Techniques to separate mycotoxins, Aflatoxins, ochratoxin, trichothecenes, cyclopiazonic acid

1. DONE BY:
SHRUTHI K (18308019)
DEPT. OF MICROBIOLOGY
PONDICHERRY UNIVERSITY

2. MYCOTOXINS
 The mycotoxins produced by fungi are able to grow in great
variety substrates and under the most diverse environmental
conditions. These have been identified as dangerous agents for
the human health.
 Humans are exposed to mycotoxins in consumption of polluted
foods, a sharp intoxication manifests for vomit, abdominal pains,
lung edema, fatty infiltration and necrosis of the liver.
 The producers of mycotoxins, are multicellular eukaryotic
organisms filamentous constituted by true mycelium.
 Mycotoxins, term derived from Greek mikes and toxicon, which
mean fungi and poison respectively, are produced during the final
stage of mold growth phase or the early stationary phase

3. SOURCE: PRESCOTT
SOURCE: Alshannaq et.al
Occurrence, Toxicity, and Analysis of
Major Mycotoxins
in Food

4. GENERAL TECHNIQUES TO SEPARATE
MYCOTOXINS
 The extraction method used to remove the mycotoxin from the
biological matrix is dependent on the structure of the toxin.
 Polar metabolites require water whereas hydrophobic toxins
require organic solvents.
 These can be direct extractions, or may be partitioned with other
solvents, such as n-hexane for partial clean up, to remove
excess components of the biological matrix.
 Extraction solvent also differs.
 This can be done by liquid-liquid extraction (polar and non-polar
solvents), Supercritical fluid extraction (CO2) or solid phase
extraction.

5. HPLC- HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
 HPLC works on the principle of separation of the material
according to their molecular weight and polarity.
 Silica is modified to make it non-polar by attaching long
hydrocarbon chains to its surface. A polar solvent is used - for
example, a mixture of water and an alcohol such as methanol.
 There will be a strong attraction between non-polar and non-polar
substances and vice versa. Here polar molecules move faster as
they don’t bind.
 Hydrophobic molecules need time to break the weak van der
Waals force.
 Detector will detect the molecules based on their time and
molecular weight.

7. GAS CHROMATOGRAPHY (GC-MS)
 Gas chromatography works on the principle of separation based on
polarity and volatility of a compound.
 The stationary phase is a high boiling liquid and the mobile phase is
compound vaporized to gas.
 Helium inert gas pressurizes the gas to move through the column.
 The retention time of the gaseous substance based on its volatility and
polarity varies.
 The retention time of a compound varies based on its solubility of the gas,
temperature, flow rate in mobile phase, length of the column and the
nature of the stationary phase.
 If the column is polar, and the gas contains polar substance its retention
time increases. If the gas is volatile, the retention time decreases.
 Based on this the detection is done by mass spectroscopy.

8. AFLATOXINS
 Aflatoxins are secondary metabolites produced by Aspergillus
flavus and A. parasiticus types of molds which grow on many
varieties of cereal grains and nuts.
 They can be detected using HPLC-fluorescence or HPLC-MS.
 Aflatoxins B2 and G2 fluoresced with enough intensity in aqueous
based mobile phases. B1 and G1 had to be treated with
oxidants like iodine to increase activity.
 So solid phase iodine reservoir is used. Upon detection
fluorescence is observed. Even ng quantitites observed.
 Post column addition of 3-cyclodextrin to enhance fluorescence of
B1 and G1.

9.  Thermo-spray (TSP) MS coupled to HPLC has been evaluated
for the confirmation of aflatoxins.
 Picogram quantities of the aflatoxins could be detected with
selected ion monitoring.
 Sodium bisulfite reacts with aflatoxins B1 and G~ to form bisulfite
adducts across the double bonds of the furofuran ring.
 Using ion-pair chromatography, the bisulfite adducts of aflatoxins
B~ and G~ were separable from aflatoxins B2 and G2 which were
unaffected by the bisulfite treatment.
 Applied to corn, milk and liver. A micro-flow particle beam interface
was used to determine aflatoxins in peanut meal by HPLC-MS.

10.  Reverse phase chromatography has been found very sensitive and
reproducible for analysis of aflatoxins M2, M1, G2, G1, B1, and B2
with the mobile phase being a polar and non-polar solvent.
 These HPLC methods differed significantly in the choice of
normal‐phase or reversed‐phase columns of different types, elution
mixtures and gradients, detection methods, and sample
preparation and purification procedures.

11. GAS CHROMATOGRAPHY:
 Flame ionization detectors or electron capture detectors with a
mass spectrometer (MS) can be used for detection of aflatoxins.
 Methyl silicone-coated fused silica columns was used for rapid
quantification of aflatoxins in corn. The limit of detection was found
to be 2 ng/g and overall recovery was 82% for this study and
the limit of quantification was 1 ng for B1 and B2.

13. OCHRATOXIN A
 Ochratoxin A is a nephrotoxic and carcinogenic mycotoxin that is
highly toxic to several animal species. It is produced by fungi of
the Aspergillus and Penicillium genera which infect cereal grains.
HPLC TECHNIQUE:
 Reversed-phase HPLC with an acidified mobile phase and
fluorescence detection has been employed for the determination
of ochratoxin A.
 Ion-pair HPLC has been successfully used for determination of
ochratoxin A in coffee products, human plasma and cheese. In this
method, the mobile phase was made basic (pH 7.5-9) or acidified
(pH 5.5) and low millimolar concentrations of ion pairing agents
such as cetyltrimethylammonium bromide, tetrabutylammonium
bromide or tetrabutylammonium hydroxide were added.

14.  Detection limits for coffee products were in the sub-ng/g range
when affinity chromatography clean-up was employed.
 The detection limits for the ion-pair HPLC plasma method were
0.02 ng/m.
 3-Cyclodextrin as a mobile phase additive resulted in a slight
increase (15%) in fluorescence intensity of ochratoxin A.
 HPLC with fluorescence detection (FD) becomes the method of
choice because of the available short and high-resolution columns
and of the sensitivity of fluorescence detectors, and its potential
for automation. Extraction is normally performed in acetonitrile-
water, methanol-water, or even chloroform.
 An early RP–HPLC–FD method coupled with solid phase
extraction (SPE) cleanup and concentration procedure was
developed for the analysis of citrinin from hydrolyzed human urine.

15.  Liquid chromatography using reversed-phase columns and
fluorescence detection was effectively used to quantify different
mycotoxins in grains, chilly, feeds, and spices.
 To obtain fine peak forms and resolution, RP-ion-pair HPLC
techniques with postcolumn fluorometric detection technique have
also been applied for the determination of mycotoxins.
 However, reversed-phase ion-pair HPLC provides good peaks,
whereas those in the native fluorescence of mycotoxins were
somewhat lost.
 This weakness can be solved by acidifying the eluate from the
HPLC column before fluorescence detection. So, RP-HPLC is
widely used because of its advantages instead of conventional
normal-phase HPLC.
 HPLC-electrospray MS/MS could detect low pg quantities of
ochratoxin A.

17. GC-MS TECHNIQUE:
 ochratoxin A was not feasible for quantitative determination that
often.
 There has been an account of GC-MS in detection of barley grain
mycotoxin. The process is as follows.
 The volatiles were thermally desorbed from the Chromosorb
adsorbent using a Perkin-Elmer ATD 400 (Perkin-Elmer,
Stockholm, Sweden) set at 170 °C and with a helium flow of 100
ml min−1 for 5 min.
 The compounds were injected directly into the gas
chromatograph, a Varian 3400 GC with FID detector, a fused silica
capillary column with chemically bound methyl-polysiloxan,
OV1CB, temperature program rising from 35 °C to 220 °C at 4 °C
per min, the carrier gas was helium at 23.1 psi at a rate of 3–4 ml
min−1.

18. TRICHOTHECENES
 Trichothecenes are mycotoxins produced by Fusarium spp. And
other types of fungi which can contaminate grains and other
plants.
 This method is carried out by Normal HPLC using photodiode
array.
 Photo Diode Array Detector operates by simultaneously
monitoring absorbance of solutes at several different wavelengths.
 Light from the broad emission source such as a deuterium lamp is
collimated by an achromatic lens system so that the total light
passes through the detector cell onto a holographic grating.
 In this way, the sample is subjected to light of all wavelengths
generated by the lamp.

19.  The dispersed light from the grating is allowed to fall on to a diode
array. The array may contain many hundreds of diodes and the
output from each diode is regularly sampled by a computer and
stored on a hard disc.
 The detection is also done by fluorescence and UV detection.
 Reagents such as coumarin-3-carbonyl chloride and anthracene-
9-carbonyl chloride have been successfully studied for several
trichothecenes.
 In UV detector, when light of a certain wavelength is directed at a
flow cell, the substance inside the flow cell absorbs the light.
 As a result, the intensity of the light that leaves the flow cell is less
than that of the light that enters it. An absorbance detector
measures the extent to which the light intensity decreases (i.e.,
the absorbance).

20.  The photolysis method involves passing the HPLC effluent through
a post-column UV irradiation unit which converts the
trichothecenes to oxidizable products.
 Direct reductive electrochemical detection (- 1.4 V) has been
studied for application to the HPLC analysis of DON in corn, rice
and wheat products.
 Supercritical fluid chromatography on HPLC columns with UV and
MS detection was applied to the separation and identification of
several trichothecenes in Fusarium culture.
 Sensitivity was poor with UV detection if no clean-up column was
used.

22. GC-MS TECHNIQUE:
 A 20 g subsample was extracted with 100 ml of methanol-1%
aqueous NaCl,1 ml of 19-nortestosterone as an internal standard
was added; the flask was shaken with a rotary shaker for 1 h at
230 rpm and filtered through a filter paper with suction.
 The filtrate was defatted with n-hexane (2 x 50 ml), and
evaporated to dryness. The residue was transferred to a Florisil
column with 2 x 2 ml methanol.
 The mycotoxins were eluted from the column with 200 ml
chloroform-methanol (90:10, vol/vol).
 The eluate was evaporated to dryness on the rotary evaporator
and the residue was dissolved in 1 ml methanol-water (60:40,
vol/vol) and filtered through Sep-Pak C18 cartridges.
 The eluate was evaporated to dryness under N2 and dissolved in
1 ml of methanol and reserved for chromatographic analysis

23.  Analysis of mycotoxins by GC-FID. A 900 μl aliquot of the
evaporated residue was treated with 100 μl silylating reagent Tri-
Sil TBT for 1 h at 45 °C.
 After reaction, 0.5 ml n-hexane and 1 ml phosphate buffer were
added to the cooled mixture.
 The sample was mixed by shaking and the organic layer was
transferred to another vial.
 Mycotoxins were detected using a Hewlett-Packard gas
chromatograph Model 6890 Series II provided with flame
ionization detection (FID) and split/splitless injector.
 helium carrier gas 1.76 ml/min; injector and detector temperature
275 °C and 300 °C, respectively; temperature programme: 150
°C (1 min)

24.  A 100 μl aliquot of the extract dissolved in methanol was
submitted to HPLC
 The mobile phase was methanol-water (70:30, vol/vol) with a flow
rate of 1 ml/min. Fluorescence was recorded at excitation and
emission wavelengths of 280 and 460 nm, respectively.

25. ZEARALENONE
 Zearalenone and its metabolites, a- and/3-zearalenol, are
biologically active mycotoxins possessing estrogenic activity.
 There is also some evidence for the carcinogenicity of
zearalenone.
 This mycotoxin is produced by Fusarium graminearum and several
other species of Fusarium fungi which can infect a variety of
agricultural crops, particularly corn and other grains.
HPLC TECHNIQUE:
 Recent HPLC methods for zearalenone determination have
employed reversed-phase chromatography with direct
fluorescence detection.
 These methods used several different sample clean-up
techniques. Liquid-liquid partitioning was used to purify aqueous
extracts of biological fluids.

26.  Solid phase extraction using an aminopropyl column (polar) was
found to be useful for clean-up of milk after an initial extraction with
basic acetonitrile and partitioning into dichloromethane.
 The partitioning was speeded up considerably using a hydrophilic
matrix to absorb the aqueous phase after removal of acetonitrile by
rotary vacuum evaporation.
 The clean-up enabled the detection of zearalenone and c~-
zearalenol in milk at levels as low as 0.2 ng/ml and for/3-
zearalenol, down to 2 ng/ml. GPC on SX-3 Biobeads was used to
purify chloroform extracts.
 The detection limit using HPLC with an amino-bonded phase and
fluorescence detection at 280 nm and 470 nm was about 2 ng/g.
 Other clean-up procedures used in methods for zearalenone are
immunoaffinity column chromatography and chromatography on
piperidinohydroxypropyl Sephadex LH-20 gel.

27.  The HPLC fluorescence detection of zearalenone in cereal
extracts was modified by adding aluminum chloride solution to the
column effluent in a heated post-column reactor and a five-fold
increase is observed.

28. CYCLOPIAZONIC ACID
 a-Cyclopiazonic acid is a toxic mycotoxin produced by certain
species of Aspergillus and Penicillium.
 It has been found in several plant products, such as corn and
peanuts, as well as in Penicillium processed cheese.
 It is also known to accumulate in certain animal tissues as a result
of the consumption of contaminated feed.
 Several different HPLC methods namely, ligand exchange
chromatography, normal-phase chromatography, normal-phase
ion-pair partition chromatography and ion exchange
chromatography using an amino-bonded phase were used.
 All methods employed UV absorbance detection at a wavelength
between 279 and 284 nm or 225 nm.

29.  The ligand-exchange methods made use of zinc acetate or zinc
sulfate (as a chelating agent) to improve the peak shape when
employing reversed-phase columns and aqueous mobile phases
 The extraction and cleanup procedures mostly involved solvent
extraction and liquid-liquid partition followed by SPE clean-up on
silica.
 Quantitation limits were in the 50-100 ng/g range for corn,
peanuts and poultry tissue using the ligand exchange systems.

30. GC-MS TECHNIQUE:
 A gas chromatograph GC 5890 [Hewlett–Packard (HP), Avondale,
PA, USA] equipped with a pressure programmable on-column inlet
and a mass spectrometer HP5971 MSD were used.
 A capillary column (30 m×0.25 μm I.D.) with Rtx-200 bonded
stationary phase film of 0.25 μm thickness
 A glass capillary tube was cut off to make a short glass tube.
 Inlet temperature was maintained at 90°C for 0.2 min,
programmed from 90–250°C at 100°C/min.
 Helium carrier gas was used at a constant flow-rate of 0.75
ml/min.
 The mass conditions were as follows: full scan mode; ionization
energy, 70 eV; ion source temperature, 180°C

31. REFERENCES
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Gas Chromatography – Mass Spectrometry for the Detection of
Mycotoxins from Trichothecene Groups A and B in Grains

32.  Alshannaq et.al Occurrence, Toxicity, and Analysis of Major
Mycotoxins in Food, International journal of Environmental
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