Dr.Y.S.R.

HORTICULTURAL UNIVERSITY
COLLEGE OF HORTICULTURE
ANANTHARAJUPETA

COURSE NO :- PHM-502

COURSE TITLE :- POSTHARVEST PHYSIOLOGY AND BIOCHEMISTRY OF

PERISHABLES.

TOPIC : BEAL BOTANY , FRUIT PHYSIOLOGY AND TECHNOLOGY .

Submitted to
Dr. K. Venkata Satish
[Link] (Hort.)
[Link] Postharvest Managemant
Sumitted by
Ch. Swethasri
AHM/ 24-25
[Link] PHM
VOLATILE COMPOUNDS PROFILING BY USING
LC-MS

INTRODUCTION:
 Profiling volatile organic compounds (VOCs) is a valuable approach in plant
pathology for detecting and understanding plant diseases. While gas
chromatography–mass spectrometry (GC-MS) is traditionally employed for
analyzing VOCs due to their volatile nature, liquid chromatography–mass
spectrometry (LC-MS) can also be utilized, particularly for semi-volatile or less
volatile metabolites.
 It refers to the combination of liquid chromatographic separation with mass
spectrometric detection for non polar , low volatility , thermally unstable
compounds.
 LC differentiates compounds by their physico-chemical properties and MS
differentiates compounds by mass .
 The mass spectrometer acts not only as the LC detector but, it provides the
capability to identify the species corresponding to each chromatographic peak
through its unique mass spectrum.
 High sensitivity of mass sprectroscopy provides the information for identification of
compound or structural elucidation of compounds.
 In the LC\MS we remove the detector from the column of LC fit the column to the
interface of MS.
 In the most cases the interface used in LC\MS are ionization source.
 As the metabolites appear from the end of the column they enter the mass detector ,
where the solvent is removed and the metabolites are ionized.
PRINCIPLE :
Thermospray method:
 The thermospray method depends on the thermal generation of a spray and further
separate heat treatment of that spray to yield desolvated ions.
 The heat creates a supersonic , expanding aerosol jet that contains a mist of droplets
of solvent vapour and sample molecules ., the droplets vapourize .
 The excess vapour is pumped away by an added mechanical pump, which is directly
coupled to the ion source.
  Ions of the sample molecules are formed in the spray either by direct desorption or
by chemical ionization when used with polar mobile phases that contain appropriate
buffers.
 A conventional electron bean is used to provide gas phase reagent , ion for the
chemical ionization of solute molecules.
 The ions are led into a quadrupole or magnetic sector mass spectrometer , to flow
rates efluents up to 2ml/min are permissible .

MONODISPERSE AEROSOL GENERATION INTERFACE:

The interface is configured in three sections:
1. Aerosol generator
2. Desolvation chamber
3. Two stage aerosol beam pressure reducer.
 In the aerosol generates the high pressure effluent from the
chromatrographic column, passes through a small diameter orifice to form
a fine liquid jet.
 The jet breaks up under natural forces to form a fine uniform drops ,
which are immediately dispersed with gas stream introduced at right
angles to the liquid flow direction .
 The solvent evaporates in the desolvation chamber . the chamber is
maintained near room temperature by heating gently to replace the latent
heat of vapourization necessary for solvent evaporation .
 The two stages aerosol beam separator consists of two nozzle and
skimmer devices.
  These reduces the pressure from an initial value close to atmospheric
pressure in the desivation chamber to a final value close to the pressure in
the ion source .
 The separator also allows solute particles to be preferentially transferred
in the ion source.
 The separator also allows solute particles to be preferentially transferred
through the system , while dispersion of gas and solvent vapor are
pumped away.

INTRUMENTATION :
HPLC SYSTEM:
ION SOURCE : Used in the vapourization , ionization of target molecule.
MASS ANALYSER: Used to separate the ga phase ions by mass to charge
ratio (m/z).
ION DETECTOR: Detection of the mass separated .
Data system.
MOBILE PHASE:
The mobile phase is the solvent that moves the solute throughout column.
General requirements:
1)Low cost , UV transparency , high purity.
2) low viscosity , low toxicity, non flammability.
3) Non corrosive to LC system component.
SOLVENT STRENGHT AND SELECTIVITY:
It is the ability of solvent to elute solutes from a column.
COLUMN:
The use of di-functional or tri-functional silanes to create bonded groups with two or three
PH and lower bleed for LCMS.
Most widely used columns for LC-MS are :
1) Fast LC column : The use of short column . (15-50mm)
2) Micro LC column: The use of large column .(20-150mm)
SAMPLE PREPARATION :
Sample preparation generally consists of concentrating the analyte and removing
compounds that can cause background ion or suppress ionization .
Example :
1) On column concentration to increase analyte concentration .
2) Desalting to reduce the sodium and potassium adduct formation that commonly
occurs in electro spray.
 3) Filtration to separate a low molecular-weight drug from proteins in plasma , milk,
or tissue.

INTERFACES:
LC-MS system include a device for introducing samples such as an( HPLC) an interface
for connecting such device , an ion source that ionizes samples , an electrostatic lens that
efficiently introduces the generated ions, a mass analyser unit that separates ions based on
their mass-to-charge (m/z) ratios, and a detector unit that detects the separated ions.
In an LC-MS unit, the liquid mobile phase would vapourize, resulting in large amounts of
gas being introduced into the MS unit.
This would decrease the vacuum level and prevent the target ions from reaching the
detector . So interfaces are to be used.

TYPES OF INTERFACES:
It is difficult to interface a liquid chromatography to a mass sprectrometer used interfaces
are:
1) Electrospray Ionization (ESI): ESI draws sample solutions to the tip of a
capillary tube , where it applies a high voltage of about 3 to 5 kv.
 A Nebulizer gas flows flows from outside the capillary to spray the sample .
This creates a fine mist of charged droplets with the same polarity as the
applied voltage.
 As this evaporation and fission cycle is repeated ,the droplets eventually
become small enough that the sample ions are liberated into the gas phase.
 ESI provides the softest ionization method available , which means it can be
used for highly polar , least volatiles , or thermally unstable compounds .
2) Atmospheric pressure chemical ionization(APCI): APCI vapourizes
solvent and sample molecules by spraying the sample solution into a heater(heated
to about 400 c) using a gas , such as N2 .
 Solvent molecules are ionized by corona discharge to generate stable reaction
ions.

3) Thermospray ionization (TSI): Heat the effluent in the capillary.

 Generate aerosol and soft ions.
a) Real Thermospray (no filament): ions form via ion evaporation as
droplets shrink or through acid/base transfer with buffer ions (e.g., NH₄⁺,
CH₃COO⁻)
b) Filament-on/Discharge mode: adds a weak discharge to create a plasma
for chemical ionization
 4)Atmospheric pressure up to ionization(APPI): The LC eluent is
vapourized using a heater at atmospheric pressure .
 The resulting gas is made to pass through a beam of photons generated by a
discharge lamp (UV LAMP) which ionizes the gas molecules .

MASS ANALYSER:
They deflections down a curved tubes in a magnetic fields base on their kinetic energy
determined by the mass, charge and velocity.
The magnetic field is scanned to measure different ions.
Types of mass analyser:
1) Qudrapole mass filter
2) Time of flight
3) Ion trap
4) Fourier transform ion cyclotron resonance (FT-ICR)

[Link] mass analyzer:

 A Quadrupole mass filter consists of four parallel metal rods with different
charges .
 Two opposite rods have an applied positive potential and the other two rods
have a negative potential .
 The applied voltages affect the trajectory of ions traveling down the flight
path.
 For given DC and AC voltages , only ions of a certain mass-to-charge ratio
pass through the quadrupole filter and all other ions are thrown out of their
original path.

[Link] (Time of Flight) Mass Analyzer:

 TOF analyser separate ions by time without the use of an electric or magnetic field.
 In a crude sense, TOF is similar to chromatography , except there is no stationary /
mobile phase, instead the separation is based on the kinetic energy and velocity of
the ions.

[Link] TRAP MASS ANALYSER:

 It uses an electric field for the separation of the ions by mass to charge ratios.
 The electric field in cavity due to the electrodes causes the ions of certain m/z values
to orbit in the space.
[Link] TRANSFORM ION CYCLONE RESONANCE (FT-
ICR):
 Uses a magnetic field in order to trap ions into an orbit inside of it.
 In this analyzer there is no separation that occurs rather all the ions of a particular
range are trapped inside , and an applied external electric fields helps to generate a
signal.

Case Study: Metabolomics of Solanum lycopersicum Infected

with Phytophthora infestans Leads to Early Detection of Late
Blight in Asymptomatic Plants

 Title: Metabolomics of Solanum lycopersicum Infected with Phytophthora

infestans Leads to Early Detection of Late Blight in Asymptomatic Plants
 Authors: Paula Liliana Galeano Garcia et al.
 Journal: Frontiers in Plant Science , Year: 2018

Objectives:
 Utilize LC–MS and MALDI-MS metabolomics to detect early-stage P. infestans
infection in tomato plants — even before visible symptoms.
 Discriminate between different infection time points (4, 12, 24, … up to 96 hours
post-inoculation).

Materials & Methods:

1. Plant & Pathogen Treatment
o Tomato plants inoculated with P. infestans and sampled at multiple time
points (4–96); control (uninfected) plants included.
2. Sample Preparation
o Leaf tissue extracts prepared for LC–MS; sample cleanup done before
injection.
3. LC–MS & MALDI-MS Acquisition

o Combined LC–MS with MALDI-MS for metabolite profiling. Multivariate

data analysis applied.

Results:
 Early detection: LC–MS profiles could differentiate infection stages—even prior to
symptoms.
  Key biomarkers identified:
o Tomatidine: significantly elevated post-infection
o Saponins: early infection markers
o Isocoumarins: present in both early and late asymptomatic phases

Conclusion:
 LC–MS metabolomics effectively detected P. infestans infection at asymptomatic
stages and distinguished different infection times.
 Identified biomarkers like tomatidine, saponins, and isocoumarins have strong
potential for:
1. Early disease detection
2. Disease monitoring
3. Targeted defense strategies in tomato crops

IN SUMMARY:
Liquid chromatography–mass spectrometry (LC-MS) has become a pivotal tool for
profiling volatile organic compounds (VOCs) in plant pathology, complementing or
overcoming some limitations of GC-MS:
 Detection of defense-related metabolites: LC-MS-based untargeted metabolomic
analyses in plants such as tomato or Arabidopsis have uncovered a variety of
induced compounds—flavonoids, phenylpropanoids, alkaloids, N-hydroxy-pipecolic
acid, and more—that correlate with plant immunity to bacterial or fungal pathogens.
 Insight into pathogen-induced metabolic changes: VOCs like (Z)-3-hexenol esters,
linalool, α-terpineol, and salicylic acid derivatives are distinctly emitted in response
to avirulent versus virulent Pseudomonas syringae strains in tomato leaves,
reflecting defense activation and metabolite pathway engagement
 Enhanced diagnostic potential: VOC fingerprinting enables early detection of plant
diseases. Panels of compounds (e.g., hexenal, methyl salicylate, benzaldehyde,
farnesene) serve as non-invasive biomarkers to differentiate diverse biotic stresses in
crops
 Increasing field applicability: While GC-MS remains common, LC-MS
complements it by capturing semi-volatile and non-volatile metabolites, with
emerging portable LC systems promising in situ diagnostics