Biomedical Chromatography

Recent advances in the detection of drugs of abuse by Dried

Blood Spots

Journal: Biomedical Chromatography

Manuscript ID BMC-22-0578.R1

Wiley - Manuscript type: Review

Fo
Date Submitted by the
n/a
Author:

Complete List of Authors: Ververi, Christina; Università degli Studi di Torino, Department of
rP

Chemistry
Vincenti, Marco; Università degli Studi di Torino, Department of
Chemistry; Centro Regionale Antidoping e di Tossicologia "A. Bertinaria",
ee

Salomone, Alberto; Università degli Studi di Torino, Department of

Chemistry

Keywords: drugs of abuse, DBS, LC-MS/MS, green chemistry, dried blood spots
rR
ev
iew

http://mc.manuscriptcentral.com/bmc
Page 1 of 18 Biomedical Chromatography

1
2
3 Recent advances in the detection of drugs of abuse by Dried Blood Spots
4
5
Christina Ververi1,2, Marco Vincenti1,2, Alberto Salomone1,2
6
7 1:
8
Department of Chemistry, University of Turin, Italy
9 2: Centro Regionale Antidoping, Orbassano (TO), Italy
10
11 * Corresponding author: Christina Ververi
12
13 Email address: [email protected]
14
15
16 Abstract
17
18 Purpose of this review study is to sum up the main recent advances reported in
19
20 the scientific literature about the detection of common drugs of abuse in biological
21 samples upon their collection by Dried Blood Spot (DBS) devices. The most recent,
Fo

22
23 innovative and fully validated methods for the qualitative and/or quantitative detection
24
of common drugs of abuse are reported, including alprazolam, clonazepam, diazepam,
rP

25
26
27
3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine
28 (MDA), 3,4-methyl- enedioxyethylamphetamine (MDEA), amphetamine,
ee

29
30 methamphetamine, cocaine, tetrahydrocannabinol (THC), 6-monoacetylmorphine,
31
rR

32 morphine, codeine, hydromorphone, hydrocodone, oxycodone, noroxycodone,

33
tramadol, methadone, buprenorphine, fentanyl, ketamine and their respective
34
35 metabolites and γ-hydroxybutyric acid (GHB). DBS proved to be extremely promising
ev

36
37 for routine analysis of forensic cases, although large-scale experiments on real samples
38
iew

39 need to be performed to confirm the emerging advantages of the technique and remove
40 the potential limitations still affecting its widespread application.
41
42
43
44
45
46
Keywords drugs of abuse, DBS, LC-MS/MS, HRMS, green chemistry
47
48
49 Introduction
50
51 The Dried Blood Spot (DBS) microsampling technique was first proposed back
52
in 1913 by Ivar Bang and used for glucose concentration monitoring (Bang I., 1913).
53
54 In 1963, Guthrie and Susi successfully utilized dried blood sampling for newborn
55
56 phenylketonuria screening (Guthrie & Susi, 1963). In the early 2000s, interest in DBS
57
58 increased, resulting in the enactment of intensive scientific research and
59 experimentation and finally leading to DBS recommendation as an accurate technique
60

http://mc.manuscriptcentral.com/bmc
 Biomedical Chromatography Page 2 of 18

Detection of drugs of abuse by DBS: recent advances

1
2
3 for HIV and hepatitis B and C diagnosis by the World Health Organization (Biondi et
4
5 al., 2019; Soulier et al., 2016). Currently, DBS is commonly used for newborn
6
7 screening for a wide range of serious genetic and metabolic disorders (Carpenter &
8
9
Wiley, 2002; Li & Tse, 2010) forensic screening of drugs of abuse and therapeutic drug
10 monitoring (Corran et al., 2008; Lee & Li, 2014; Wilhelm et al., 2014). In 2021, the use
11
12 of DBS also proved successful for COVID-19 epidemiological research using ELISA-
13
14 based assays (Fontaine & Saez, 2021).
15
16
17 According to the global overview of drug demand and drug supply, issued by
18 the United Nations Office on Drugs and Crime this year, more young people are using
19
20 drugs compared with previous generations while cocaine production reaches its record
21
Fo

22 high, and seizures of amphetamine and methamphetamine have dramatically increased

23
24
(Global Overview Drug Demand Drug Supply, 2022). Furthermore, drug-related
rP

25 diseases and disorders keep rising among users while the illicit drug trade continues to
26
27 hold back the economic and social development of several Countries. These combined
28
ee

29 plagues challenge the forensic toxicology laboratories to develop new, easy, accurate,
30 and quick-turnaround methods to detect drugs of abuse in seizures and biological
31
rR

32 samples and help law officers to tackle these inherent ever-growing problems. Forensic
33
34 toxicology involves a variety of professionals - clinicians, chemists, law officers - that
35
ev

36 must work together to achieve successful outcomes (Watterson, 2015). Within this
37 context, forensic toxicology is traditionally distinguished between human-health
38
iew

39 toxicology, that analyses human specimens (blood, urine, hair, oral fluids) to assess
40
41 whether drugs or toxins may have affected the donor’s performances/abilities or may
42
have caused him/her serious health disorders, and postmortem toxicology, whose goal
43
44 is to assess whether drugs, alcohol, or toxins may have contributed or caused death of
45
46 the investigated subject (Watterson, 2015). Moreover, Stove et al. aptly distinguished
47
48 the DBS samples between the ones obtained from adult donors, addressed to investigate
49 typical forensic toxicology inquiries, and those collected from newborns for the mere
50
51 assessment of drug “exposure” prior to birth (Stove et al., 2012). Through the years,
52
53 several publications appeared suggesting the use of DBS for drugs of abuse detection
54
55
in a variety of circumstances, including performance tests, physical disorders diagnosis,
56 abstinence compliance, early exposure assessment, and postmortem investigations.
57
58 The classes of analytes that gained significant interest and whose positive detection has
59
60 been demonstrated include benzodiazepines (e.g., alprazolam, clonazepam, diazepam,

http://mc.manuscriptcentral.com/bmc
Page 3 of 18 Biomedical Chromatography

Detection of drugs of abuse by DBS: recent advances

1
2
3 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine
4
5 (MDA), 3,4-methyl- enedioxyethylamphetamine (MDEA), amphetamine,
6
7 methamphetamine, cocaine, tetrahydrocannabinol (THC), opiates (6-
8
9
monoacetylmorphine, morphine, codeine, hydromorphone, hydrocodone, oxycodone,
10 noroxycodone), tramadol, methadone, buprenorphine, fentanyl, ketamine and their
11
12 respective metabolites, and γ-hydroxybutyric acid (GHB) (Stove et al., 2012).
13
14
15 DBS is repeatedly cited as a simple and rather inexpensive sample collection
16
17 procedure in which the filter paper can retain many blood components, allowing an
18 easier and even automated blood extraction procedure (Déglon et al., 2012; Versace et
19
20 al., 2013). DBS can solve several limitations involved in routine blood analysis that
21
Fo

22 concern the intrusiveness of intravenous blood sampling, the long-term analytes’

23
24
stability in blood, and the complex and time-consuming sample preparation and
rP

25 handling. On the other hand, the limitations of DBS sampling include the problems
26
27 posed by the hematocrit effect, the difficulty to perform repeated analysis due to the
28
ee

29 small blood volume collected, and the referability of the results obtained with DBS to
30 the conventional cut-off values and normal ranges defined in whole blood analysis
31
rR

32 (Jantos & Skopp, 2011).

33
34 The analysis of target analytes after DBS collection may be performed by
35
ev

36 several analytical techniques, according to the application: immunoassays, gas

37 chromatography (GC), liquid chromatography (LC) coupled to ultraviolet (UV) or
38
iew

39 fluorescence detectors, to mass spectrometry (MS) or tandem mass spectrometry

40
41 (MS/MS). Due to the technological advances over the last decade, LC-MS/MS
42
instruments currently achieve the highest levels of sensitivity and specificity in targeted
43
44 drug analysis. Extreme sensitivity is obtained by triple quadrupole detectors operating
45
46 in the multiple reaction monitoring mode (MRM), while the newest TOF (time-of-
47
48 flight) and Orbitrap analyzers additionally allows high-resolution mass analysis and
49 accurate determination of elemental composition, resulting in accurate qualitative and
50
51 quantitative analysis of substances present in blood even at extremely low concentration
52
53 (Antelo-Domínguez et al., 2013; Joye et al., 2019; Kacargil et al., 2020; Keevil, 2011;
54
55
Li & Tse, 2010; Sadler Simões et al., 2018; Stove et al., 2012). The present review
56 provides an overview of the most recent studies that propose easy and highly sensitive
57
58 procedures for the detection of drugs of abuse after DBS sampling.
59
60

http://mc.manuscriptcentral.com/bmc
 Biomedical Chromatography Page 4 of 18

Detection of drugs of abuse by DBS: recent advances

1
2
3 Advantages of DBS
4
5 The greatest assets of DBS are the ease of sampling and the fast sample
6
7 handling, both being crucial aspects in forensic toxicology. For example, rapid
8
9
sampling is essential in cases of drug-facilitated sexual assault (DFSA) and of driving
10 under the influence of drugs (DUID). In both cases, the sample needs to be collected as
11
12 soon as possible, since the target substances are generally metabolized in a few hours
13
14 rapidly becoming undetectable in the primary matrix. On the other hand, alternative
15
matrices such as oral fluid and urine suffer from drawbacks, including controversy
16
17 about the convertibility between the detected drug concentration and the original dosage
18
19 and their proneness to alteration (mouth washing or urine exchange) (Huestis et al.,
20
21 2011). DBS provides reliable estimation of blood concentration without requiring
Fo

22 dedicated personnel and a medical office for blood collection.

23
24 Typical forensic casework involves extraction of blood volumes ranging from
rP

25
26 100–2000 μl depending on the chosen procedure (liquid-liquid extraction or solid-phase
27
28
extraction) and instrumental analysis (GC/MS or LC-MS/MS), while DBS needs only
ee

29 10-100 μl of blood sample which can be collected by just pricking a finger or a heel
30
31 (Watterson, 2015).
rR

32
33 Another important aspect of the use of DBS for blood sampling is the improved
34
stability of the contained drugs after being spotted and dried, because of the blood
35
ev

36 immobilization within the paper matrix (Alfazil A. A. & Anderson R. A., 2008; Garcia
37
38 Boy et al., 2008). DBS substrates consist of paper chemically treated to inhibit bacterial
iew

39
40 growth and enzyme activity that would possibly drive analytes’ degradation. As a
41 matter of fact, most drugs have shown better stability when dried onto a filter paper
42
43 than in whole blood, which represents a key issue when time elapses between sampling
44
45 and analysis, and transportation or storage are needed before the sample can be
46
processed (Cone, 1995; Rook et al., 2006). This is particularly crucial for some drugs
47
48 of abuse, such as cocaine, heroin, and GHB, that have short a half-life time and limited
49
50 stability. In particular, cocaine intake can be confirmed by identifying its main
51
52 metabolite, benzoylecgonine (BZE), whereas heroin abuse cannot be simply deduced
53 from the presence of its main metabolite and hydrolysis product, morphine, because the
54
55 latter may also arise from a licit pharmaceutical drug, codeine. The case of GHB, also
56
57 produced by endogenous biochemical processes, is even more complex, since its abuse
58
59
or unaware administration is ascertained by measuring its excess in blood with respect
60 to the physiological level, which rapidly levels off either by metabolism or degradation

http://mc.manuscriptcentral.com/bmc
Page 5 of 18 Biomedical Chromatography

Detection of drugs of abuse by DBS: recent advances

1
2
3 (Stove et al., 2012). For these reasons, both drugs and their intake-suggestive
4
5 metabolites need to be stabilized. For example, stabilization of cocaine and of 6-
6
7 acetylmorphine (the first product of heroin metabolism) under DBS conditions has been
8
9
demonstrated (Sadler Simões et al., 2018).
10 DBS is also offered for checking drug abuse epidemiology in nightlife
11
12 environments, resulting in a powerful tool for large-scale investigation of the diffusion,
13
14 trends, addiction rates, and other physical and social effects of drugs intake and abuse
15
together with statistical analysis, provided that individual consent is mandatory
16
17 (Sañudo et al., 2015; Wood et al., 2009).
18
19 Another demonstrated application of DBS is the follow-up of drug and alcohol
20
21 addicts’ treatment. DBS is used to control abstinence from drugs and/or intake of
Fo

22 substitution medication offering an alternative to routine urine testing in which no risk

23
24 of sample falsification/alteration is possible (Stove et al., 2012). Obviously, DBS
rP

25
26 involves minimal patient discomfort, unlike intravenous blood sampling, making it
27
28
admissible on a routine basis. Moreover, DBS involves minimal sample handling which
ee

29 is also an important feature to protect the nursing personnel from the risk of HIV
30
31 infection, hepatitis or other blood viruses, more frequently affecting drug addicts
rR

32
33 (Brambilla et al., 2003; Mei et al., 2001).
34
As far as newborn screening is concerned, DBS has been proposed for studying
35
ev

36 the prevalence of cocaine and tobacco products intake among pregnant women near
37
38 childbirth, via the analytical determination of their respective metabolites,
iew

39
40 benzoylecgonine and cotinine (Stove et al., 2012).
41
42
43 Limitations of DBS
44
45 Although DBS solves all problems associated with venipuncture, including also
46
excessive blood volume sampling, the opposite problem combined with the need of
47
48 controlling the blood distribution homogeneity in the spot emerges from DBS
49
50 collection. In particular, it is not obvious that the blood deposition onto the card is
51
52 adequate and uniform. Older studies have tested DBS using around 100 μl (Garcia Boy
53 et al., 2008; Jantos & Skopp, 2011) even if this volume corresponds to intentionally
54
55 spotted samples after intravenous collection. As observed from recent applications to
56
57 real samples, the blood volume obtained by a finger prick and directly deposited onto
58
59
the card does not exceed 30-40 μl. Moreover, the area where the analyte expands to is
60

http://mc.manuscriptcentral.com/bmc
 Biomedical Chromatography Page 6 of 18

Detection of drugs of abuse by DBS: recent advances

1
2
3 possibly smaller than the spot itself, drawing attention on the correct procedures for
4
5 spotting the card, estimating the collected volume, and recovering the blood spot.
6
7 These variables are connected to other blood parameters that should be
8
9
considered, for example the hematocrit effect, that mainly affects the deposition area
10 and the analytes distribution in it. A clarifying study has been published from Veghle
11
12 et al. (Velghe et al., 2019) concerning the hematocrit effect and the manageable
13
14 practices that can be used to circumvent the inherent analytical problems. Notably, the
15
hematocrit bias can be overcome either by using controlled and fixed volumetric
16
17 application of blood or by correcting it with commercially available automated DBS
18
19 analyzers.
20
21 Further risks arise from manual cutting/punching the spot, that are avoided
Fo

22 when an automated DBS extractor is used. In the manual procedure, the paper
23
24 undergoes several treatment steps (cutting, transfer between tubes, extraction with
rP

25
26 solvents, centrifugation) which require skilled operators not to makes the whole
27
28
procedure prone to avoidable mistakes. For example, it is important to punch the correct
ee

29 DBS area, in order not to reduce the available material and consequently the sampled
30
31 volumes, the analytical results, and their comparability.
rR

32
33 Lastly, the application of DBS method to any sort of forensic cases (drug
34
monitoring, workplace drug-testing, newborn screening, etc.) consistently requires
35
ev

36 instrumental methods provided with high sensitivity, to support conclusions with

37
38 unequivocal data, and bearing in mind the limited amount of analyte present on the card
iew

39
40 and typically arising from less than 30 μl blood sampling.
41
42
43 Recently proposed methods and applications
44
45 In the last few years, significant laboratory development in DBS preparation,
46
extraction, and results acquisition has been observed. For the data acquisition stage,
47
48 hyphenation of UHPLC with either MS/MS or HRMS proved to provide adequate
49
50 sensitivity to detect analytes at trace level in the tiny amount of blood sampled by DBS.
51
52 The most persuasive studies are those that include real samples analysis and compare
53 the results obtained after DBS sampling with the ones arising from traditional routine
54
55 methods. The desirable final goal of these studies is actually to prove that DBS-based
56
57 methods are suitable for the detection of the most common drugs of abuse and their
58
59
metabolites under standard routine conditions and provide results similar to
60

http://mc.manuscriptcentral.com/bmc
Page 7 of 18 Biomedical Chromatography

Detection of drugs of abuse by DBS: recent advances

1
2
3 conventional analysis so as to be complementary to it whenever DBS is preferable to
4
5 intravenous sampling.
6
7 Sadler Simões et. al (Sadler Simões et al., 2018) developed and validated an
8
9
easy and quick method for the analysis of 11 illicit drugs of abuse based on DBS
10 sampling and using UPLC-MS/MS for the instrumental determination. The preparation
11
12 is simple: 50 μl of whole blood are spotted on a DBS card, allowed to dry off; then, 3
13
14 ml of methanol/acetonitrile (3:1 v/v) and 25 μl of deuterated internal standards (ISTD)
15
are added as an extraction solution. After stirring, sonication and centrifugation, the
16
17 solvent was dried under a nitrogen flow at 35oC. The residue was reconstituted with
18
19 100 μl mobile phase and 10 μl were injected into the UPLC-MS/MS system. The
20
21 method includes validation of several parameters, including LOD, LOQ, matrix effect
Fo

22 and recovery, accuracy, and precision. The results show LOD and LOQ values at 0.5-
23
24 5 ng/ml and 1-5 ng/ml for all analytes. In the discussion section, this study offers a
rP

25
26 detailed comparison of LOD and LOQ values for drugs of abuse, obtained in previous
27
28
studies that followed similar approaches but used slightly different experimental
ee

29 parameters (Alfazil A. A. & Anderson R. A., 2008; Ambach et al., 2013; Clavijo et al.,
30
31 2011; Garcia Boy et al., 2008; Jantos R. et al., n.d.; Langel K. et al., 2011; Lauer E. et
rR

32
33 al., 2011; Sosnoff et al., 1996; Thomas et al., 2012; Versace et al., 2013). The method
34
used is similar to the one proposed by Odoardi et al. (Odoardi et al., 2014), which
35
ev

36 demonstrated the detection of drugs of abuse and their metabolites with DBS and
37
38 subsequent UHPLC-MS/MS analysis obtaining LODs at the range of 0.05–1 ng/ml.
iew

39
40 Moreover, this work tests and demonstrates the stability of the analytes into different
41 storage temperatures (-10oC, 2-8oC and room temperature) for 8 months and compares
42
43 these results with those reported in previous literature focused on 6-AM, MDMA,
44
45 amphetamine, cocaine, and their metabolites. Finally, the new method was applied to
46
64 real samples and the results were compared to those from validated routine whole
47
48 blood analysis using linear regression analysis (paired t-test and the Bland-Altman
49
50 statistical method) from which no significant differences were revealed.
51
52 In a similar context, Kacargil et al. (Kacargil et al., 2020) presented the
53 development, validation, and application to real samples of a DBS method based on
54
55 LC-MS/MS and aimed at the detection of illicit drugs. This approach used higher
56
57 volumes of spotted blood (100 μl) and extraction solvent (4 ml), and involved higher
58
59
extraction time and longer instrumental analysis than that previously cited. Method
60 validation and stability tests were accomplished similarly. From real sample testing, a

http://mc.manuscriptcentral.com/bmc
 Biomedical Chromatography Page 8 of 18

Detection of drugs of abuse by DBS: recent advances

1
2
3 significant difference between the two approaches is that in Sadler Simões et al. paper,
4
5 morphine yielded the lowest extraction efficiency, while in Kacargil et al. work
6
7 morphine provided the best results, even if the Authors themselves admitted the
8
9
possibility that degradation may have produced some biases in their data. For example,
10 only in 3 out of 13 real samples, which had previously tested positive for codeine, were
11
12 confirmed by DBS testing. Codeine degradation may in turn increase the morphine
13
14 positivity rate. In general, it is confirmed that the original drugs stability is increased
15
when blood is spotted on a DBS card and allowed to dry.
16
17 Chepyala et al. (Chepyala et al., 2017) developed an ultra-high-performance
18
19 liquid chromatography - ion booster - quadrupole time-of-flight mass spectrometry
20
21 (UHPLC-IB-QTOF-MS) method for the analysis of 57 compounds recovered from
Fo

22 DBS cards and including the most abused drugs, namely amphetamines, opioids,
23
24 cocaine, benzodiazepines, barbiturates, and other emerging drugs of abuse and their
rP

25
26 metabolites. 25 μl of spiked whole blood was spotted on the filter paper and dried. The
27
28
dried blood spot was extracted with 200 μl 80%/20% acetonitrile/water mixture and
ee

29 centrifuged. 170 μl of the supernatant was evaporated under nitrogen flow and finally
30
31 reconstituted with 150 μl of deionized water, vortexed, filtered, and injected into the
rR

32
33 LC system. The ion booster (IB) source was used to improve the detection sensitivity.
34
Based on the ESI principles, the IB source comprises a heated vaporizer, a charge-HV
35
ev

36 transfer tube, and the additional use of nitrogen as a sheath gas to improve the mobile-
37
38 phase evaporation and the analytes’ ionization. After optimization of the experimental
iew

39
40 parameters (including ion polarity) for all the analytes, improvements ranging from 1.5
41 to 14-fold with respect to conventional ESI was observed, allowing the detection of
42
43 95% of the target analytes below the respective therapeutic intervals. Such an
44
45 improvement makes the method particularly suitable for toxic and forensic drug
46
screening.
47
48 Recently, Joye et. al (Joye et al., 2019) developed a LC-HRMS procedure for
49
50 the screening of drugs of abuse after DBS sampling. Three main differences with
51
52 respect to previous methods were proposed: i) the target analytes panel was expanded
53 by adding new anticonvulsants and THC-COOH to the common drugs of abuse, ii) the
54
55 extraction method was diversified, and iii) the mass detection was based on high-
56
57 resolution data. The extraction was performed on 2 spots of 10 μl each: the first was
58
59
extracted with 100 μl methanol while the second with 100 μl borate buffer 0.5 M at pH
60 9.5 and 300 μl dichloromethane/hexane/ethyl acetate mixture (5:4:1). The two-

http://mc.manuscriptcentral.com/bmc
Page 9 of 18 Biomedical Chromatography

Detection of drugs of abuse by DBS: recent advances

1
2
3 extraction solution were mixed, then the solvent was evaporated, the residue was
4
5 solubilized into 50 μl water, and injected into the LC-HRMS instrument. Validation of
6
7 the method was performed on the target analytes (for example, LODs and limits of
8
9
identification (LOIs) were calculated for 30 substances), but the full-scan MS data
10 acquisition allowed by Orbitrap technology enables the qualitative detection of more
11
12 than one thousand drugs. This is particularly important for new psychoactive substances
13
14 (NPS) which are normally not tested in the targeted methods addressed to conventional
15
drugs but are increasingly abused to circumvent testing and to evade national and
16
17 international legislation. The possibility of large-scale screening for all the reported
18
19 classes of drugs was confirmed by testing 104 real samples and comparing the present
20
21 method to other routinely applied methods.
Fo

22 The use of DBS for the detection of drugs of abuse is also applicable when
23
24 postmortem samples are available, particularly when small blood volume is available.
rP

25
26 Moretti et al. (Moretti et al., 2018) developed and validated a LC-MS/MS method for
27
28
the identification of cocaine and its metabolites in DBS using postmortem samples. 45
ee

29 real postmortem cases were evaluated during the period of one year and the results were
30
31 compared to those arising from routine whole blood analysis showing good qualitative
rR

32
33 and quantitative correlation. Few years later, Moretti et al. (Moretti et al., 2021)
34
developed a study to compare the behavior of two different paper substrates for DBS
35
ev

36 sampling used in qualitative and quantitative analysis of postmortem cardiac blood

37
38 specimen. Twenty different cases were examined, involving antidepressants,
iew

39
40 antipsychotics, benzodiazepines, and their metabolites, from which it was concluded
41 that both types of filter papers were equally applicable, whereas specific attention have
42
43 to be paid to the type of extraction solvent used, possibly resulting in different
44
45 extraction yields from the two cards for a few analytes.
46
An innovative proposal that solves the main limitations of DBS sampling is the
47
48 fully automated DBS method that couples a DBS autosampler with an LC-MS/MS
49
50 system. Luginbühl and Gaugler (Luginbühl & Gaugler, 2020) presented step by step
51
52 the workflow of such a proposal. The DBS autosampler is a system in charge of all the
53 steps preceding injection: from the sample information recovery and quality control
54
55 check to internal standard application and spot extraction. At the end, a start signal
56
57 triggers the sample injection and instrumental run from the autosampler to the LC-
58
59
MS/MS system. Specifically, Gaugler et al. (Gaugler et al., 2018) reported a fully
60 automated screening for 12 drugs of abuse with DBS and online LC-MS/MS analysis.

http://mc.manuscriptcentral.com/bmc
 Biomedical Chromatography Page 10 of 18

Detection of drugs of abuse by DBS: recent advances

1
2
3 A stock standards solution was mixed with the donor blood in four different
4
5 concentrations to prepare the calibration curve. 15 μL aliquots were spotted onto the
6
7 card, dried at room temperature, and extracted into a 20 μL sample loop with a mixture
8
9
of methanol and water (70:30, v/v) and then injected. Another forensic study from the
10 same Authors (Gaugler et al., 2019) was focused on routine DBS screening in
11
12 workplace testing, and included 28 compounds. The method robustness was confirmed
13
14 on real blood samples. The major advantages of the automatic procedure is that the
15
direct sample elution from the DBS card eliminates the need of several consumables,
16
17 such as sampling tubes, pipette tips, and glass vials and exclude the chance of errors
18
19 due to human sample handling. These advantages, combined with high performance
20
21 MS instrumentation (either HRMS or MS/MS) is likely to make the DBS method fast,
Fo

22 efficient, and reliable.

23
24
rP

25
26
27
28
Conclusions
ee

29 The methods of toxicological analysis applied on samples collected by DBS

30
31 have already gone far beyond the stage of proof-of-concept and are earning a chance to
rR

32
33 progressively substitute for traditional methods carried out on whole blood from
34
intravenous sampling. The DBS methods have been validated for quantitative
35
ev

36 determinations, their results have been cross-checked with respect to conventional

37
38 methods, and successfully applied to real samples. Moreover, improved sample and
iew

39
40 analyte stability had been demonstrated on DBS cards. Still, DBS collects a very limited
41 amount of blood and produces spatially inhomogeneous samples. Therefore, DBS
42
43 methods for toxicological analysis are required to go through large-scale trials on real
44
45 samples to standardize further the experimental conditions and normalize the possible
46
limitations that these methods may pose. Also, wider “on-field” sampling campaigns
47
48 (e.g., road-side testing, drug testing in nightclubs) are needed to definitely prove the
49
50 routine applicability of DBS sampling in contexts where traditional methods of blood
51
52 sampling are totally impracticable. In parallel, DBS practices and results should be
53 compared with those using different alternative matrices in “on-field” controls (for
54
55 example, oral fluid in road-side testing, hair or urine in drug withdrawal controls) to
56
57 verify the respective advantages and limitations and choose the most effective approach
58
59
for each circumstance.
60

http://mc.manuscriptcentral.com/bmc
Page 11 of 18 Biomedical Chromatography

Detection of drugs of abuse by DBS: recent advances

1
2
3 Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS)
4
5 still represents the preferred instrumental method among those commonly available in
6
7 forensic laboratories, because of its inherent high speed, sensitivity, accuracy, and
8
9
precision, especially in targeted operating mode (selected reaction monitoring - SRM).
10 In alternative, LC-HRMS offers an equally selective detection mode but frequently
11
12 combined with the availability of full-scan data, of unique usefulness whenever
13
14 postponed reevaluation of the analytes’ panel becomes necessary. This is an
15
increasingly common scenario in the rapidly evolving market of drugs of abuse and
16
17 novel psychoactive substances, that are habitually not included in the targeted SRM
18
19 methods of analysis but have to be prospectively integrated into the forthcoming wide
20
21 screening procedures.
Fo

22 The HRMS offers the ability to perform both qualitative and quantitative
23
24 analysis and this opportunity is appealing for forensic perspective as the information
rP

25
26 obtained is not only about the target analyte but also for its metabolites and potential
27
28
biomarkers of interest. The greater mass range of HRMS in comparison to triple-
ee

29 quadrupole instruments offers the possibility of analyzing large molecules, for both
30
31 identification and direct quantification. HRMS provides highly specific
rR

32
33 characterization of the analytes, reducing or eliminating the interferences that
34
occasionally affect the determinations made by triple quadrupole instruments.
35
ev

36 Moreover, the use of electrospray ionization (ESI) or ion booster (IB) source can further
37
38 improve the MS sensitivity for a wide range of drug candidates and metabolites, as
iew

39
40 Chepyala et al. proposed. As demonstrated by Joye et al., more than satisfactory results
41 were obtained when a quadrupole-orbitrap HRMS operating in DDA full-scan approach
42
43 was used. Indeed, 99% of the DBS cases were identified by the quadrupole-orbitrap
44
45 HRMS, whereas at concentrations between 0 and 10 ng/ml 40-50% of the substances
46
detected by the HRMS were not identified by LC-MS/MS. Remarkably, broad-
47
48 spectrum HRMS screening methods can become of particular interest owing to the
49
50 limitations to targeted LC-MS/MS methods presented by the progressive introduction
51
52 of new drugs of abuse in the illegal market. The triple quadrupole tandem mass
53 spectrometer will likely remain the preferred instrument for quantitative detection of
54
55 traditional drugs of abuse, usually predetermined as target analytes. However, the
56
57 retrospective investigation represents an added value for investigation of DBS samples,
58
59
especially when the small amount allows only one analysis, and especially in forensic
60

http://mc.manuscriptcentral.com/bmc
 Biomedical Chromatography Page 12 of 18

Detection of drugs of abuse by DBS: recent advances

1
2
3 labs where there is a greater need to maximize the range of detectable compounds
4
5 (Massano et al., 2022).
6
7
8 Several forensic laboratories are currently increasing their interest in DBS and
9
10
consider it the potential procedure of choice for several reasons. DBS guarantees an
11 unquestionable ease of sampling, as the collection of capillary blood is minimally
12
13 invasive and there is no need for trained medical staff to perform its sampling. DBS
14
15 also assures fast result turnaround, and early stabilization effects on drugs by inhibiting
16
their degradation. The whole procedure also complies with the aims of Green Analytical
17
18 Chemistry and Sustainable development (The Sustainable Development Goals Report,
19
20 2022), that nowadays represents a must, as it involves a small sample volume and
21
consequently demands a lower amount of extraction solvent and less energy
Fo

22
23 consumption, both leading to lower waste production and environmental impact.
24
rP

25 Likewise, DBS cards shipping is significantly simplified, requiring no special

26
27 conditions and reduced storage space, making the technique applicable for testing and
28
ee

29
research in every part of the world, also under uncomfortable conditions. In conclusion,
30 it is predictable that DBS will play a rapidly increasing role in the toxicological testing
31
rR

32 activity toward illicit substances including old and new drugs and doping agents, so as
33
34 to expand the feasibility of blood testing and effectively tackle the proliferating problem
35
ev

of illicit substances abuse.

36
37
38
iew

39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60

http://mc.manuscriptcentral.com/bmc
Page 13 of 18 Biomedical Chromatography

Detection of drugs of abuse by DBS: recent advances

1
2
3 REFERENCES
4
5
6 Alfazil A. A., & Anderson R. A. (2008). Stability of Benzodiazepines and Cocaine in
7 Blood Spots Stored on Filter Paper. Journal of Analytical Toxicology, 32(7),
8
9
511–515. https://doi.org/10.1093/jat/32.7.511
10 Ambach, L., Hernández Redondo, A., König, S., & Weinmann, W. (2013). Rapid and
11 simple LC-MS/MS screening of 64 novel psychoactive substances using dried
12 blood spots. Drug Testing and Analysis, 6(4), 367–375.
13 https://doi.org/10.1002/dta.1505
14 Antelo-Domínguez, Á., Ángel Cocho, J., Jesús Tabernero, M., María Bermejo, A.,
15
Bermejo-Barrera, P., & Moreda-Piñeiro, A. (2013). Simultaneous determination
16
17 of cocaine and opiates in dried blood spots by electrospray ionization tandem
18 mass spectrometry. Talanta, 117, 235–241.
19 https://doi.org/10.1016/j.talanta.2013.09.010
20 Bang I. (1913). Der Blutzucker. https://doi.org/https://doi.org/10.1007/BF01347215
21 Biondi, M. J., van Tilborg, M., Smookler, D., Heymann, G., Aquino, A., Perusini, S.,
Fo

22 Mandel, E., Kozak, R. A., Cherepanov, V., Kowgier, M., Hansen, B., Goneau, L.
23
24
W., Janssen, H. L. A., Mazzulli, T., Cloherty, G., de Knegt, R. J., & Feld, J. J.
(2019). Hepatitis C Core-Antigen Testing from Dried Blood Spots. Viruses,
rP

25
26 11(9). https://doi.org/10.3390/v11090830
27 Brambilla, D., Jennings, C., Aldrovandi, G., Bremer, J., Comeau, A. M., Cassol, S.
28 A., Dickover, R., Jackson, J. B., Pitt, J., Sullivan, J. L., Butcher, A., Grosso, L.,
ee

29 Reichelderfer, P., & Fiscus, S. A. (2003). Multicenter evaluation of use of dried

30 blood and plasma spot specimens in quantitative assays for human
31
immunodeficiency virus RNA: Measurement, precision, and RNA stability.
rR

32
33 Journal of Clinical Microbiology, 41(5), 1888–1893.
34 https://doi.org/10.1128/JCM.41.5.1888-1893.2003
35 Carpenter, K. H., & Wiley, V. (2002). Application of tandem mass spectrometry to
ev

36 biochemical genetics and newborn screening. Clinica Chimica Acta , 322.

37 https://doi.org/https://doi.org/10.1016/S0009-8981(02)00135-3
38
iew

Chepyala, D., Tsai, I. L., Liao, H. W., Chen, G. Y., Chao, H. C., & Kuo, C. H. (2017).
39
40 Sensitive screening of abused drugs in dried blood samples using ultra-high-
41 performance liquid chromatography-ion booster-quadrupole time-of-flight mass
42 spectrometry. Journal of Chromatography A, 1491, 57–66.
43 https://doi.org/10.1016/j.chroma.2017.02.037
44 Clavijo, C. F., Hoffman, K. L., Thomas, J. J., Carvalho, B., Chu, L. F., Drover, D. R.,
45 Hammer, G. B., Christians, U., & Galinkin, J. L. (2011). A sensitive assay for
46
47
the quantification of morphine and its active metabolites in human plasma and
48 dried blood spots using high-performance liquid chromatography-tandem mass
49 spectrometry. Analytical and Bioanalytical Chemistry, 400(3), 715–728.
50 https://doi.org/10.1007/s00216-011-4775-z
51 Cone, E. J. (1995). Pharmacokinetics and Pharmacodynamics of Cocaine. Journal of
52 Analytical Toxicology, 19(6), 459–478.
53 https://doi.org/https://doi.org/10.1093/jat/19.6.459
54
55
Corran, P. H., Cook, J., Lynch, C., Leendertse, H., Manjurano, A., Griffin, J., Cox, J.,
56 Abeku, T., Bousema, T., Ghani, A. C., Drakeley, C., & Riley, E. (2008). Dried
57 blood spots as a source of anti-malarial antibodies for epidemiological studies.
58 Malaria Journal, 7. https://doi.org/10.1186/1475-2875-7-195
59 Déglon, J., Versace, F., Lauer, E., Widmer, C., Mangin, P., Thomas, A., & Staub, C.
60 (2012). Rapid LC-MS/MS quantification of the major benzodiazepines and their

http://mc.manuscriptcentral.com/bmc
 Biomedical Chromatography Page 14 of 18

Detection of drugs of abuse by DBS: recent advances

1
2
3 metabolites on dried blood spots using a simple and cost-effective sample
4
5
pretreatment. Bioanalysis, 4(11), 1337–1350. https://doi.org/10.4155/bio.12.42
6 Fontaine, E., & Saez, C. (2021). Analysis of SARS-CoV-2 antibodies from dried
7 blood spot samples with the Roche Elecsys Immunochemistry method. Practical
8 Laboratory Medicine, 25. https://doi.org/10.1016/j.plabm.2021.e00234
9 Garcia Boy, R., Henseler, J., Mattern, R., & Skopp, G. (2008). Determination of
10 Morphine and 6-Acetylmorphine in Blood With Use of Dried Blood Spots. Ther
11
Drug Monitoring, 30(6), 733–739.
12
13
https://doi.org/10.1097/FTD.0b013e31818d9fdb
14 Gaugler, S., Al-Mazroua, M. K., Issa, S. Y., Rykl, J., Grill, M., Qanair, A., & Cebolla,
15 V. L. (2019). Fully Automated Forensic Routine Dried Blood Spot Screening for
16 Workplace Testing. Journal of Analytical Toxicology, 43(3), 212–220.
17 https://doi.org/10.1093/jat/bky074
18 Gaugler, S., Rykl, J., Grill, M., & Cebolla, V. L. (2018). Fully automated drug
19
screening of dried blood spots using online LC-MS/MS analysis. Journal of
20
21 Applied Bioanalysis, 4(1), 7–15. https://doi.org/10.17145/jab.18.003
Fo

22 GLOBAL OVERVIEW DRUG DEMAND DRUG SUPPLY. (2022).

23 www.unodc.org/unodc/en/data-and-analysis/world-drug-report-2022.html
24 Guthrie, R., & Susi, A. (1963). A simple phenylalanine method for detecting
rP

25 phenylketonuria in large populations of newborn infants. Pediatrics, 32, 338–

26 343. http://www.ncbi.nlm.nih.gov/pubmed/14063511
27
28
Huestis, M. A., Verstraete, A., Kwong, T. C., Morland, J., Vincent, M. J., & de La
ee

29 Torre, R. (2011). Oral Fluid Testing: Promises and Pitfalls. Clinical Chemistry,
30 57(6), 805–810. https://academic.oup.com/clinchem/article/57/6/805/5621125
31 Jantos, R., & Skopp, G. (2011). Comparison of drug analysis in whole blood and
rR

32 dried blood spots. Toxichem Krimtech , 78, 268–275.

33 Jantos R., Veldstra, J. L., Mattern R., Brookhuis K. A., & Skopp G. (n.d.). Analysis of
34
3,4-Methylenedioxymetamphetamine: Whole Blood Versus Dried Blood Spots.
35
ev

36
J. Anal. Toxicol. 35 (5) (2011) 269–273.
37 Joye, T., Sidibé, J., Déglon, J., Karmime, A., Sporkert, F., Widmer, C., Favrat, B.,
38 Lescuyer, P., Augsburger, M., & Thomas, A. (2019). Liquid chromatography-
iew

39 high resolution mass spectrometry for broad-spectrum drug screening of dried

40 blood spot as microsampling procedure. Analytica Chimica Acta, 1063, 110–116.
41 https://doi.org/10.1016/j.aca.2019.02.011
42
Kacargil, C. U., Daglioglu, N., & Goren, I. E. (2020). Determination of Illicit Drugs
43
44 in Dried Blood Spots by LC–MS/MS Method: Validation and Application to
45 Real Samples. Chromatographia, 83(7), 885–892.
46 https://doi.org/10.1007/s10337-020-03907-x
47 Keevil, B. G. (2011). The analysis of dried blood spot samples using liquid
48 chromatography tandem mass spectrometry. Clinical Biochemistry, 44(1), 110–
49 118. https://doi.org/10.1016/j.clinbiochem.2010.06.014
50
51
Langel K., Uusivirta H., Ariniemi K., & Lillsunde P. (2011). Analysis of drugs of
52 abuse by GC-MS in dried blood spot sample matrix. 49th Annual Meeting of The
53 International Association of Forensic Toxicologists (TIAFT), 25–30.
54 Lauer E., Déglon J., Versace F., Thomas. A., Mangin P., & Staub C. (2011). Target
55 screening of drugs from dried blood spot samples based on LC-MS/MS and on-
56 line desorption. 49th Annual Meeting of The International Association of
57
Forensic Toxicologists (TIAFT), 25–30.
58
59
Lee, M. S., & Li, W. (2014). Dried blood spots: Applications and Techniques.
60 https://doi.org/10.1002/9781118890837

http://mc.manuscriptcentral.com/bmc
Page 15 of 18 Biomedical Chromatography

Detection of drugs of abuse by DBS: recent advances

1
2
3 Li, W., & Tse, F. L. S. (2010). Dried blood spot sampling in combination with LC-
4
5
MS/MS for quantitative analysis of small molecules. In Biomedical
6 Chromatography (Vol. 24, Issue 1, pp. 49–65). https://doi.org/10.1002/bmc.1367
7 Luginbühl, M., & Gaugler, S. (2020). The application of fully automated dried blood
8 spot analysis for liquid chromatography-tandem mass spectrometry using the
9 CAMAG DBS-MS 500 autosampler. Clinical Biochemistry, 82, 33–39.
10 https://doi.org/10.1016/j.clinbiochem.2020.02.007
11
Massano, M., Incardona, C., Gerace, E., Negri, P., Alladio, E., Salomone, A., &
12
13
Vincenti, M. (2022). Development and validation of a UHPLC-HRMS-QTOF
14 method for the detection of 132 New Psychoactive Substances and synthetic
15 opioids, including fentanyl, in Dried Blood Spots. Talanta, 241.
16 https://doi.org/10.1016/j.talanta.2022.123265
17 Mei, J. v, Richard Alexander, J., Adam, B. W., Harry Hannon, W., & Finley, A.
18 (2001). Use of filter paper for the collection and analysis of human whole blood
19
specimens. The Journal of Nutrition, 131(5), 1631S-1636S.
20
21 https://doi.org/https://doi.org/10.1093/jn/131.5.1631S
Fo

22 Moretti, M., Manfredi, A., Freni, F., Previderé, C., Osculati, A. M. M., Grignani, P.,
23 Tronconi, L., Carelli, C., Vignali, C., & Morini, L. (2021). A comparison
24 between two different dried blood substrates in determination of psychoactive
rP

25 substances in postmortem samples. Forensic Toxicology, 39(2), 385–393.

26 https://doi.org/10.1007/s11419-020-00567-2
27
28
Moretti, M., Visonà, S. D., Freni, F., Tomaciello, I., Vignali, C., Groppi, A., Tajana,
ee

29 L., Osculati, A. M. M., & Morini, L. (2018). A liquid chromatography–tandem

30 mass spectrometry method for the determination of cocaine and metabolites in
31 blood and in dried blood spots collected from postmortem samples and
rR

32 evaluation of the stability over a 3-month period. Drug Testing and Analysis,
33 10(9), 1430–1437. https://doi.org/10.1002/dta.2399
34
Odoardi, S., Anzillotti, L., & Strano-Rossi, S. (2014). Simplifying sample
35
ev

36
pretreatment: Application of dried blood spot (DBS) method to blood samples,
37 including postmortem, for UHPLC-MS/MS analysis of drugs of abuse. Forensic
38 Science International, 243, 61–67.
iew

39 https://doi.org/10.1016/j.forsciint.2014.04.015
40 Rook, E. J., van Ree, J. M., van den Brink, W., Hillebrand, M. J. X., Huitema, A. D.
41 R., Hendriks, V. M., & Beijnen, J. H. (2006). Pharmacokinetics and
42
Pharmacodynamics of High Doses of Pharmaceutically Prepared Heroin, by
43
44 Intravenous or by Inhalation Route in Opioid-Dependent Patients. Basic &
45 Clinical Pharmacology & Toxicology, 98, 86–96.
46 https://doi.org/https://doi.org/10.1111/j.1742-7843.2006.pto_233.x
47 Sadler Simões, S., Castañera Ajenjo, A., & Dias, M. J. (2018). Dried blood spots
48 combined to an UPLC–MS/MS method for the simultaneous determination of
49 drugs of abuse in forensic toxicology. Journal of Pharmaceutical and
50
51
Biomedical Analysis, 147, 634–644. https://doi.org/10.1016/j.jpba.2017.02.046
52 Sañudo, A., Andreoni, S., & Sanchez, Z. M. (2015). Polydrug use among nightclub
53 patrons in a megacity: A latent class analysis. International Journal of Drug
54 Policy, 26(12), 1207–1214. https://doi.org/10.1016/j.drugpo.2015.07.012
55 Sosnoff, C. S., Ann, Q., Bernert, J. T., Powell, M. K., Miller, B. B., Henderson, L. O.,
56 Hannon, W. H., Fernhoff, P., & Sampson, E. J. (1996). Analysis of
57
Benzoylecgonine in Dried Blood Spots by Liquid Chromatography-Atmospheric
58
59
Pressure Chemical Ionization Tandem Mass Spectrometry. Journal of Analytical
60 Toxicology, 20, 179–184. https://doi.org/https://doi.org/10.1093/jat/20.3.179

http://mc.manuscriptcentral.com/bmc
 Biomedical Chromatography Page 16 of 18

Detection of drugs of abuse by DBS: recent advances

1
2
3 Soulier, A., Poiteau, L., Rosa, I., Hézode, C., Roudot-Thoraval, F., Pawlotsky, J. M.,
4
5
& Chevaliez, S. (2016). Dried blood spots: A tool to ensure broad access to
6 hepatitis C screening, diagnosis, and treatment monitoring. Journal of Infectious
7 Diseases, 213(7), 1087–1095. https://doi.org/10.1093/infdis/jiv423
8 Stove, C. P., Ingels, A. S. M. E., de Kesel, P. M. M., & Lambert, W. E. (2012). Dried
9 blood spots in toxicology: From the cradle to the grave? Critical Reviews in
10 Toxicology, 42(3), 230–243. https://doi.org/10.3109/10408444.2011.650790
11
The Sustainable Development Goals Report. (2022).
12
13
https://doi.org/https://unstats.un.org/sdgs/report/2022/
14 Thomas, A., Geyer, H., Schänzer, W., Crone, C., Kellmann, M., Moehring, T., &
15 Thevis, M. (2012). Sensitive determination of prohibited drugs in dried blood
16 spots (DBS) for doping controls by means of a benchtop quadrupole/Orbitrap
17 mass spectrometer. Analytical and Bioanalytical Chemistry, 403(5), 1279–1289.
18 https://doi.org/10.1007/s00216-011-5655-2
19
Velghe, S., Delahaye, L., & Stove, C. P. (2019). Is the hematocrit still an issue in
20
21 quantitative dried blood spot analysis? Journal of Pharmaceutical and
Fo

22 Biomedical Analysis, 163, 188–196. https://doi.org/10.1016/j.jpba.2018.10.010

23 Versace, F., Déglon, J., Lauer, E., Mangin, P., & Staub, C. (2013). Automated DBS
24 extraction prior to hilic/RP LC-MS/MS target screening of drugs.
rP

25 Chromatographia, 76(19–20), 1281–1293. https://doi.org/10.1007/s10337-012-

26 2377-3
27
28
Watterson, J. (2015). Analysis of dried blood spots in forensic toxicology. New
ee

29 Sampling Strategies in Toxicology and Therapeutic Drug Monitoring, 80–92.

30 https://doi.org/10.4155/fseb2013.14.395
31 Wilhelm, A. J., den Burger, J. C. G., & Swart, E. L. (2014). Therapeutic Drug
rR

32 Monitoring by Dried Blood Spot: Progress to Date and Future Directions.

33 Clinical Pharmacokinetics, 53(11), 961–973. https://doi.org/10.1007/s40262-
34
014-0177-7
35
ev

36
Wood, D. M., Nicolaou, M., & Dargan, P. I. (2009). Epidemiology of Recreational
37 Drug Toxicity in a Nightclub Environment. Substance Use & Misuse, 44(11),
38 1495–1502. https://doi.org/10.1080/10826080802543580
iew

39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60

http://mc.manuscriptcentral.com/bmc
Page 17 of 18 Biomedical Chromatography

Detection of drugs of abuse by DBS: recent advances

1 Table 1: Summary table of the main parameters reported by each study
2
3
4 SAMPLE RUN
FILTER EXTRACTION LOD STABILITY
5 ANALYTES VOLUME TECHNIQUE EQUIPMENT TIME REFERENCES
PAPER SOLVENTS (ng/ml) TEST
6 (μl) (min)
7
8 Amphetamine,6-
9 AM,
10 Benzoylecgonine,
11 Cocaine, Codeine, Waters Acquity UPLC

Fo
12 EDDP, MDA, coupled with Waters TQ
13 MDMA, Whatman Detector, Waters Sadler Simões

rP
14
15
Methadone, BFC 180 Acquity UPLC® HSS RT, 2-8oC et al. (Sadler
Methamphetamine bloodstain UPLC- T3 column (100 × 2.1 and -10oC Simões et al.,

ee
16
17 and Morphine cards 50 Methanol/acetonitrile MS/MS mm, 1.8 μm) 10 0.5-1 for 8 months 2018)
18

rR
19 Amphetamine, Shimadzu CBM-20A
20 Methamphetamine, UFLC* coupled with

ev
21 MDMA, MDA, Sartorius Shimadzu 8040 Mass
22 Morphine, Stedim Spectrometry,

iew
23 Codeine, Cocaine, Biotech Allure® RT, 4oC, -
24 Benzoylecgonine Sample Pentafluorophenylpropyl 20oC Kacargil et al.
25
26
and Carrier (PFPP) column (50 mm for 15, 35 (Kacargil et al.,
27 Methylecgonine Paper 100 Ethylacetate/methanol LC-MS/MS × 2.1 mm, 5 μm) 20 0.1-6.52 and 95 days 2020)
28
Amphetamines, Agilent 1290 UHPLC
29
30
opioids, cocaine, system coupled with a
31 benzodiazepines, Bruker maXis QTOF
32 barbiturates, and with IB source,
33 metabolites Agilent Poroshell EC- RT, -80oC Chepyala et al.
34 (total of 57 Whatman UHPLC-IB- C18 column (2.1 × 100 for 1 and 6 (Chepyala et
35 compounds) 903 card 25 ACN 80% QTOF-MS mm, 2.7 μm) 20 0.2-35.7 months al., 2017)
36
37 Amphetamines, Methanol, Borate Thermo Scientific LC-Q
38 benzodiazepines, Whatman buffer 0.5 M pH 9.5 Exactive Plus system,
39
cocaine, Protein and DCM: Hexane: Phenomenex 2.6 mm Joye et al.
40
41
antidepressants, saver 10 × 2 Ethyl Acetate (5:4:1) C18 column (2.1 × 50 (Joye et al.,
42 neuroleptics, DBS (2 spots) (2 spots extraction) LC-HRMS mm) 0 <1-10 no 2019)
43 http://mc.manuscriptcentral.com/bmc
44
45
46
 Biomedical Chromatography Page 18 of 18

Detection of drugs of abuse by DBS: recent advances

1
2 opioids, NPS,
3 anticonvulsants
4 and THC-COOH
5 (total of 30
6 compounds)
7
8 Alprazolam,
9 Amphetamine, CAMAG DBS-MS 500
10
Cocaine, Codeine, system, Shimadzu
11

Fo
12 Diazepam, UHPLC system coupled
13 Fentanyl, LSD, with 8060 triple

rP
14 MDMA, quadrupole,
15 Methadone, Ahlstrom Shim-pack GIST

ee
16 Methamphetamine, TFN filter column (2.3 x 50 mm, 5 Gaugler et al.
17 Morphine and paper Methanol/water μm C18, PN 227-30017- (Gaugler et al.,
18

rR
19
Oxycodone CAMAG 15 (70:30 v/v) LC-MS/MS 3) 10 0.05-30 no 2018)
20 CAMAG DBS-MS 500

ev
21
system, Shimadzu
22
UHPLC system coupled

iew
23
24 with 8060 triple
25 Amphetamines, quadrupole,
26 benzodiazepines, Shim-pack GIST (2.1 ×
27 cocaine, heroin, 50 mm, 2 μm C18) and
28 cannabinoids Ahlstrom Kinetex (2.1 × 100 mm, Gaugler et al.
29
30
(total of 28 TFN filter Methanol/water 2.6μm, XB-C18) (Gaugler et al.,
31 compounds) paper 20 (70:30 v/v) LC-MS/MS columns 5 10-100 no 2019)
32
33
34
35
36
37
38
39
40
41
42
43 http://mc.manuscriptcentral.com/bmc
44
45
46