptic and pentobarbital as an anesthetic [360]. Besides depression. In such cases, intoxications by drugs used in
the widespread abuse due to the high addictive potential, treatment of these illnesses occur frequently [373], such
barbiturate overdose became a common method in suicide as tricyclic antidepressants (e.g., amitriptyline [374]), the
attempts. Famous victims were the writer Stephan Zweig selective serotonin reuptake inhibitor citalopram [375],
(1881–1942), the actress Marilyn Monroe (1926–1962), or the mood stabilizer and antiepileptic carbamazepine
the Beatle manager Brian Epstein (1934–1967). A lethal

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4 Page 68 of 95 ChemTexts (2025) 11:4

Fig. 61  Structure of sleeping drugs occurring in lethal poisonings

[376], or the neuroleptic clozapine [377, 378] (structures [381]. The antimalarial chloroquine in combination with
in Fig. 62). benzodiazepines was recommended as the so-called Kusch
Cardiovascular medication is a further group frequently method of suicide because of its fast absorption and its low
occurring in poisonings with therapeutic drugs, e.g., car- therapeutic margin [382, 383].
diac glycosides [379] or beta adrenoreceptor blockers [380]. Among analgesics, opioids play a dominant role in poi-
Public knowledge about the toxicity of the only two beta- soning statistics (structures in Fig. 63). Although intoxica-
blockers available in East Germany, propranolol and talino- tions with therapeutic opioids were never seldom, an “opioid
lol, led to a high prevalence of suicides with these drugs, a epidemic” has occurred in recent years in the USA with,
trend which rapidly diminished after German reunification for instance in 2018, altogether 46,802 fatalities as a result

Fig. 62  Examples of psycho-

therapeutic and other drugs
frequently occurring in lethal
poisoning

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ChemTexts (2025) 11:4 Page 69 of 95 4

of opioid overdose, including prescription opioids, heroin, cases [392]. A geriatric nurse killed at least six patients out of
and illicitly manufactured fentanyl [384–386]. An extended- greed with chlorprothixene [393]. Lethal concentrations of this
release formulation of oxycodone which was marketed antipsychotic were detected in brain tissue of exhumed corpses
to physicians as a safe and effective opioid pain reliever after between 2 and 5.5 years in an earth grave.
with very low risk of iatrogenic addiction contributed to Serial killing of verifiably 91 patients was accomplished
the problem. The ensuing overprescription led to annually between 1999 and 2005 by a male nurse in two different hos-
4967–6199 death cases involving oxycodone between 2011 pitals in Northern Germany, who gave patients cardiovascular
and 2016. drugs (ajmaline, amiodarone, sotalol, or lidocaine) to put them
Non-opioid analgesics (acetyl salicylic acid, metamizole, in a state requiring resuscitation, with the aim of distinguish-
paracetamol, ibuprofen) occur frequently in poisonings. ing himself when the ensuing resuscitation attempt took place
Overdose of paracetamol is particularly treacherous because [394]. Although the high number of death cases during his
of the lethal hepatic coma after a symptom-free interval of shifts was suspicious previously, he was only caught in the
up to 24 h [387]. Therefore, testing for paracetamol is part act when he added ajmaline without indication to the injec-
of every toxicological analysis in emergency cases. tion pump of a patient. Analysis of tissue samples from 134
Serial murder by medical drugs is often committed by corpses exhumed from 67 graveyards and comparison of the
healthcare professionals who treacherously misuse the trust of results with the patient charts was decisive for the evidence.
their patients [388, 389]. The most infamous example was the Further series of murders in medical clinics and care homes
English family doctor Harold Shipman who killed at least 284 and methods for their prevention were reviewed by Beine [395]
elderly patients by injection of heroin (which was approved and included, among others, use of antidiabetics, muscle relax-
for pain treatment in Great Britain), morphine, or pethidine (a ants, or anticoagulants.
synthetic opioid, Fig. 63) between 1974 and 1998 [390, 391]. Medical drugs were also misused in drug-facilitated
In most cases, he injected the lethal dose during a home visit, crimes, mainly drug-facilitated sexual assault (DSFA) or
later issued the certificate of death himself, added false entries robbery [396–398]. A urine or blood sample of the victim
about preexisting life-threatening diseases in the patient charts, should be collected as soon as possible after the incident.
and managed to circumvent autopsies. The crimes remained After a delay of weeks or months, hair analysis can be useful
undiscovered until suspicions were raised that he had forged [399]. Medical knockout drugs detected in such cases are
a will in one case in September 1998. The detection of mor- benzodiazepines, analgesics, z-drugs, antipsychotics (e.g.,
phine in the exhumed corpses was important evidence in these clozapine), but also hypotensive agents (e.g., clonidine),

Fig. 63  Structure of medi-

cal drugs misused in lethal
poisonings. Oxycone in the
“opioid epidemic” [384–386],
heroin and pethidine used in
at least 284 killings by Harold
Shipman [390], chlorprothixene
used in at least 6 killings by the
geriatric nurse Marianne Nölle Oxycodone Heroin Pethidine
[393], and ajmaline, sotalol, and
amiodarone used in at least 91 CH3
killings by the male nurse Niels
Högel [394] CH3

Sotalol

Chlorprothixene Ajmaline

Clonidine Amiodarone

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4 Page 70 of 95 ChemTexts (2025) 11:4

often in combination with alcohol. A typical phenomenon pill contains 19.8 mg of the substance, the child must have
is the retrograde and anterograde amnesia of the victim, that ingested at least three or four of them.
she or he cannot remember any details before or after the
unconscious phase of the incident. Acute and lethal poisonings by pesticides The majority of
Accidental poisonings with a wide variety of medical pesticides are organic compounds with low volatility and
drugs are abundant in children [400, 401]. Pediatric expo- they are by definition toxic to living organisms. Acute and
sures are typically unintentional and involve medications left lethal poisonings of humans with pesticides are not seldom.
within reach of the exploring toddler, or administered by an It followed from a systematic review that there were world-
adult in a manner that leads to a dosing error. It is commonly wide approximately 110,000 pesticide self-poisoning deaths
believed that ingestion of one or two tablets by a toddler is each year from 2010 to 2014, comprising 13.7% of all
a benign act and not expected to produce any significant global suicides [402]. However, the proportion of suicides
toxicity. Although this is true for most exposures, certain due to pesticide self-poisoning varies considerably between
common medications have the potential to cause life-threat- countries and is much lower in Europe, which has regulated
ening toxicity or death despite the ingestion of only one or access to the most toxic pesticides and high degree of urban-
two tablets. An example investigated by the author of this ization (0.9%), than in India, which has a more rural popula-
paper may demonstrate the careless handling of potentially tion and daily access to pesticides (estimated 38%).
lethal medication by adults (Pragst, unpublished results). A Organophosphorus esters and carbamates (acetylcho-
3.5-year-old girl was found dead in her bed in late evening linesterase inhibitors) are most prevalent in pesticide poi-
by the parents. The analysis by HPLC–DAD identified the sonings. The mechanism of action is through the inhibition
antiarrhythmic detajmium with 11.6 µg/mL in blood and of the enzyme acetylcholinesterase, leading to the accumula-
38 mg in 150 mL gastric content. The brother of the girl then tion of acetylcholine at cholinergic synapses [403–405]. The
told the parents that they had found pink pills in the bin in excess acetylcholine causes constant acetylcholine recep-
the backyard and had played doctor with them. Since one tor triggering, resulting in malfunction of the autonomic,
somatic, and central nervous systems. Symptoms are miosis,

Fig. 64  Structure and oral L

­ D50 (rat) of toxic organophosphorus compounds (insecticides and chemical warfare)

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ChemTexts (2025) 11:4 Page 71 of 95 4

Fig. 65  Mechanism of inhibi-

tion, reactivation, and aging of
acetylcholinesterase (AChE) of
parathion

vision disorders, headache, lachrymation, hypersalivation, kg are moderately hazardous. Deliberate poisonings with
bronchoconstriction, dyspnea, cyanosis, nausea, desire to insecticides occur usually via oral intake. Beside frequent
void, myasthenia, bradycardia, cramps, respiratory paralysis, suicides, homicides with organophosphorus insecticides
and death by asphyxiation. were also reported. One of the first cases was the serial mur-
The general chemical structure of organophosphorus der by Christa Lehmann of her husband, her father-in-law,
insecticides and warfare agents (“nerve gases”) is described and inadvertently her best friend with parathion between
by the Schrader formula (Fig. 64) with the phosphorus atom 1952 and 1954 [406]. A natural death (stomach perforation
bound to oxygen or sulfur, to a leaving group X (e.g., –F, and cardiac death) was erroneously certified in the first two
–CN, –O-Aryl), and to two variable substituents ­R1 and ­R2. cases. In the third case, a piece of chocolate filled with the
Figure 65 shows schematically the molecular action mech- poison was not intended for her friend but for her friend’s
anism of the insecticide parathion with the leaving group mother. Because of the bitter taste, the victim immediately
p-NO2–C6H4–O. A serine hydroxy group of the esterase spat it out but nevertheless died within minutes as did her
which is essential in the complex mechanism of acetylcho- dog who swallowed the residue of the chocolate. The detec-
line hydrolysis is selectively bound to the phosphorus atom tion of the poison was based on a color test for the hydrolysis
with liberation of the leaving group (p-nitrophenol). This product p-nitrophenol and paper chromatography, in the first
leads to deactivation of the enzyme. At this step, its regen- two cases after exhumation. A biological test of the gas-
eration by cleaving off the organophosphorus group with tric content with flies (Drosophilidae) was also performed
specific antidotes is still possible, but slow alteration of one [407]. Today, all essential insecticides and many of their
of the –OC2H5 groups (“aging”) by hydrolysis leads to per- degradation products are included in HPLC–DAD, GC–MS,
manent deactivation. and LC–MS/MS databases. Because of the instability of
Several thousand phosphoric esters with different X, R ­ 1, organophosphorus insecticides, the analysis of the hydro-
and ­R2 were synthesized and tested for use as pesticides a well lytically formed and more stable dialkylphosphates, e.g.,
as warfare agents, but fortunately, only about 100 substances ­(CH3O)2POOH or ­(C2H5O)2POOH, is recommended [408].
have reached practical importance and occur in poisonings. Organophosphorus warfare agents are by orders of mag-
Thiophosphoric esters are inactive and are metabolically con- nitude more toxic than the insecticides (Fig. 64). They were
verted to the corresponding active phosphoric esters (e.g., specifically designed for fast resorption by inhalation and
parathion to paraoxon in Fig. 65). The toxicity varies widely through intact skin and were subject to extensive research for
and depends on the reactivity of the P=O moiety (electron- development, toxicological evaluation, analytical detectabil-
withdrawing effect of X), steric effects, and lipophilicity of R
­ 1 ity, antidotes, and clinical treatment [409, 410]. Although
and ­R2. The structure and ­LD50 values of some examples of available in thousands of tons, sarin, tabun, and soman
insecticides and chemical warfare agents are shown in Fig. 64. were not utilized in World War 2 but later in the Iraq–Iran
From the insecticides, parathion and phorate with war [411], in military conflicts in Syria [412, 413], and in
­L D 50 ≤ 5 mg/kg are classified as extremely hazardous terrorist attacks in Japan [414, 415]. The extremely toxic
whereas chlorpyriphos or dimethoate with ­LD50 ≥ 50 mg/ nerve agent Novichok was developed in the Soviet Union

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4 Page 72 of 95 ChemTexts (2025) 11:4

in the 1970s to 1990s as an answer to the development of toxic diquat can be easily determined in human samples by
VX in the USA [416–418] and was obviously used in the HPLC–DAD or LC/MS. A specific test in urine is based on
poison attacks on the former Russian spy Sergey Skripal the reduction with sodium dithionite in basic medium leading
and his daughter [419] as well as on the oppositionist Alexei to the typical violet color of the formed radical cation.
Navalny [420]. These incidents occurred even though the Rodenticides with coumarin structure such as warfarin and
transport and deployment of nerve agents are banned by the superwarfarins (e.g., brodifacoum; structures in Fig. 66) are
Chemical Weapons Convention (CWC) [421]. Suspicion of another group of dangerous pesticide poisons [431–433]. The
the use of these agents in living victims arises as a result strong anticoagulant effect of these substances is based on the
of the typical symptoms (see above). Tests for butyrylcho- blockade of the regeneration of vitamin K which is crucial for
linesterase activity, which are widely available in clinical the activation of the inactive precursors of several coagulation
routine practice, are usually the only laboratory parameter factors. This leads to bleeding symptoms such as gross hema-
to confirm a clinical diagnosis of organophosphorus poison- turia (bloody urine), mucosal bleeding, gastrointestinal bleed-
ing [422]. This enables a specific antidote therapy without ing, soft tissue bleeding, or coughing up blood. Intracranial
knowing the exact identity of the active substance. Because hemorrhage is a frequent cause of death in poisoning cases
of the extremely low concentrations involved, the inacces- with these substances. The superwarfarins were developed
sibility of reference substances, and of probable political in the 1970s when warfarin-resistant rodent strains emerged.
implications, these substances are not included in the ana- These drugs are more than 100 times more toxic than war-
lytical routine of most toxicological laboratories and corre- farin, extremely lipophilic, and have an elimination half-life
sponding samples are passed on to specialized military labo- typically longer than 20 days. The lethal dose of brodifacoum
ratories. There, sophisticated mass spectrometric techniques is about 15 mg. However, it is normally provided in bait at
are employed to identify the specific agent via long-lived 0.005%, a formulation considered “safe,” since consumption
adducts on acetylcholinesterase, butyrylcholinesterase, or of at least 300 g of bait is needed to deliver a lethal dose to
serum albumin in blood after protein isolation, purification, an adult human.
and pepsin-catalyzed hydrolysis [423]. For the Novichok Nevertheless, despite safety regulations, the potential use
group, unambiguous and sensitive identification of specific of superwarfarins in accidental, surreptitious, or deliberate
degradation products from urine and plasma after derivatiza- poisoning has became increasingly evident since the early
tion by GC–MS/MS was described [424]. 1980s [434, 435]. Mass anticoagulant poisoning using a
Carbamate pesticides with the general structure lethal chemical warfare toxin was deployed by the all-white
­R1–NH–CO–O–R2 are the second group of acetylcholinest- Rhodesian military during the 1970s in an attempt to regain
erase inhibitors which bind in the same way as the organo- control of Rhodesia from rebel forces [436]. An epidemic
phosphorus agents on the serine OH group with liberation of exposure to superwarfarins was also reported to occur owing
HO–R2 [403]. Typical examples are aldicarb and carbofuran to lacing of drugs (e.g., synthetic cannabinoids) to enhance
(structures in Fig. 66) which occurred also in murder cases and extend euphoric effects [437, 438]. The rationale is that
[425, 426].
The toxic effects of the total herbicide paraquat
(1,1′-dimethyl-4,4′-bipyridinium dichloride, structure in
Fig. 66) at the cellular level result from its ability to generate
intracellular reactive oxygen species (free radicals) through
redox cycling and to disrupt the mitochondrial electron trans-
port chain [427–429]. Moreover, an imbalance in the cellular
redox state leads to significant mitochondrial damage, lipid
peroxidation, and cellular toxicity. High doses (50–100 mL
of liquid 20% concentrate) lead to fulminant organ failure,
pulmonary edema, cardiac, renal, and hepatic injury, CNS
toxicity, and finally death due to multiorgan failure within
several hours to a few days. At lower dose, death may occur
from progressive pulmonary fibrosis with a delay up to several
weeks. There is no specific antidote, and despite the pres-
ence of supportive treatments, fatality is very high in toxi-
cology centers. In the EU, paraquat has been banned since
2007, but in developing countries it is still highly present in
suicidal and accidental poisonings. Homicides using the poi- Fig. 66  Structure and L
­ D50 of further pesticides frequently occurring
son were repeatedly reported [430]. Paraquat and the equally in poisonings

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ChemTexts (2025) 11:4 Page 73 of 95 4

saturation of liver enzymes with superwarfarins reduces the A new possibility which can also work if diagnostic
metabolism of the drug, thus prolonging its effects. morphological features have been degraded is the DNA
Although superwarfarins are included in all databases for barcoding of plant residues from gastric or intestinal
toxicological analysis, they are difficult to detect in general content [443–447]. Using a plant extraction kit, one can
screenings by HPLC–DAD or LC–MS/MS because of their extract DNA from the sample. Polymerase chain reac-
extremely long retention time on the usual reversed-phase tion (PCR) and amplified fragment length polymorphism
columns. Therefore, special methods were developed for (AFLP) are used to gain a DNA profile. For identification,
these poisons [439]. sequence catalogues have been prepared for poisonous
plants that are relevant for human intoxications [445] and
Poisons from toxic plants The most important plant poisons applications on veterinary and human death cases were
are used either as illicit drugs, such as cocaine, mescalin, described [443, 447].
and psilocybin, or as medical drugs, such as atropine, mor- However, the analytical determination of the poison in
phine, or digitoxin, and are discussed in Sects. “Illicit drug severe survived and lethal cases is inevitable for unam-
abuse and fatalities” or “Suicide, murder, drug-facilitated biguous clarification. Special LC–MS/MS methods were
crimes, and accidental poisoning by medical drugs”. They developed for identification of plant poisons in blood [448]
usually have a complicated chemical structure and, from an and most of them are present in toxicological analytical
analytical point of view, are nonvolatile organic compounds. databases.
Poisonings occur usually by accidental but also by inten- Figure 67 shows the structural formulas of the major toxic
tional intake of leaves, fruits, seeds, or roots of the plant. substance of some extremely poisonous plants. As a rule,
Reports from poison information centers show that this is toxic plants contain more than one poisonous substance.
not a rare event [440–442]. Accidental poisonings occur For instance, more than 600 substances were identified
often in children who ingested berries, capsules, pods, or in yew (Taxus baccata) [449], from which mainly pacli-
stone fruits from toxic plants. Thus far, the fast identifica- taxel, 10-deacetylpaclitaxel, baccatine III, 10-deacetylbac-
tion of the poison in such cases has mainly been based on catine III, cephalomannine, and 3,5-dimethoxyphenol are
the symptoms and on the description of the plants or inspec- included in analysis of poisoning cases with yew needles
tion of plant residues in vomit or gastric lavage rather than or yew tea [450, 451]. At autopsy, plant residues in stom-
on analytical detection of the toxic ingredient. Nowadays, ach or duodenum are indications of the poison. The less
apps on smartphone can support the identification of the toxic 3,5-dimethoxyphenol serves as an analytical marker of
botanical species. yew poisoning and can be easily measured by HPLC–DAD

Fig. 67  Structure and molecular

mass of the major toxic sub-
stances of some extremely poi-
sonous plants. Paclitaxel in yew
(Taxus baccata), aconitine in
blue monkshood, hyoscyamine
and scopolamine in nightshades
(Solanaceae), cicutoxin in
water hemlock (Cicuta virosa),
coniine in poison hemlock
(Conium maculatum), colchicin
in autumn crocus, and digitoxin
in foxglove (Digitalis purpurea)

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4 Page 74 of 95 ChemTexts (2025) 11:4

or GC–MS whereas the more structurally complex taxines However, it is contained in GC–MS and LC–MS/MS or LC-
require LC–MS/MS. TOF libraries and lethal concentrations in blood (35–410 ng/
Atropine (hyoscyamine) and scopolamine are the active mL) can be easily detected.
substances of nightshades (Solanaceae) [452]. As a result Although water hemlock (Cicuta virosa) to a certain
of their anticholinergic activity (antagonistic actions with degree resembles poison hemlock, the toxic substances and
acetylcholine at central and peripheral muscarinic recep- poisoning mechanisms are completely different [447, 474,
tors), these substances can cause life-threatening symptoms 475]. The toxins of water hemlock, cicutoxin and oenantho-
primarily associated with the cardiac, respiratory, and nerv- toxin, have a polyine structure and act as antagonists of the
ous systems. They are hallucinogenic, a reason for the rec- γ-aminobutyric acid (GABA) receptor in the central nerv-
reational abuse of these plants. Intoxicated victims are often ous system with cramps as the essential primary symptom.
in a long-lasting delirium. HPLC–DAD and LC–QTOF-MS were applied for their
Poisoning with Atropa belladonna (deadly nightshade) detection in plant residues, gastric content, and blood. The
takes place primarily as a result of the berries being mis- analysis is hampered by the instability of the toxins against
taken for edible fruits [453–455]. Datura stramonium (jim- air and heat, and quantitative data is rare.
sonweed, thorn apple, leaves, seeds, tea from roots) is often Accidental poisonings with autumn crocus were described
intentionally ingested for its hallucinogenic effects but also as a result of erroneous ingestion of its leaves instead of
mistaken for vegetables [456, 457]. The poisonings were ramson (Allium ursinum) in the spring but also by suicidal
mostly survived under medical care but fatalities were also intake of the tubers or plant extracts [476, 477]. Since the
reported [458, 459]. Also, Hyoscyamus niger (black hen- toxic constituent colchicine is also used as a medical drug,
bane) was found to be largely ingested to produce a pleasant its toxicological properties were thoroughly studied. It is a
experience or when mistaken for other plants [460]. This mitosis inhibitor with multiple organ dysfunction and cardio-
applies furthermore to Brugmansia (angel’s trumpet) which vascular collapse at lethal doses (about 25 g tuber for adults).
is widely abused by adolescents [461, 462]. In one case, an A typical late symptom is alopecia. Colchicine occurs also
18-year-old high school student without prior mental disor- in flame lily (Gloriosa superba), a tropical climbing flower
ders amputated his penis and his tongue in a delirious state plant [478–480]. Toxic levels (> 25 ng/mL in blood) can be
after having consumed angel’s trumpet tea [463]. Atropine detected by HPLC–DAD, GC–MS, and LC–MS/MS tech-
and scopolamine are included in toxicological-analytical niques using the corresponding analytical databases.
databases and toxic or lethal concentrations in blood can be There are several plants with cardiac glycosides as the
determined by GC–MS and LC–MS methods. toxic component. This includes especially foxglove (Digi-
The active ingredient of blue monkshood aconitine talis purpurea), but also oleander (Nerium oleander) and lily
belongs to the most toxic plant poisons with a minimal lethal of the valley (Convallaria majalis), with digitoxin, olean-
dose of 1–2 mg. The poisoning mechanism is deactivation of drin, and convallatoxin as the main toxic substances. They
sodium channels with cardiotoxic and neurotoxic effects and act by inhibition of the N ­ a+/K+-ATPase leading to hyper-
fatal respiratory paralysis or cardiorespiratory arrest [464]. kalemia and atrioventricular block at higher doses. Acci-
Beside the accidental intake of plant parts, extracts were dental poisoning occurred by confusing comfrey leaves with
prepared in suicide and homicide cases [465–467]. Blood foxglove leaves when the plants are not in bloom or with
concentrations in fatalities were mainly > 10 ng/mL, and borage [481–485]. Both accidental and suicidal poisonings
HPLC–DAD and preferentially LC–MS techniques were were reported for oleander leaves [486–488]. Lily of the
applied for its determination. valley in non-blossoming state was confused with wild gar-
Coniine, the classical poison of Socrates’ execution [468], lic [489, 490]. Because of assay cross-sensitivity, both con-
is formed in hemlock (Conium maculatum). It has, as com- vallatoxin and oleandrin are detectable by commonly used
pared to other herbal poisons, a relatively simple structure digoxin immunoassays [491]; however, exact identification
((S)-2-propylpiperidine, M = 127). It is a nicotinic-type and quantitation requires LC–MS/MS methods. Lethal blood
acetylcholine receptor agonist and doses above 6–7 mg/kg concentrations are in the range of 10–100 ng/mL.
(about 100 g plant material) can lead to death by respira- Ricin (M = 60–65 kDa) and abrin (M = 65 kDa) are both
tory paralysis. Accidental hemlock poisoning by ingestion extremely toxic lectins of castor beans [492–495] and abrus
is described in humans who have mistaken the leaves of seeds [496–499], respectively, and act through inhibition of
the plants for parsley, the roots for parsnips, or the seeds the protein synthesis at the ribosomes. Both toxins are white
for anise, and suicides by intake of the plant or injection of powders, but poisoning occurred mostly by the seeds or seed
extracts were reported [469–473]. Since hemlock poison is extracts. Injection of doses as low as 100 µg are lethal for
a common weed, it is particularly dangerous for livestock. human. Particularly ricin had found interest as a warfare or
Because of its saturated structure, it has no UV absorption terrorist agent. The resulting very low concentrations can only
above 200 nm and cannot be detected by HPLC–DAD. be detected by special immunological techniques as reviewed

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ChemTexts (2025) 11:4 Page 75 of 95 4

Fig. 68  Structure and molecular

mass of highly toxic mushroom
poisons. Orellanine in fools
webcap (Cortinarius orel-
lanus), α-amanitin in death
cap (Amanita phalloides),
muscarine in Clitocybe and Ino-
cybaceae mushroom families,
ibotenic acid and muscimol in
fly agaric (Amanita muscaria),
and gyromitrin in false morel
(Gyromitra esculenta)

by Musshoff [495]. However, the accompanying pyridine α- and β-amanitin, muscarine, muscimol and ibotenic acid,
alkaloid ricinine (3-cyano-4-methoxy-N-methyl-2-pyridone, and gyromitrin (structures in Fig. 68) [503].
M = 164) of castor beans occurs in poisonings with these Orellanine occurs in the fools webcap (Cortinarius orel-
seeds in concentrations well measurable with LC–MS/ lanus) and other Cortinarius species in concentrations of
MS (0.08–10 ng/mL in urine). In the same way, l-abrine 0.45% to 1.4%. It is strongly nephrotoxic and six mushrooms
(N-methyl-l-tryptophan, M = 218) is used as a biomarker can lead to acute and lethal renal failure. Irreversible renal
of poisonings with seeds of Abrus precatorius. A MALDI- damage and the need for dialysis or transplantation was
TOF-MS/MS method for detection of ricin in real cases was described [504, 505]. Orellanine was first assayed in plasma,
described [500]. In a spectacular case in 1978, the Bulgarian urine, and kidney biopsies by spectrofluorimetry on two-
dissident Georgi Markov was assassinated by a ricin injection dimensional thin-layer chromatograms [506, 507]. In a newer
to his thigh via a specially modified umbrella [501]. method, it can be quantified by liquid chromatography with
high-resolution mass spectrometry (LOQ 0.5 ng/mL) [508].
Amanitines, the poisons of the death cap (Amanita phal-
Mushroom poisons Mushrooms are neither plants nor ani- loides), are the most toxic mycotoxins [509]. They are bicy-
mals but constitute their own kingdom with a huge diver- clic octapeptides with three unusual hydroxylated amino
sity, the fungi. Poisonings by mushrooms were reviewed acids (Fig. 68). The content of α-amanitin in dry mushroom
by Wennig et al. [502] and Flament et al. [503]. There are is in the order of 1.5 mg/g. It is extremely stable against high
about 5000 species of so-called higher fungi, from which a temperature, pH, and digestive enzymes. The lethal dose
few dozen contain mycotoxins, which, when ingested, could is 0.1 mg/kg body weight (one or two mushrooms). It is a
cause poisoning of varying degrees of severity, and may potent inhibitor of RNA polymerases that blocks the produc-
even result in death. The poisonings are mostly seasonal, tion of mRNA and protein synthesis in liver and kidney cells
between August and November in Europe, and are not sel- which leads to necrotic damage and death within 5–7 days
dom. For instance, in France, an average of 1300 poisoning after ingestion of the mushrooms. Early unambiguous iden-
cases per year was reported between 2010 and 2017 [504]. tification of the poison is very important [510]. Specialists
However, the cause of poisoning is mainly based on clini- were able to identify the mushroom in food residue on the
cal signs and case history and is only seldom confirmed by basis of the spore morphology and size using the traditional
toxicological analysis. Nevertheless, there are only a limited microscopic method. Also, a special color test was applied.
number of mycotoxins involved in the most serious cases Later, immunoassays (RIA, ELISA, see Sect. “Immunoas-
which are typical nonvolatile organic substances: orellanine, says for drugs and poisons”) enabled detection of amanitines

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4 Page 76 of 95 ChemTexts (2025) 11:4

in urine. Today, chromatographic methods dominate and ingestion, it is sequentially hydrolyzed by the gastric acid to
can determine the amanitines in serum or plasma with a form N-methyl-N-formylhydrazine and more slowly mono-
limit of detection of 20 pg/mL [511]. The concentration of methylhydrazine which is the actual toxic species and causes
α-amanitin in plasma of nine poisoning cases varied between cytotoxicity via alkylating free radicals leading to oxida-
37 and 2980 pg/mL. LC–MS/MS methods for determination tive stress. Poisoning symptoms range from gastrointestinal
in urine, liver tissue, and bile were developed [512, 513]. disorder to hepatotoxicity in more severe cases, and neuro-
However, tests for amanitines are not yet well established in logical symptoms including seizures. Although accidental
systematic toxicological analysis. A considerable dark figure poisoning due to confusion between false and true morels
of poisonings can be assumed, and, surprisingly, no murder including death cases were repeatedly reported [524, 525],
cases were reported in scientific literature. no analytical methods for detection of gyromitrin or its deg-
Muscarine is actively present in several mushroom fami- radation products in human samples were described.
lies, among others in Clitocybe and Inocybaceae with con- In summary, mushroom poisons and plant poisons are
centrations of 0.15% to 0.43%. Muscarine takes its name not yet sufficiently present in procedures of the systematic
from Amanita muscaria (fly agaric) from which it was iso- toxicological analysis.
lated for the first time. But the content in this species is
very low (0.0002–0.0003%) which makes its toxic action
insignificant compared with other active compounds [503]. Interpretation of the results:
Muscarine stimulates postganglionic acetylcholine recep- the toxicological expertise
tors in the autonomic nervous system, causing a cholinergic
toxidrome that may include symptoms such as diarrhoea, General aspects
vomiting, miosis, bradycardia, bronchorrhea, lacrimation,
and hypersalivation. Since the lethal human dose is about After completion of the analytical investigations, the results
500 g of raw mushrooms, poisonings are mostly survived but must be interpreted and reported to the medical or juridical
lethal outcome was also described [514, 515]. Liquid chro- institution. Usually, prosecutors, judges, or lawyers are not
matography with high-resolution mass spectrometry detec- specialized in toxicology. Therefore, a simple list of identi-
tion led to muscarine concentrations from 0.12 to 14 μg/L fied substances and determined concentrations is not suf-
in whole blood, < LOQ to 43 μg/L in plasma, and < LOQ ficient and a detailed explanation is expected.
to 1537 μg/L in urine from ten suspected poisoning cases The toxicological expertise should start with the case his-
[516]. tory, which contains all relevant information as described
Fly agaric (Amanita muscaria) has a red or orange cap in Sect. “Indications from case history, clinical symptoms,
with small white plaques. The distinctive appearance of this or autopsy” and known before starting the analysis. This
mushroom and the widespread belief in its toxic proper- is followed by a short description of the used analytical
ties make accidental consumption very rare. However, it is techniques and the substance groups which, in principle,
abused for its psychedelic effect through drinking infusions are detectable by these methods. Then, the qualitative and
made from those mushrooms or eating the whole fruiting quantitative analytical results are presented in a table. For
bodies [517, 518]. Ingestion causes euphoria and psycho- reasons of unambiguity:
tropic effects owing to the neurotoxins ibotenic acid and
muscimol (structure in Fig. 68) which act on the glutamic – Substances are indicated by the international non-propri-
acid receptors and GABA receptors, respectively. Muscimol etary drug names or IUPAC names and not by the trade
is formed from ibotenic acid via decarboxylation. The dose names of the manufacturers.
required to induce hallucinations is 30–60 mg of ibotenic – Metabolites of the substances are included as far as iden-
acid and 10–15 mg of muscimol—this is less than the fresh tified.
weight content of a single mushroom. Both substances are – Concentrations are given as weight/weight (e.g., µg/g) for
also the toxins of the panther cap (Amanita pantherina) tissue samples or weight/volume (e.g., µg/mL) for liquid
which can be easily mistaken for edible species [519, 520]. samples. The use of molar concentrations (e.g., mmol/L)
Despite the relatively simple structure, ibotenic acid and is in this branch of analytical chemistry unusual.
muscimol are not easily included in general toxicological
screening procedures by LC–MS/MS but require specific Then, each identified substance is interpreted concerning
sample preparation for analysis in urine or blood [519, 521, the following aspects:
522].
Gyromitrin (acetaldehyde N-methyl-N-formylhydrazone), – Essential effect and/or use of the substance.
the poison of the false morel (Gyromitra esculenta), has an – Interpretation of the determined concentration by use
even more simple organic structure (Fig. 68) [523]. Upon of databases described in literature, own cases previ-

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ChemTexts (2025) 11:4 Page 77 of 95 4

ously investigated, and case reports described in litera- Pharmacokinetic/toxicokinetic considerations

ture. The blood concentration list of Schulz et al. [280,
281] and the famous collection of toxicological mono- Sometimes it is necessary in interpretation to estimate the
graphs of more than 2000 substances by Randall C. blood concentration at the time of an incident from the value
Baselt (1944–2021) [152] are very helpful and should of a later collected blood sample or from the reported dose
be at hand in every toxicological laboratory. Generally, applied some time before. For alcohol, there are well-estab-
the determined concentrations are ascribed to one of lished procedures of calculation and back-calculation [527].
the following three ranges: However, for toxic substances in general, it is more compli-
cated. On its way through the human body, the concentration
Therapeutic∕natural − Toxic − Comatose∕lethal
of a substance in body fluids and tissues changes during
absorption, distribution, metabolism, and elimination. Phar-
These three ranges overlap widely and there are no macokinetics/toxicokinetics is the study of the time course
sharp limits. Individual features of the victim and circum- of these processes [528, 529]. For calculations of concentra-
stances play an essential role. Therefore, the following tions in blood after oral intake and for living individuals, the
specifics of the case must be considered: following parameters are used:

– Age, gender, health state, and diseases of the victim. – Half-life of absorption t1/2,a
– Combined effect of the different identified substances – Half-life of elimination t1/2,e
including alcohol. – Bioavailability Fb
– Kind of poison exposure: oral, inhalation, or injection. – Volume of distribution Vd
– Time span after poison exposure and death or sampling – Body mass of the individual m
in survived cases. – Administered dose D
– Adaptation to the poison in case of opioids.
– Postmortem changes of concentrations. Generally, a first-order kinetics is assumed for t1/2,a and
t1/2,e. The bioavailability is set at 100% for intravenous injec-
In the final conclusion of the toxicological expertise, tion. For oral administration, it is the portion in % of the
only a probability can be stated, for instance: “The death administered substance which reaches the systemic circula-
of the person can be caused by the oral intake of a high tion. It is reduced by incomplete absorption from the gastro-
overdose of the antidepressant amitriptyline in combina- intestinal tract and by the first-pass metabolism in the liver
tion with a high alcohol concentration.” The final deci- (see Sect. “Comparison between different samples and con-
sion about the cause of death is made by the physician in sideration of metabolites”). The volume of distribution (liter/
consideration of the results of autopsy and circumstances. kg body mass) is the theoretical volume that would be neces-
This can be other than poisoning even if the concentra- sary to contain the total amount of administered drug at the
tion is in the upper comatose/lethal range. There are many same concentration as it is measured in the blood plasma.
examples of competing causes of death with comatose- Neutral drugs with a high lipid solubility are accumulated in
lethal concentrations of poisons but the final cause of fat tissue and have high volumes of distribution (e.g., chlo-
death being drowning, hanging, suffocation, or traumatic roquine, Vd = 116–228 L/kg), whereas it is low for polar and
injury and the poison has only incapacitated the victim. highly ionized drugs (e.g., caffeine, Vd = 0.4–0.6 L/kg).
Beside the qualitative and quantitative interpretation of t1/2,e, Fb, and Vd of many medical drugs were determined
the results as the possible cause of illness or death, other in clinical trials in the context of drug development and
information may be requested in forensic cases, such as: can be found in the summary of product characteristics (in
Germany “Fachinformation”) of the manufacturers. Mean-
– Estimation of the ingested or administered dose from while, these data were included also in Wikipedia (English
the concentration in blood and the interval between language edition). For many other toxic substances, they
intake/administration and sampling or estimation of are available in Baselt reference works [152] or in the sci-
this interval from dose and blood concentration. entific literature via PubMed. For calculations using these
– Assessment of the capability of a victim under a certain parameters, an Excel-based program was developed [530].
drug concentration to fight back in a homicide case. Although this allows only a rough approximation because
– Assessment of the criminal responsibility of a perpetra- of the large interindividual variation of Fb, Vd, and t1/2,e, it
tor under the influence of a certain drug concentration appeared to be very helpful in practical cases, also of the
in blood in murder case. author of this paper. However, it is only to a very limited
extent applicable to postmortem concentrations as shown
in the next section.

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4 Page 78 of 95 ChemTexts (2025) 11:4

Postmortem changes of substance concentration the esophageal sphincter and passive regurgitation. Some
death cases with extremely high concentrations in heart
The concentrations of natural or therapeutic and toxic ranges blood and lung and low or negative results in venous blood
measured for living subjects are not necessarily applicable were interpreted in this way by the author of this paper
to postmortem samples because of the following reasons [33, 368].
[531]: in the last hours before death, premortem factors such To sum up, the toxicological interpretation in post-
as decreased motility of the gastrointestinal tract, reduced mortem cases should be based primarily on the venous
function of liver or kidney, and centralized blood circula- blood concentration which is least affected by the changes
tion can cause changes in the poison distribution within the described above. If venous blood is not available, results
body. As a perimortem factor, the continued metabolism from heart blood, tissue samples, and gastric content
during a longer agony phase can lead to disproportionate should be critically evaluated considering the aforemen-
concentrations. tioned effects and the case history. In most cases, a state-
However, the most drastic changes are the postmortem ment about the possibility of an intoxication as the cause
factors which are summarized as “postmortem redistri- of death can be given.
bution” [531–535]. These are caused on the one hand by
biochemical changes after death and by physical processes
(diffusion and other kinds of substance transport) on the Comparison between different samples
other. On the cellular level, oxygen deficit leads to lactic and consideration of metabolites
acidosis with accumulation of basic substances and degra-
dation of membranes. The ATP deficit induces rigor mortis Additional information about the poisoning can be
with change of pressure in the blood vessels and correspond- obtained from the concentration ratio between the different
ing substance movement. The destruction of lysosomes and samples (e.g., blood, urine, and gastric content). In case
liberation of lysozymes enables autolytic self-destruction of oral intake, high concentration in the gastric content
and finally putrefaction by invading microorganisms which with very low or negative results in urine point to a short
metabolically may degrade or change the substance. Toxic survival time whereas high concentration in urine with
substances which are often accumulated in liver, lung, and low concentration in blood can be an indication of a longer
myocardium by protein binding or in fat tissue are liberated survival time. However, a positive result in gastric fluid
during these biochemical processes. Furthermore, metabo- is not always evidence of oral intake since basic poisons
lization of the poison can be continued in the liver but also are excreted with the gastric acid and accumulate in this
in body fluids and other tissues [33]. acidic medium.
Postmortem redistribution in the narrower sense means Metabolites of the poison can be detected in almost every
the movement of the toxic substances from organs with systematic toxicological analysis. They are an additional
high concentrations and from the gastrointestinal tract to proof of the identified drug. In case of injection or inhala-
body fluids and tissues with lower concentrations. Dif- tion, the metabolite/parent substance concentration ratio can
fusion to heart blood along the concentration gradient is be helpful for estimation of the survival time. Missing or
believed to play an essential role. However, since diffusion very low metabolite concentration is typical for a very short
alone is a very slow process, it can be supported by a flow time. However, this is limited to non-hydrolytic metabolites
within the blood vessels caused by compression or dilata- since the hydrolases in blood are still active after death and
tion if the corpse is moved, e.g., from the site of detection in the collected sample.
to the autopsy table. In the same way, some of the liquid For oral intake, metabolites are present in blood to a
gastric contents can be moved through the esophagus and different degree at the very beginning since everything
larynx to the trachea. Pounder and Yonemitsu have shown absorbed in the gastrointestinal tract passes the portal vein
in an experiment with human corpses that deposition of to the liver as the main organ of metabolization before it
60 mL of gastric content which contained drugs and alco- enters the systemic circulation (first pass effect). Therefore,
hol into the trachea lead to extremely high concentrations the use of metabolite/drug ratio for estimation of the sur-
in the pulmonary artery and pulmonary vein but also to vival time is limited. An exception is metabolites which
high concentrations in right and left heart blood and in the are formed over several steps and need more than one liver
aorta [536]. However, blood from femoral veins remained passage. An example is the formation of diphenylmethoxy-
negative. The authors concluded that the artifactual eleva- acetic acid from diphenhydramine [368]. For this substance,
tion of cardiac blood concentration due to agonal aspira- the metabolite/drug ratio could be used for an approximate
tion of drug-laden vomitus is possible and that even with- estimation of the time between drug intake and sampling in
out agonal vomiting the drug-laden gastric fluid might clinical cases or of the survival time after drug ingestion in
enter the airways because of postmortem relaxation of death cases.

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ChemTexts (2025) 11:4 Page 79 of 95 4

Pharmacodynamic/toxicodynamic considerations bewilderment, hallucinations, and paranoia to exercise

appropriate judgment, to make calm and rational choices,
Pharmacodynamics refers to the effects of (therapeutic) or to think clearly. The drug can interfere with the ability
drugs in the body and the mechanism of their action [529]. to form intent. There are no fixed rules in such cases, and
In case of other toxic substances, the term “toxicodynam- usually a forensic psychiatrist is called in by the court as an
ics” is more appropriate. It includes the interaction of the expert to analyze the personality of the offender in general
substance on the cellular level, specific binding to receptors, and during the crime. Whether and to what extent reduced or
and therapeutic effects as well as toxic symptoms and their even lacking culpability influences the sentence on court is
dependence on the dose or concentration in blood. very complicated and depends on the country [545, 546]. A
The essential effect of the poison belongs generally to the similar situation appears if a patient with schizophrenia fails
toxicological report in clinical and lethal cases. However, in to take the prescribed psychiatric medication and commits
forensic cases, sometimes a more specific interpretation of a crime [547, 548]. Toxicological analysis must prove the
the detected substance and its concentration on the physi- absence of antipsychotic drugs in blood and urine, and hair
cal performance and the psychical behavior is requested. analysis should confirm previous chronic intake.
Illegal as well as therapeutic drugs may impair conscience, Concerning the victim of a crime, it must be assessed
alertness, balance, and eyesight. Typical for illegal drugs are whether the detected concentration of a poison could have
euphoria, decrease of criticism and self-criticism, risk-taking caused unconsciousness, amnesia, or reduced their alertness,
propensity, confusion, paranoia, inclination to aggression, vigilance, and ability to fight back. This is particularly com-
illusions, and hallucinations. These symptoms are incompat- mon in cases of drug-facilitated sexual assault [549] but also
ible with driving safety and jobs with a high danger poten- other drug-facilitated crimes [550, 551].
tial. In case of an accident, the toxicological expert must Overall, the toxicological interpretation of the analytical
interpret the detected concentrations concerning a causal results is very complex and in many cases can only propose
relevance. For therapeutic drugs, such undesirable side probabilities. However, it is an important part in solving the
effects are indicated in the patient information leaflet. For case together with the physician doing the autopsy and the
road traffic delicts, respective paragraphs in national road investigating criminal officer. Despite all information from
traffic acts (e.g., §24a of the German road traffic act [537]) literature and the internet, it depends to a high degree on the
define in an annexure limits of illegal drug concentrations experience of the forensic toxicologist and a critical check-
above which fitness to drive can be excluded. For example, ing of the plausibility of all results.
a limit of 3.5 ng/mL THC in blood of drivers was set in
the context of cannabis liberalization. Occupational acci-
Author contributions F.P. did all work of this paper.
dents may be treated analogously. If human lives are lost
or very high material damage is caused, a comprehensive Funding Open Access funding enabled and organized by Projekt
expertise must assess the probable impairing influence of DEAL.
the drug in the context of all concomitant circumstances.
Data availability No datasets were generated or analysed during the
This involves often a profound study of the actual literature current study.
including reports about similar cases. Writing an expertise
about death or heavy injury by medical malpractice can take Declarations
several weeks including the thorough study of all clinical
reports. Examples from the author’s own practice are a fatal Conflict of interest The authors declare no competing interests.
injection of zinc oxide lotion instead of propofol [538], dis- Open Access This article is licensed under a Creative Commons Attri-
regard for the deadly interaction between a herpes zoster bution 4.0 International License, which permits use, sharing, adapta-
medication and a 5-fluorouracil-based chemotherapeutic in tion, distribution and reproduction in any medium or format, as long
cancer treatment [539], or depot injection of testosterone as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
enanthate despite chronic treatment with the oral anticoagu- were made. The images or other third party material in this article are
lant phenprocoumon as a clear contraindication which led included in the article's Creative Commons licence, unless indicated
to severe embolisms in that case (Pragst, unpublished case). otherwise in a credit line to the material. If material is not included in
The assessment of psychic or behavioral drug effects the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
plays a crucial role in crimes such as murder or sexual need to obtain permission directly from the copyright holder. To view a
assault. This can concern offenders as well as victims of the copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/.
crime. For the offender, the possible effect of the detected
drug concentration at the time of the crime may reduce or
deny criminal responsibility [540–544]. In a drug-induced
psychosis, an offender’s culpability may be reduced by

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4 Page 80 of 95 ChemTexts (2025) 11:4

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544. Stevenson R, Tuddenham L (2014) Novel psychoactive substance Fritz Pragst (*1941) studied
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25:60–61. https://​doi.​org/​10.​1016/j.​jflm.​2014.​04.​007 versity of Berlin (Germany) and
545. Fischer B, Rehm J (1998) Intoxication, the law and criminal worked there in the Institute of
responsibility—a sparkling cocktail at times: the case studies of Organic Chemistry from 1966 to
Canada and Germany. Eur Addict Res 4:89–101. https://​doi.​org/​ 1987 in student education and
10.​1159/​00001​8931 research in organic electrochem-
546. Mellsop G, Choi WK, Every-Palmer S, Green B, Heffernan E, istry (voltammetry in aprotic
Kachaeva M, Shiina A, Wang X (2016) Drug driven psychoses solvents and superacidic media,
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2016.​02.​037 organic compounds. In 1987, he
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O (2018) Schizophrenia and criminal responsibility: a systematic toxicological analysis and
review. J Nerv Ment Dis 206:370–377. https://​doi.​org/​10.​1097/​ became head of the Department
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criminal responsibility: is the insanity defense legitimate? J in 1989. Main areas of his scientific work there were development and
Psychiatry Law 40:219–242 application of methods for systematic toxicological analysis and hair
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lence of selected licit and illicit drugs in drug facilitated sexual clinical and death cases. After his official retirement in 2006, he con-
assaults. Forensic Sci Int Synerg 9:100545. https://​doi.​org/​10.​ tinues working as a guest scientist at the Institute of Legal Medicine.
1016/j.​fsisyn.​2024.​100545 Prof. Pragst published more than 230 papers in scientific journals and
550. Volonnino G, La Russa R, Di Fazio N, Ottaviani M, Zamponi book chapters. His work was recognized by a honorary doctorate of the
MV, Spadazzi F, Umani-Ronchi F (2023) Z-Drugs and their use Forest Technical University Saint Petersburg (Russia), the Alan Curry
in drug-facilitated crimes: a review of the literature. Clin Ter Award of The International Association of Forensic Toxicologists
174:451–468. https://​doi.​org/​10.​7417/​CT.​2023.​2465 (TIAFT), the Jean Servais Stas Medal of the Society of Forensic and
551. Singh SP, Kaur S, Singh D, Aggarwal A (2015) Lorazepam: a Toxicological Chemistry (GTFCh), and the Konrad Händel Prize of
weapon of offence. J Clin Diagn Res. https://​doi.​org/​10.​7860/​ the German Society of Legal Medicine (DRM).
JCDR/​2015/​11677.​5667

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