Order Manual Analysis Mycotoxins 20 07 2021
Order Manual Analysis Mycotoxins 20 07 2021
Name Organization
Dr. Rajan Sharma ICAR-National Dairy Research Institute, Karnal
Dr. Ajit Dua Punjab Biotechnology Incubator, Mohali
Dr. AK Dikshit Retd. from Division of Agricultural Chemicals, Indian
Agricultural Research Institute (IARI), Pusa, New Delhi
Dr. Alok Kumar Srivastava CSIR-Central Food Technological Research Institute
(CFTRI), Mysuru
Dr. Jagan Mohan Rao Retd. from CSIR-Central Food Technological Research
Institute (CFTRI), Mysuru
Dr. Kiran N Bhilegaonkar ICAR-Indian Veterinary Research Institute Regional
Station, Pune
Dr. Lalitha Ramakrishna Retd. from CSIR-Central Food Technological Research
Gowda Institute (CFTRI), Mysuru
Dr. Mohana Krishna Reddy CSIR- Indian Institute of Chemical Technology, Hyderabad
Mudiam
Dr. Rajesh R Nair CALF, National Dairy Development Board, Anand
Dr. Raju Khan CSIR-Advanced Materials & Processes Research Institute,
Bhopal
Dr. Reena Das Postgraduate Institute of Medical Education and Research,
Chandigarh
Dr. Kaushik Banerjee ICAR-National Research Centre for Grapes, Pune
Dr. Sanu Jacob Food Safety and Standards Authority of India
Dr. Dinesh Kumar Food Safety and Standards Authority of India
Shri Shailender Kumar Food Safety and Standards Authority of India
Ms. Kanika Aggarwal Food Safety and Standards Authority of India
Shri Ratish Ramanan Food Safety and Standards Authority of India
TABLE OF CONTENTS
Note: The test methods given in the manual are standardised/ validated/ taken from national or
international methods or recognised specifications, however it would be the responsibility of the
respective testing laboratory to verify the performance of these methods onsite and ensure that it gives
proper results before putting these methods in to use.
List of Abbreviations
AF Aflatoxin
DON Deoxynivalenol
ELISA Enzyme Linked Immunosorbent Assays
FLD Fluorescence detector
HPLC High Performance Liquid Chromatography
HP-TLC High Performance Thin Layer Chromatography
IAC Immuno-Affinity Chromatography
LC Liquid Chromatography
LC-MS/MS Liquid chromatography tandem mass
OTA Ochratoxin A
PAT Patulin
PBS Phosphate Buffered Saline
PHRED Photochemical Reactor Enhanced Detection
TLC Thin Layer Chromatography
UPLC Ultra Performance Liquid Chromatography
1.0 Introduction
Mycotoxins—toxic secondary metabolites of filamentous fungi—are biological in origin. Only a few of
the thousands of mycotoxins present significant food safety challenges to the farm-to-fork food
continuum. The natural fungal flora associated with food safety are dominated by three genre:
Aspergillus, Fusarium, and Penicillium.
These fungal metabolites when present in sufficiently high levels in food, can have toxic effects that
range from acute (for example, liver or kidney deterioration), to chronic (for example, liver cancer),
mutagenic, and teratogenic; and resulting symptoms range from skin irritation to immunosuppression,
neurotoxicity, and death (ICMSF 1996). Aflatoxin B1, fumonisins, and patulin are suspected human
carcinogens.
The chemical structures of some important mycotoxins are shown in Figure 1.
Figure 1: Chemical structures of a few mycotoxins that are of food safety concern.
Aflatoxins
Aflatoxin, ahighly toxic secondary metabolite derived from polyketides produced by fungal species
Aspergillus flavus, A. parasiticus, and A. nomius,is probably the most common and widely known
mycotoxin contaminant. Aflatoxin-producing fungi can contaminate crops in the field, at harvest, and
during storage.Some of the more common crops susceptible to contamination with aflatoxins are cereals
(e.g. maize, rice and wheat), tree nuts (e.g. pistachios, walnuts and Brazil nuts), cottonseed and
groundnuts and can lead to serious threats to human and animal health. Unrefined vegetable oils made
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from contaminated seeds or nuts usually contain aflatoxin. However, during the refining process aflatoxin
is destroyed therefore, refined oils are safe. The most ambient climates for aflatoxin-production are high
temperature and humidity typically found in tropical and subtropical regions of the world including sub-
Saharan Africa and Southern Asia.
There are more than 20 known aflatoxins, but the four main ones are aflatoxin B1, aflatoxin B2, aflatoxin
G1 and aflatoxin G2. Aflatoxin M1 and M2 are the mono-hydroxylated derivatives of B1 and B2,
respectively, and occur in the milk of lactating mammals including humans, after ingestion of food or
feed contaminated with the toxins. The chemical structures of the aflatoxins are show in Figure 2. The
level of toxicity associated with aflatoxin varies with the types present, with the order of toxicity
beingB1> G1> B2>G2
Aflatoxin B1, B2, G1, and G2 refer to toxins which fluoresce blue (B) or green (G) under ultraviolet light
and are separable by thin layer chromatography (TLC). The only structural difference between B and G
toxins is the inclusion of an oxygen in the cyclopentanone ring.
The stringent regulations worldwide place more emphasis on estimating the aflatoxin content in food and
feed. The current methods for quantitative aflatoxin suitable for use in regulatory laboratories include 1)
thin layer chromatography (TLC), 2) high performance thin layer chromatography (HP-TLC)3) high
performance liquid chromatography (HPLC), and4) the more recent liquid chromatography tandem mass
spectrometry (LC-MS/MS). Several semiquantitative and qualitative methods including Enzyme Linked
Immunosorbent Assays (ELISA) and immunoaffinity column followed by fluorescence spectrometry are
also used.Rapid in-field and laboratory involve the lateral flow dip-stick kits, hyperspectral imaging and
electronic nose.
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Deoxynivalenol (DON)
Deoxynivalenol (DON) also known as vomitoxin is a trichothecene mycotoxin mainly produced by
Fusarium fungi (Fusarium molds). Major producing fungi include Fusarium species F. graminearum and
F. culmorum, one of plant pathogens that cause scab mainly in wheat and barley etc., and damages cereals
the most widely by contamination in the field. The main commodities affected are cereals such as wheat,
rice, barley, oats and maize etc.
Trichothecene mycotoxins are classified into three groups by structural characteristics, and
deoxynivalenol is classified into Group B.
Trichothecene mycotoxins act on serotonin-mediated neurons and induce anorexia and vomiting. FSSA(I)
has established a level of restriction.
The current methods suitable for use in regulatory laboratories for DON estimation include 1) thin layer
chromatography (TLC), 2) high performance liquid chromatography (HPLC), and 3) the more recent
liquid chromatography tandem mass spectrometry (LC-MS/MS).
Patulin
Patulin(Figure 4) is a mycotoxin that is produced by certain species of Penicillium, Apergillus, and
Byssochylamys molds that may grow on variety of foods including fruit, grains, and cheese.
Patulin is a furopyran (Figure 4)Patulin has been found to occur in a number of foods including apple
juice, apples, and pears. Patulin contamination is primarily associated with damaged and rotting fruits and
fruit juicesmade from poor quality fruits.The amount of patulin in apple products is generally viewed as a
measure of the quality of the apples used in production. It is not a particularly potent toxin, but a number
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of studies have shown that it is genotoxic, which has led to some theories that it may be a carcinogen,
though animal studies have remained inconclusive.
Ochratoxin A
Ochratoxin A (OTA) is a naturally occurring foodborne mycotoxin found in a wide variety of agricultural
commodities worldwide, ranging from cereal grains to dried fruits to wine and coffee. Ochratoxins A, B,
and C contain a phenylalanine moiety attached to a dihydroisocoumarin group via an amide bond (Figure
5). OTA is the most prevalent, most important from an animal and human health standpoint, while
ochratoxins B and C are of lesser importance. It is produced by several fungal species including
Aspergillus ochraceus, A. carbonarius, A. niger and Penicillium verrucosum. Contamination generally
occurs as a result of poor storage of commodities and suboptimal agricultural practices during the drying
of foods. Ingestion is the main source of exposure to OTA. OTA is a chemically stable compound; hence,
ordinary food processing measures fail to substantially reduce its presence in foods and beverages. OTA
has been shown to be toxic and carcinogenic in animals. It is nephrotoxic to multiple species, and is a
potent renal carcinogen in rodents. The kidney is the main target organ.
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2.0The regulatory limits for the presence of these contaminant is listed in Table 1 below:
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3.0Safety requirements while handling mycotoxins
All food samples suspected of being contaminated with mycotoxins must be handled with extreme care.
Aflatoxins are potent carcinogenic substances. Refer to MSDS for specific information.
I.Personal Safety precautions
a) Use disposable gloves and protective face masks while grinding the food creates dust.
b) Prepare samples in area separate from analytical laboratory.
c) Wear a full sleeved lab coat, safety goggles, closed shoes and gloves when carrying out analyses.
d) The laboratory coat or apron must be soaked in 5% sodium hypochlorite solution over-night and
washed in water
e) All work must preferably be carried out in a hood
f) While handling pure aflatoxin reference material, extreme precautions must be taken as they are
electrostatic.
g) Weighing and transferring mycotoxins in dry form should be avoided; they should be dissolved in
a solvent. The electrostatic nature of a number of the mycotoxins in dry form results in a tendency
for them to be easily dispersed in the working area, and to be attracted to exposed skin and
clothes. Their concentrations should be determined spectrophotometrically.
h) Protect eyes with UV-absorbing filter when using UV-viewing chamber.
i) Swab any accidental spill of toxin with 1% sodium hypochlorite bleach (NaOCl), leave 10 min
and then add 5% aqueous acetone.
II. Precautions during analysis
a) Reactive vapors i.e. O2, SO2, HCl can affect adsorbents used in TLC as well as the stability of
adsorbed spots. TLC must, therefore, be performed only in a laboratory free of volatile reagents.
b) Always dry TLC plates thoroughly before exposure to UV light.
c) UV light from sunlight or fluorescent lamps can catalyse changes to compounds being examined
when exposed on adsorbent surface, particularly in the presence of solvent.
d) Avoid exposing to UV light underdeveloped spots and expose developed plates to UV light for
the minimum time needed for visualization.
e) Protect analytical material adequately from light and keep aflatoxin standard solutions protected
from light by using amber vials or cover with aluminium foil. Put a warning note on the label.
III. Handling glassware for aflatoxin analysis
a) Use of non-acid washed glassware for aflatoxin aqueous solutions may cause loss of aflatoxin.
b) Before use soak new glassware in dilute acid (carefully add 105 mL concentrated Sulphuricacid
to water and make upto 1 L) for several h, then rinse extensively with distilled water to remove
all traces of acid. (Check with pH paper).
c) Rinse all glassware exposed to aflatoxin with methanol, add 1% sodium hypochlorite (NaOCl)
solution and after 2 h add acetone to 5% of total volume. Let it react for 30 min and then wash
thoroughly.
Reference:FAO Manuals of Food Quality Control 14 /7, 1986, page 185 / AOAC 17th edn, 2000,
Chapter 49, subchapter 1 Mycotoxins /Sub chapter 2 Aflatoxins).
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Preparation of a Homogenous Laboratory Sample for Analysis of
Aflatoxin
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Preparation of Aflatoxin Standards for Thin Layer Chromatography
Method
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Where A350 = the absorbance of the aflatoxin at 350 nm,
Mw = molecular weight of the aflatoxin (Table below),
ε = the molar absorptivity of the aflatoxin in benzene-acetonitrile
solution. The Mw and molar absorptivity values are provided in the Table
below
Aflatoxin Molecular Solvent Ε
weight
B1 312 Benzene-acetonitrile 19800
(98+2)
Toluene-acetonitrile (9+1) 19300
Methanol 21500
Acetonitrile 20,700
B2 314 Benzene-acetonitrile (98+2) 20900
Toluene-acetonitrile (9+1) 21000
Methanol 21400
Acetonitrile 20,700
G1 328 Benzene-acetonitrile (98+2) 17100
Toluene-acetonitrile (9+1) 16400
Methanol 1717700
Acetonitrile 17600
G2 330 Benzene-acetonitrile (98+2) 18200
Toluene-acetonitrile (9+1) 18300
Methanol 19200
Acetonitrile 18900
M1 328 Benzene-acetonitrile (9+1) 18000
Acetonitrile 19000
M2 330 Acetonitrile 21000
Preparation and storage 1. Dilute portions of stock solution to a spotting concentration (0.5
of working standards μg/mL) with the same solvent used to prepare aflatoxin standards.
2. Use benzene–acetonitrile (9+1) to dilute Aflatoxin M1 solution.
3. Before storage, weigh flasks to nearest mg and record mass for future
reference.
4. Wrap flasks tightly with aluminum foil and store at 0 °C. When the
solution is to be used after storage, reweigh flask and record any
change.
5. To avoid incorporation of water by condensation, bring all standards
to room temperature (25 ±2 °C) before use.
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6. Do not remove aluminum foil until contents have reached room
temperature. Standard solutions of aflatoxins B1, B2, G1, G2 are
stable for more than one year.
7. The criteria of purity of the standards can be checked by determining
chromatographic purity and molar absorption.
8. The absorbance close to 350 nm is determined and concentration
calculated.
Preparation of Prepare resolution reference standards by mixing B1, B2, G1 and G2 to
Resolution Reference give a final spotting concentration of 0.5 μg/mL for each aflatoxin.
Standards
Reference AOAC 17th Edn 2000, Official Method 971.22 Standards of aflatoxin, sub
Para E, Preparation and storage of TLC Standards)
Approved by Scientific Panel on Methods of Sampling and Analysis
10 | M o M - M y c o t o x i n s
TLC methodfor Determination of AflatoxinsBF Method
(Applicable for groundnuts and groundnut products, oilseeds and
food grains)
Method No. FSSAI 07.003:2020 Revision No. & Date 0.0
Caution Follow all safety precautions described earlier.
Inhalation of chloroform vapors can cause headaches, drowsiness,
dizziness, and nausea. Disorientation, anesthetic effects, and loss of
consciousness can occur at high concentrations. Wear laboratory
safety goggles and mask. Perform work in a fume hood when using
solvents.
Protect eyes with UV-absorbing filter when using UV-viewing
chamber.
Refer to MSDS for specific information.
Principle Aflatoxins are extracted with aqueous methanol, concentrated and
subjected to Thin Layer Chromatography. The resolved toxins are
visualized using long wavelength UV lamp.
Apparatus/Instruments 1. Stoppered Conical Flask
2. Measuring Cylinders – 25, 50, 250 mL
3. Chromatography column – 25 mm (i. d.) 300 mm length
4. High speed blender
5. Funnel – 7.5 cm diameter or Buchner Funnel with Whatman No1
filter paper or equivalent
6. Wrist action shaker
7. Rotary evaporator
8. UV light Chamber equipped with Longwave UV lamp with an
intensity of 430 mwatt/cm2 at 15 cm at 365 nm
9. Adjustable Micropipette– 5-100 μL,
10. Vials, Borosilicate – screw cap lined with foil or Teflon
11. Microsyringe
12. TLC chamber
Materials and Reagents Note: Refer to Material Safety Data Sheets and ensure that safety
guidelines are applied before using chemicals
1. Acetone
2. Aflatoxin Standard
3. Sodium chloride
4. Methanol
5. Chloroform (CHCl3)
6. Diatomaceous earth (Celite)
7. Glass Wool
8. Hexane
9. Methanol
10. Nitrogen Gas for Drying
11. Silica Gel (60 Mesh) or Precoated silica gel 60 (0.25 mm
11 | M o M - M y c o t o x i n s
thickness) plates
12. Screw capped borosilicate vial
Preparation of Reagents 1. Methanol: Water (55: 45): Add 55 mL of methanol to 45 mL of
water in a glass conical flask and mix by inversion.
2. Acetone: Chloroform (1: 9): Add 20 mL of acetone to 180 mL of
chloroform in a glass conical flask and mix by inversion Note:
Prepare reagent fresh daily and in the fume hood.
3. Aflatoxin standard solution: As described earlier under
‘Preparation of Standards’
4. Silica gel for column chromatography: Silica Gel 60 (0.063–0.2
mm) for 50 g test portions. Activate by drying 1 h at 105 °C. Add
H2O,1 mL/100 g, seal, shake until thoroughly mixed, and store
15 h in air-tight container
Sample Preparation Peanut butter and peanut meal need no preparation unless they
contain large particles, in which case reduce extraction by milling.
Use hammer mill, rotary cutter, or disk (burr) type mill for meals.
Grind raw materials and roasted peanuts and peanut butter with
pieces of pea nuts to paste with disk (burr) type mill before
extraction.
Alternatively, prepare peanut samples by H2O slurry method: Blend
1100 g peanuts comminuted in subsampling mill with 1.5L H2O and
22 g NaCl 3 min at medium speed in 1 gal. blender cup.
Extraction
1. Weigh 100 g of peanut meal or powder or 50 g peanut butter into
a blender jar.
2. Add: 1) 250 mL methanol–water (55+45) and 100 mL hexane to
peanut butter 2) 500 mL methanol–water (55+45), 200 mL
hexane and 4 g NaCl to peanut powder.
3. Blend for one min at high speed.
4. Transfer to 250 mL centrifuge bottles and centrifuge for 5 min at
2000 rpm. Alternatively let mixture stand undisturbed in blender
jar wherein separation will occur within 30 mins.
5. Pipette 25 mL of lower aqueous methanol phase into a separating
funnel, add 25 mL chloroform, stopper and shake for 30–60 s.
6. Let layers separate and drain bottom chloroform layer through
anhydrous Na2SO4 into a 250 mL beaker.
7. Repeat extraction with two 25 mL portions of chloroform.
8. Evaporate all combined chloroform extracts on a steam bath with
a stream of N2 to between 2 mL and just dryness or as soon as
condensing vapor is no longer visible on beaker lip.
9. Do not leave beaker on hot plate after solvent has evaporated.
10. Transfer extract with careful washing to a screw capped
borosilicate vial and evaporate to dryness under gentle stream of
nitrogen Seal vial with hollow polyethylene stopper and cap.
12 | M o M - M y c o t o x i n s
Save for TLC.
11. Re-dissolve the residue just prior to TLC.
Method of analysis Thin Layer Chromatography
Preparation of TLC plates
1. Weigh 30 g silica gel, into 300 mL glass-stoppered Erlenmeyer,
add H2O as recommended by manufacturer, shake vigorously for
1 min, and pour into applicator. Adjust amount of H2O to obtain
best consistency of slurry for spreading.
2. Immediately coat five 20×20 cm glass plates with 0.25 mm
thickness of silica gel slurry.
3. Rest the plates undisturbed until gelled (ca 10 min). Adjusting
thick ness of spread to 0.5 mm, provides good resolution of
aflatoxins and tightness of spots.
4. Dry coated plates 2 h at 80 °C or 1 h at 110 °C, and store in
desiccating cabinet with active silica gel until further use.
5. Alternatively, Precoated silica gel 60 0.25 mm thickness, TLC
plates of appropriate size may be used.
Preliminary TLC:
1. Uncap vial containing the extract, add 200 μL benzene–
acetonitrile and reseal with a polythene stopper.
2. Shake vigorously to dissolve.
3. Puncture polythene stopper to accommodate the needle of a 10
μL syringe.
4. Under subdued incandescent light and as rapidly as possible spot
2, 5 and 10 μL on an imaginary line 4-5 cm from bottom of the
TLC plate. Keep vial for quantitative analysis.
5. On the same plate spot 2.5 and 10 μL of aflatoxin standards. Spot
at least one 5 µL resolution reference standard, to show whether
adequate resolution is attained.
6. Add 50 mL acetone–chloroform (10:90) to trough of unlined
developing tank. Allow the chamber to be saturated with solvent
before use.
7. Use only one plate per tank, placing trough to one side to permit
maximum exposure of the coated surface to tank volume.
Immediately insert spotted plate into the tank and seal tank.
8. Develop plate for 40 min 23°–25 °C or until aflatoxins reach a Rf
0.4-0.7.
9. Remove plate from the TLC chamber, evaporate solvent at room
temperature.
10. View the plate using long wavelength UV lamp in a viewing
chamber.
11. Observe pattern of the four fluorescent spots. Protect eyes with
UV-absorbing filter
Note: Composition of acetone–CHCl3 can be varied from (5 + 95) to
13 | M o M - M y c o t o x i n s
(15 + 85) to compensate for variations in Silica gel and developing
conditions.
Quantitative TLC:
If preliminary TLC shows the need for further dilution/concentration
of test solution, evaporate to dryness on a steam bath and re-dissolve
in a calculated volume of benzene–acetonitrile. Spot successively 3.5,
5.0, and 6.5 µL of test solution. All spots should be approximately of
the same size and ~ 0.5 cm in diameter. On the same plate spot 3.5,
5.0, 6.5 µL aflatoxin standard. Spot 5.0 µL of each standard used on
top of one of the two 6.5 mL test solution origin spots as internal
standard. To see whether adequate resolution is achieved. Spot at
least one 5.0 µL resolution reference standard. After developing the
plate, dry in subdued light. Compare fluorescent intensities of the
sample spot with those of the standard aflatoxins and determine
which of the sample spot matches the standards. If the spots of the
smallest quantity of sample are too intense to match standards, the
sample should be further diluted and re-chromatographed.
Inference Four clearly identifiable spots should be visible in resolution
(Qualitative Analysis) reference standard. Examine pattern from test solution spot
containing internal standard for aflatoxin spots. Rf values of
aflatoxins used as internal standards should be same as or only
slightly different from those of respective standard aflatoxin spots.
(Since spots from test solution are compared directly with standard
aflatoxins on same plate, magnitude of Rf is not important. These
may vary from plate to plate.)
Compare test solution patterns with pattern containing internal
standard. Fluorescent spots in test solution thought to be aflatoxins
must have Rf values identical to and color similar to aflatoxin
standard spots when un known spot and internal standard spot are
super imposed. Spot from test solution and internal standard
combined should be more intense than either test solution or standard
alone
Calculation with units of Calculate the concentration of Aflatoxin B1 from the formula
expression
Where,
S = μL Aflatoxin standar
d, which matches the test solution
Y = Concentration of Aflatoxin B1 standard (μg/mL)
V = μL of final dilution of test extract applied
X = μL of sample extract spotted giving a fluorescent
intensity equivalent to S (B1 standard)
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W = mass of the sample (in g) contained in final extract
(10 g if 50 mL Chloroform extract is used)
Calculate Aflatoxin B2, G1, and G2 similarly
Reference Official Method 968.22 ‗Aflatoxins in Peanuts and Peanut Products
CB Method‘, AOAC 17th edn, 2000
Approved by Scientific Panel on Methods of Sampling and Analysis
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TLC Method for Determination of Aflatoxins in Food and Feeds:
Romer Mini Column Method
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12. Sodium Hydroxide Solution – 0.02 M – 8.0 g NaOH/L
13. 0.03% Sulphuric acid Solution– Add 0.3 mL of concentrated
Sulphuric acid in 1000 mL
14. Precipitating reagents – (1) Copper carbonate – Basic (2) Ferric
Chloride Slurry – Mix 20 g of FeCl3 with 300 mL water
15. Column packing (a) Florisil (100– 200 mesh) (b) Silica gel 60
(70-230 mesh) for column chromatography (c) Alumina Neutral,
(80–200 mesh)- activate for two h at 110 °C (d) Calcium Sulfate
anhydrous (20–40 mesh).
Dry all packing material for 1-2 h at 110 °C. Store all packing
materials and packed columns in vapour-tight containers.
Aflatoxin solution for spiking - Dilute solutions of B1 and G1 to final
concentration of 2 μg/mL
Preparation of mini column Trap a small plug of glass wool into the tapered end of a column. To
the column add to the height indicated in the following order: 1) 5-7
mm, Calcium Sulfate, 2) 5-7 mm, Florisil, 3) 18-20 mm, Silica gel, 4)
8-10mm, neutral alumina, and 5) 5-7 mm, Calcium Sulfate. Finally
trap the column top with a small plug of glass wool. Tap column after
each addition to settle packing and maintain uniform interfaces levels
as possible. After packing apply pressure to top glass wool plug with
a 5 mm dia. glass rod. Packed mini-columns are available
commercially.
Sample Preparation Extraction:
1. Weigh 50 g test sample into a blender jar, add 250 mL acetone–
water (85+15) and blend for three min. Alternatively use a 500
mL glass stoppered Erlenmeyer flask and shake for 45 min on a
mechanical shaker.
2. Filter through Whatman filter paper No 4 or equivalent into a 250
mL graduated cylinder.
3. Collect 150 mL filtrate and transfer to 400 mL beaker.
Purification:
1. Quantitatively add 170 mL of 0.02 N Sodium hydroxide and 30
mL Ferric chloride slurry to a 600 mL beaker and mix well.
2. To the filtrate in the 400 mL beaker add about three grams basic
Copper carbonate, mix well and add to the mixture in the 600 mL
beaker.
3. To this add 150 mL diatomaceous earth and mix well.
4. Filter using a 160 mm funnel or Buchner funnel using Whatman
No 4-filter paper or equivalent.
5. Quantitatively transfer 150 mL filtrate to a 500 mL separator, add
150 mL 0.03% Sulphuric acid and 10 mL Chloroform.
6. Shake vigorously for about two mins and let separate.
7. Transfer lower Chloroform layer (13-14 mL) to 125 mL
separator.
17 | M o M - M y c o t o x i n s
8. Add 100 mL Potassium hydroxide wash solution swirl gently for
30 s and let separate.
9. If emulsion occurs drain emulsion into 10 mL glass stoppered
flask, add about one g anhydrous Sodium Sulfate, stopper shake
30 s and let separate (Chloroform phase need not be completely
clear).
10. If emulsion is not broken, transfer emulsion to 125 mL separator
and wash with 50 mL 0.03% Sulphuric acid.
11. Collect 3 mL of Chloroform layer in a 10 mL glass stoppered
cylinder for chromatography
Method of analysis 1. Transfer two mL of Chloroform solution to a mini-column using
a 5 mL syringe with 5-inch, 15- gauge needle.
2. Allow to drain by gravity (15–30 min).
3. When solvent reaches top of adsorbent, add 3mL elution solvent,
Chloroform – acetone (9+1).
4. Allow to drain by gravity until solvent reaches the top of
adsorbent.
5. Do not let columns run dry during determination.
6. Examine columns in darkened chamber using a UV lamp. Look
for a blue fluorescent band at the top of the Florisil layer (ca 2.5 cm
from bottom of column), which is indicative of aflatoxin.
7. Perform analysis with ―clean‖ test portion and with test portion
spiked with known amounts of aflatoxin to obtain comparison
standards.
8. Some uncontaminated products show white, yellow or brown
fluorescence at top of Florisil in sample column. If band has no
definite bluish tint test portion is negative.
Reference AOAC 17th edn, 2000 Official Method 975.36. Aflatoxins in Food
and Feed – AACC- AOAC Method
Approved by Scientific Panel on Methods of Sampling and Analysis
18 | M o M - M y c o t o x i n s
Thin Layer Chromatographic Method for Determination
Aflatoxins in Corn and Peanuts (Groundnuts)
19 | M o M - M y c o t o x i n s
6. UV-Viewing cabinet: 270×270 mm base minimum, equipped
with 15 W long wave ultraviolet (UV) lamp.
7. Fluoro-densitometer (TLC/HPTLC scanner): Capable of scanning
in reflectance mode by fluorescence, equipped with high-pressure
Hg lamp, monochromator for adjustment to excitation 366 nm,
and emission cutoff filter 420 nm.
Preparation of Reagents 1. Solvents: Methanol, hexane, chloroform, anhydrous ethyl ether
(100%), dichloromethane, acetone and isopropanol.
2. Aflatoxin standard solution: Prepared in benzene-acetonitrile
(98+2) to contain 0.5 μg/mL each B1 and G1 and 0.15 μg/mL
each B2 and G2.
Sample Preparation Extraction:
1. Weight 50 g (ground to pass No. 20 sieve) corn or peanuts into
500 mL glass-stoppered Erlenmeyer flask.
2. Add 200 mL methanol-H2O (85+15) and secure stopper with
masking tape.
3. Shake vigorously by hand until samples show no clumps.
4. Shake 30 min on wrist-action shaker and filter mixture through
medium fluted paper. Collect 40 mL filtrate in 50 mL graduated
cylinder.
5. Transfer filtrate to 125 mL separatory funnel.
6. Add 40 mL 10% Sodium chloride solutions, mix, and add 25 mL
hexane.
7. Shake one min. Let the phases separate, drain lower (aqueous)
phase into second 125 mL separatory funnel, and discard upper
phase.
8. Extracts aflatoxins from aqueous phase with two 25 mL portions
chloroform
9. Shake one min each time.
10. Combine chloroform fractions in 125 mL Erlenmeyer flask and
evaporate to dryness on steam bath
Method of analysis Silica Gel Column Chromatography:
1. Attach silica gel column, to extraction system, (or clamp to stand
if using gravity flow only).
2. Condition the column by washing with three mL hexane,
followed by three mL dichloromethane using vacuum (flow rate 6
mL/min), or let drip freely unassisted by suction.
3. Check column suitability by adding aflatoxin B1 standard (three
mL dichloromethane containing 100 ng aflatoxin B1) to 0.5 g
silica gel column. Recovery must be >90% by this method.
4. Dissolve residue of extracted sample, in 3mL dichloromethane
and add to column. Let drip freely (flow rate ca 3 mL/min, apply
vacuum if needed).
5. Rinse residue container with two × one mL portions of
20 | M o M - M y c o t o x i n s
dichloromethane and add rinses to column.
6. Wash column with 3 mL hexane, 3 mL anhydrous ethyl ether,
and then 3 mL dichloromethane. (Use vacuum, flow rate 6
mL/min, or use syringe and adapter to apply pressure to increase
solvent flow if necessary. Do not pull up syringe plunger while it
is still attached to column.)
7. Turn off vacuum, remove extraction system cover, and place vial,
under each column (test tube rack can be used to hold vials).
8. Elute aflatoxins (without vacuum) with two to four 3 mL portions
(according to results of column suitability test) of chloroform-
acetone (9+1).
9. Evaporate eluate to dryness on steam bath under stream of
nitrogen.
Thin-Layer Chromatography: Fluro-densitometry:
1. Dissolve residue from above in 250 µL chloroform.
2. Spot plate, with 5 µL chloroform test solution in duplicate and 2,
5, 10, and 20 µL aflatoxin standard solution.
3. Randomize standard and test solution spots across plate so
duplicate test solution spots are not next to each other and
standard spots are dispersed evenly.
4. To avoid errors, prepare spotting plan, either on plate or in
notebook, prior to spotting.
5. Develop plate for one h with chloroform-acetone (9 + 1).
6. Evaporate solvent for five min in fume hood followed by 2 min at
50 °C forced draft oven.
7. Examine plate under long wave UV light to determine presence
or absence of aflatoxins.
8. Quantitate by fluoro-densitometric measurement. Scan test and
aflatoxin reference spots (transmission or reflectance mode,
excitation 365 nm and emission cutoff 430 nm).
9. At end of plate scan, rescan 1st or 2nd lane. Scans of test spots
should be within +5%; if not, rescan entire plate.
Calculation with units of Calculate concentration of aflatoxin B1 in test portion, using
expression following formula:
21 | M o M - M y c o t o x i n s
standard spots;
10 = g corn or peanut represented by extract.
Calculate concentrations of aflatoxins B2, G1, and G2 similarly.
Reference AOAC Official Methods of Analysis (2000), Ch.49.2.15 Method,
993.17
Approved by Scientific Panel on Methods of Sampling and Analysis
22 | M o M - M y c o t o x i n s
Determination of Aflatoxin in Corn and
PeanutPowder/ButterLiquid Chromatographic Method
23 | M o M - M y c o t o x i n s
and G1.
III. Clean Up Column – 20 cm × 1cm i. d. with Teflon stopcock and
coarse frit bed support, detachable glass solvent reservoir with
24/40 fitting
IV. Adjustable autopipettes – 10-100 and100–200 μL with disposable tips
V. Filter tube – glass 15 × 2.5 cm i. d. with coarse frit bed support
(glass wool not recommended)
Materials and Reagents 1. Solvents: HPLC grade: methanol, hexane, methylene chloride,
benzene, acetone, acetonitrile. Anhydrous ethyl ether stored in
metallic container (Glass bottled ether forms peroxides soon after
opening which degrades aflatoxins)
2. Hydrochloric acid (0.1 M): Prepare in a fume hood. Dilute 5.0 mL
of concentrated HCl (11.6M) to 580 mL with distilled water.
Caution: Add acid to water.
3. LC elution solvents – Water: acetonitrile: methanol (700:170:170).
Adjust ratio of water to obtain baseline resolution of aflatoxin B2
and G2. Note: Mix the solvents do not makeup volume.
4. Silica gel for Column chromatography – Silica gel 60, (0.063-0.2
mm). activated by drying at 110 °C. Cool to room temperature.
Weigh desired quantity (100 g) into glass stoppered container. Add
one ml water in small increments, agitate silica gel between
additions. Shake or tumble mechanically 4-6 h. Let stand 16 h
5. Trifluoroacetic acid (TFA) – 98.5% pure. Transfer 1-2 mL TFA
to a one-dram vial with a Teflon lined cap. Keep in freezer when
not in use. Discard if discoloration appears.
Anhydrous Sodium sulfate: Sift out fines to obtain 20–40 mesh. Heat
for 2-3 h at 600 °C to remove organic impurities
Preparation of Reagents Aflatoxin standard solutions:
Aflatoxin stock solution – 10 μg/mL. Prepare individual stock solution
in benzene-acetonitrile (98+2) and determine concentration of each by
measuring UV absorption if desired.
Working standard solutions - Use an autopipette (Pipetman) to transfer an
appropriate quantity stock solution to each 4-dram vial (15 mL) to obtain the
final concentrations of aflatoxins in each vial as indicated in Table below
Table Working Aflatoxin Final concentration of
Standards Aflatoxins
Vial B1& G1 B2 & G2 B1& G1 B2 & G2
Number (ng) (ng) (µg/10.05 (µg/10.05
mL) mL)
1 250 125 0.25 0.125
2 500 250 0.50 0.25
3 1000 500 1.0 0.50
4 2000 1000 2.0 1.
24 | M o M - M y c o t o x i n s
Evaporate solutions to dryness under a gentle stream of nitrogen
(drying may be facilitated by warming to 40 °C). Using Eppendorf
pipette add 200 μL hexane and 50 μL of TFA to each vial, cap and
vortex for 30 s. Let solutions stand 5 min, then add 10 mL water:
acetonitrile (9+1) and vortex for 30 s. Let layers separate for 5 -10 min
or centrifuge at 1000 rpm for 30 s. Final concentration of aflatoxins
shall be as shown in the table above.
Sample Preparation Extraction and partition:
1. Transfer 50 g prepared corn, or peanut powder or peanut butter to a
jar (Capacity 1L)
2. Add 200 mL of methanol followed by 50 mL of 0.1 M HCl and
blend for three min at high speed.
3. Filter through 24 cm Whatman No 1 filter paper or equivalent.
Filtrate may not be completely clear.
4. Collect 50 mL filtrate.
5. Transfer to 250 mL separatory funnel.
6. Add 50 mL 10% Sodium chloride solution, swirl.
7. Add 50 mL hexane and shake gently for about 30 s.
8. Let phases separate then drain lower aqueous layer into another
250 mL separator funnel. Discard hexane layer.
9. Add 25 mL methylene chloride and shake moderately for 30 s. If
emulsion occurs break up with clean pipette.
10. Let phases separate then drain lower methylene chloride layer
through coarse granular anhydrous sodium sulfate in glass filter
tube.
11. Collect elute in a 250 mL beaker.
12. Evaporate elute, on steam bath under a gentle stream of nitrogen to
2-3 mL.
Method of analysis Column Chromatography:
1. Make a slurry of two g silica gel with about 10 mL ether–hexane
(3+1) in a 30 mL beaker.
2. Pour slurry into a clean-up column and wash beaker with
additional 5 mL ether–hexane solvent to effect complete transfer.
3. Keep stop cock closed and let silica gel settle without tamping.
4. Wash sides of column with 2-3 mL ether–hexane using squeeze
bottle.
5. After gel settles, open stop cock and while column drains, add
about 1 cm anhydrous sodium sulfate.
6. Transfer eluate collected after extraction to column.
7. Wash beaker with about 2 mL of methylene chloride and add wash
to column. Do not use more than 5-6 mL methylene chloride to
transfer eluate to column.
8. With stop cock fully open, add 25 mL benzene–acetic acid (9+1)
and the 30 mL ether–hexane (3+1) to column, draining each wash
25 | M o M - M y c o t o x i n s
to top of sodium sulfate.
9. Discard washes.
10. Elute aflatoxin with 100 mL methylene chloride–acetone (90+10)
11. Collect elute in 250 mL beaker.
12. Evaporate elute on steam bath under a gentle stream of nitrogen to
about 6 mL. Quantitatively transfer to 3-dram vial.
13. Evaporate elute to dryness using a steam bath or an aluminum
block under a gentle stream of nitrogen.
14. Evaporate remaining 200 μL just to dryness under a gentle stream
of nitrogen by holding vial in palm of hand and slowly rotating vial
Derivatization:
1. Add 200 μL hexane to the residue obtained above.
2. Then add 50 μL of TFA using Eppendorf pipette, cap the vial and
vortex vigorously for 30 s (exactly). This procedure must be
followed closely to ensure consistent reaction yields.
3. Let mixture stand 5 min.
4. Using transfer pipette add 1.950 mL water-acetonitrile (9+1).
5. Vortex vigorously for exactly 30 s and let layers separate 10 min.
Concentration is 10 g/2 mL aqueous acetonitrile.
[Note: Post column derivatization with Kobra Cell may also be used]
HPLC:
1. Using a HPLC equipped with a fluoresce detector and C-18
column set at a flow rate of 1 mL/min equilibrate the column with
solvent (Water: acetonitrile: methanol (700:170:170).
2. Inject 25 μL of derivatized standard solutions.
3. Prepare standard curve to check linearity of responses.
4. Inject 25 μL of derivatized test solution (lower aqueous phase).
If test peaks are outside the dynamic linear range, dilute aliquot of
derivatized test solution to suitable volume with water –acetonitrile,
remix on vortex mixer and inject another 25 μL portion.
Calculation with units of Calculate individual aflatoxin concentration as follows:
expression Use responses of standard containing 500 ng B1 and G1, and 250 ng
B2 and G2 for calculations.
Aflatoxins, ng/g = (P/P‵ ) ×C × (2/10) ×1000×D
where P and P‵ = peak areas (integrator counts) or height for test
solution and standard, respectively, per 25 µL injection;
C =concentration of individual aflatoxins in standard solution (0.5 or
0.25 mg/10.05 mL);
D = dilution factor if 2 mL test solution for injection is diluted.
Reference AOAC 17th edn, 2005 Official Method 990.33 Aflatoxins in Corn and
Peanut Butter, Liquid Chromatographic Method)
Approved by Scientific Panel on Methods of Sampling and Analysis
26 | M o M - M y c o t o x i n s
Determination of Aflatoxins B1, B2, and G1 in Corn, Cottonseed,
Peanuts, and Peanut Butter
Enzyme-Linked Immunosorbent (Immuno-dot Screen Cup)
Screening Assay
27 | M o M - M y c o t o x i n s
6. Cross-reactivity to aflatoxin B1 for antibody should be 100, 70,
75, and <10% for aflatoxins B1, B2 and G1 and G2, respectively.
All other toxins tested should show no cross-reactivity.
Sensitivity of ELISA Reagent (a) Negative control test solution: Use 100 µLofbuffer solution in the
cup. Follow procedure in enzyme immunoassay steps 7-9
(b) Thresh old-level standard solution: Used to define lower limit of
determination. Dispense 100 mL working standard into test tube. Add
350 mL methanol–buffer, (30 + 70), and mix.
Follow procedure in enzyme immunoassay steps 7-9
(c) Positive control test solution: Use working standard solution;
follow procedure for enzyme immunoassay steps 7-9
Negative control test solution should develop blue color; positive
control test solution should have no color development. Threshold
standard solution should show no color development
Preparation of Reagents Reagents from commercial suppliers can be used provided
requirements listed below are met.
1. Antibody-coated solid support: Antibody-coated filter
material attached to analytical cup made of porous polyethylene
(3.2 cm diameter, 2.5 cm high, capacity 4 mL). Coated cup is
specified by manufacturer to be stable for 6 months stored at4-
8°C. Coated 8/12/96 well strips or plates can be used.
2. Aflatoxin-enzyme conjugate- Aflatoxin B1-horseradish
peroxidase conjugate at toxin-enzyme molar ratio of 10-15:1.
Conjugate is specified by manufacturer to be stable for 6 months
at 4-8°C.
3. Wash solution (Phosphate-buffer saline (PBS) solution). Dissolve
0.23 g NaH2PO4.H2O, 1.95 g K2HPO4.3H2O, 8.70 g Sodium
chloride, 0.125 mL Tween 20 (polyoxyethylenesorbitan
monolaurate), and 10 mg thimerosal (Ethylmercurithiosalicylic
acid, sodium salt), in 900 mL H2O adjust pH to 7.2, and dilute to
1 L.
4. Buffer –Bovine serum albumin (0.1% w/v) in PBS containing
0.05% thimerosal.
5. Substrate solution A – Tetramethylbenzidine (TMB) (0.4 g/L
H2O), pH 8.3.
6. Substrate solution B – Hydrogen peroxide (0.02% H2O2 in 0.13%
aqueous citric acid solution), pH 3.0.
7. Methanol, hexane, and chloroform – Reagent grade.
8. Standard aflatoxin B1 – Approximately 28 μg as dry film.
Apparatus/Instruments Equipment specified is not restrictive; other suitable and compatible
equipment may be used.
1. High- speed blender – With 500 mL jar
2. Micropipette and tips- recommended range 100-1000 μL; with
disposable polypropylene tips.
28 | M o M - M y c o t o x i n s
3. Glass culture (test) tubes- 10×75 mm; 3 mL.
4. Microplates (96-well)/ 8/16 well strips
5. Filters- Whatman No. 4 or equivalent.
6. Timer- Graduated in 1 s intervals.
7. Carborundum boiling chips.
General Instructions 1. Store all kit components at 4-8 °C. Do not freeze.
2. Before use, allow one h for antibody coated cups/ plates/strips
and reagents to reach room temperature (23-29 °C).
3. Use separate disposable pipet tips for each solution to avoid cross
contamination.
4. Include one negative control with each group (20 cups/wells) of
test samples. Negative control must be functioning properly
(must develop blue color in center of cup/wells) for test to be
valid.
5. Positive controls must be used with each group of test portions
and must show no color in the center of the cup/well.
6. Threshold level standard should also be used and must show no
color development. If color develops, repeat the test. Color
development in more than 2 tests indicates a defective kit.
7. Reagents are stable for 6 h at room temperature. To ensure shelf
life of kit components promptly return reagents to refrigerator
after use.
8. Addition of reagents to cups/wells must be successively spaced at
convenient time intervals e.g. 60 s or higher for making
observations.
Sample Preparation (a) Corn, raw peanuts, and whole cottonseed: Weigh 50 g test portion
into blender jar. Add 100 mL methanol-water (8+2). Blend for three
min at high speed. Filter mixture and recover filtrate. Alternatively,
let mixture stand 10-15 mins and recover supernatant liquid. Dilute
extract in ratio 1:1 with extraction solvent.
(b) Peanut butter-: Weigh 50 g test portion into blender jar. Add 100
mL hexane and 250 mL methanol-water (55+45). Blend for three min
at high speed. Filter mixture and transfer filtrate to separator funnel.
Let layers separate for 10 mins. Place 20 mL lower layer in 150 mL
beaker. Add minimum of 15 boiling chips and heat in steam bath or
on hot plate. Boil for 3 mins and let cool.
Preparation of Aflatoxin B1 Standard Solutions:
(a) Stock solution- Add 3 mL chloroform to vial containing 28 μL
aflatoxin B1 standard (ca 9 ng/μL). Cap vial, mix contents, and store
vial in refrigerator.
(b) Working solution- Prepare fresh daily. Dispense 300 μL stock
solution into vial. Add 2400 μL methanol (1 ng/μL), mix and store
solution in refrigerator. Dispense 10 μL diluted standard (1 ng/μL)
into test tube. Add 300 μL methanol and 700 μL buffer, Prepare ≤2h
29 | M o M - M y c o t o x i n s
before use.
Method of analysis Enzyme Immunoassay# for Corn, raw peanuts and whole cottonseed:
1. Allow 1 h for all reagents to reach room temperature (23-29 °C).
2. Prepare fresh substrate in a small culture (test) tube by mixing
500 μL substrate solution A with 500 μL substrate solution B for
each cup/well being used. Do not combine substrate solution A
with solution B more than 15 min before use.
3. Run 1 negative control and 1 positive standard control each day
to ensure that all reagents are functional. Threshold-level
standard should be run with each set of new reagents. Negative
control should be run by using 100 μL buffer. For positive
standard control, using working standard.
4. Add 200 μL test extract to 400 μL PBS (600 μL total).
5. Thoroughly mix diluted test extract and apply one 150 μL aliquot
to cup/well.
6. Using timer, after exactly 60 s add second 150 μL aliquot of
diluted test extract to same well cup/well. Using timer, wait
additional 1 min before proceeding to next step.
7. Apply 100 μL enzyme solution to center of cup/well. Using
timer, wait one min.
8. Wash with 1.5 mL wash solution added drop wise. If more than 1
cup is being used, wash successively with 500 μL per cup 3
times.
9. Add entire contents of substrate solution 1.0 mL from each test
tube to each cup. (Start time as soon as substrate mixture is added
to cup.). Wait one min and immediately observe the disk (center
of cup) for blue color development (negative) or no color
development (positive).
Enzyme Immunoassay# for Peanut butter:
1. Allow 1 h for all reagents to reach room temperature (23-29 °C).
2. Prepare fresh substrate solution in small culture (test) tube by
mixing 500 μL (10 drops) substrate solution A with 500 μL (10
drops) substrate solution B for each cup being used. Do not
combine substrate solution A with substrate solution B more than
15 min before use.
3. Add 500 μL test extract to 500 μL PBS (1000 μL total).
4. Thoroughly mix diluted test extract and apply one 200 μL aliquot
to center of cup. Using timer, after exactly 60 s add second 200
μL aliquot of diluted test extract. After exactly additional 60 s
third 200 μL aliquot of diluted test extract and after 60 s add
fourth 200 μL aliquot of diluted test extract before proceeding to
next step.
5. Proceed as for corn steps 7-9.
30 | M o M - M y c o t o x i n s
Inference Observe well/cup for blue color or no color development at exactly
(Qualitative Analysis) after 60 s of adding substrate A and B mixture.
Negative- If it turns light blue or darker, test sample total aflatoxin
B1, B2 and G1 is < 20 ng/g (cottonseed, butter).
Positive- If no color is observed in disk (center of cup/plate) and disk
remains completely colorless (no color change) for at least 60 s, test
sample contains total aflatoxin B1, B2 and G1 at >20 ng/g.
Negative control- Negative control cup must develop blue color in
center of cup.
Positive control-Positive standard cup must remain completely white
(no color change) for at least 60 s.
Threshold-level standard- Cup must remain completely white (no
color change) for 60 s.
#
Note The ELISA kits are meant for primary screening purposes and results
obtained must be confirmed with other analytical methods. Various
manufacturers have different protocols for using their kits. It would
be the responsibility of the lab to validate these kits prior to use.
Reference AOAC Official Methods of Analysis (2000), Method, 990.34.
Ch.49.2.07
Approved by Scientific Panel on Methods of Sampling and Analysis
31 | M o M - M y c o t o x i n s
Method for Determination of Aflatoxins B1, B2, and G1 in Corn:
Enzyme-Linked Immunosorbent Assay method
(Afla-20 cup Test)
Method No. FSSAI 07.008:2020 Revision No. & Date 0.0
Scope Applicable to the detection of 20 ng total aflatoxins /g of corn
(maize).
Caution Follow all personal safety procedures while handling and disposing
solution described earlier.
Grinding of dry samples may result in airborne dust. Even if no toxin
is present, there is potential harm from inhalation of mold spores or
from allergic response to inhaled dust. Use protective mask and/or
dust collector. Prepare samples in area separate from analytical
laboratory.
Inhalation of solvent vapors can cause headaches, drowsiness,
dizziness, and nausea. Disorientation, anesthetic effects, and loss of
consciousness can occur at high concentrations. Wear laboratory coat,
gloves, safety goggles and mask. Perform work in a fume hood when
using solvents.
Refer to MSDS for specific information.
Principle Antibodies specific to aflatoxins B1, B2, and G1 are immobilized on
a filter, and toxin (aflatoxin B1) is labeled with an enzyme
(horseradish peroxidase). Binding of toxin-enzyme conjugate by
immobilized antibodies is inhibited by addition of free toxin present
in test sample. Since fixed number of antibody reaction sites are
available, enzyme activity is proportional to amount of bound toxin-
enzyme conjugate. Antibody-toxin-enzyme complex concentration is
inversely proportional to concentration of free toxin added. Bound
enzyme catalyzes oxidation of substrate to form blue complex.
Development of color indicates that test sample contains aflatoxins at
<20 ng/g; no color development indicates that test sample contains
aflatoxins at ≥ 20 ng/g.
Determining the specificity Antibodies have specific ability to bind structurally related
of antibodies compounds, namely, aflatoxins B1, B2, and G1. Determine
specificity of purified rabbit anti-aflatoxin B1 polyclonal antibodies
by direct competitive ELISA method.
1. Coat serially diluted antibodies on microtiter plates.
2. Prepare standard solutions of aflatoxins B1, B2, G1, G2, and M1;
zearalenone; T-2 toxin; and deoxynivalenol, and add to individual
microtiter well.
3. Add solution of aflatoxin B1 conjugated to horseradish
peroxidase to each well.
4. Add substrate solution of tetramethylbenzidine and hydrogen
peroxide, and measure development of color with scanner.
32 | M o M - M y c o t o x i n s
5. Least color development indicates highest reactivity of toxin-
antibody reaction.
6. Cross-reactivity to aflatoxin B1 for antibody should be 100, 70,
75, and <10% for aflatoxins B1, B2 and G1 and G2, respectively.
All other toxins tested should show no cross-reactivity.
Sensitivity of ELISA Reagent (a) Negative control test solution: Use 100 µLofbuffer solution in the
cup. Follow procedure in enzyme immunoassay steps 7-9
(b) Thresh old-level standard solution: Used to define lower limit of
determination. Dispense 100 mL working standard into test tube. Add
350 mL methanol–buffer, (30 + 70), and mix.
Follow procedure in enzyme immunoassay steps 7-9
(c)Positive control test solution: Use working standard solution;
follow procedure for enzyme immunoassay steps 7-9
Negative control test solution should develop blue color; positive
control test solution should have no color development. Threshold
standard solution should show no color development.
Materials and Reagents Reagents from commercial suppliers can be used provided
requirements listed below are met.
1. Antibody-coated solid support: Antibody-coated filter material
attached to analytical cup made of porous polyethylene (3.2 cm
diameter, 2.5 cm high, capacity 4 mL). Coated cup is specified by
manufacturer to be stable for 6 months stored at 4-8 °C. Coated
8/12/96 well strips or plates can be used.
2. Aflatoxin-enzyme conjugate- Aflatoxin B1-horseradish peroxidase
conjugate at toxin-enzyme molar ratio of 10-15:1. Conjugate is
specified by manufacturer to be stable for 6 months at 4-8 °C.
3. Wash solution (Phosphate-buffer saline (PBS) solution). Dissolve
0.23 g NaH2PO4.H2O, 1.95g K2HPO4.3H2O, 8.70 g Sodium
chloride, 0.125 mL Tween 20 (polyoxyethylenesorbitan
monolaurate), and 10 mg thimerosal (Ethylmercurithiosalicylic
acid, sodium salt), in 900 mL H2O adjust pH to 7.2, and dilute to 1
L.
4. Buffer –Bovine serum albumin (0.1% w/v) in PBS containing
0.05% thimerosal.
5. Substrate solution A – Tetramethylbenzidine (TMB) (0.4 g/L
H2O), pH 8.3.
6. Substrate solution B – Hydrogen peroxide (0.02% H2O2 in 0.13%
aqueous citric acid solution), pH 3.0.
7. Methanol, hexane, and chloroform – Reagent grade.
8. Standard aflatoxin B1 – Approximately 25 μg as dry film.
Apparatus/Instruments Equipment specified is not restrictive; other suitable and compatible
equipment may be used.
1. High- speed blender – With 500 mL jar
2. Micropipette and tips- recommended range 100-1000 μL; with
33 | M o M - M y c o t o x i n s
disposable polypropylene tips.
3. Glass culture (test) tubes- 10×75 mm; 3 mL.
4. Microplates (96-well)/ 8/16 well strips
5. Filters- Whatman No. 4 or equivalent.
6. Timer- Graduated in 1 s intervals.
7. Carborundum boiling chips
General Instructions 1. Store all kit components at 4-8 °C. Do not freeze.
2. Before use, allow one h for antibody coated cups/ plates/strips and
reagents to reach room temperature (23-29 °C).
3. Use separate disposable pipet tips for each solution to avoid cross
contamination.
4. Include one negative control with each group (20 cups/wells) of
test samples. Negative control must be functioning properly (must
develop blue color in center of cup/wells) for test to be valid.
5. Positive controls must be used with each group of test portions and
must show no color in the center of the cup/well.
6. Threshold level standard should also be used and must show no
color development. If color develops, repeat the test. Color
development in more than 2 tests indicates a defective kit.
7. Reagents are stable for 6 h at room temperature. To ensure shelf
life of kit components promptly return reagents to refrigerator after
use.
Addition of reagents to cups/wells must be successively spaced at
convenient time intervals e.g. 60 s or higher for making observations.
Sample Preparation 1. Weigh 50 g test portion into blender jar.
2. Add 100 mL methanol-water (8+2).
3. Blend for three min at high speed.
4. Filter mixture and recover filtrate.
5. Alternatively, let mixture stand 10-15 mins and recover
supernatant liquid.
6. Dilute extract in ratio 1:1 with extraction solvent.
Preparation of Aflatoxin B1 Standard Solutions:
1. Stock solution: Add 2.5 mL methanol to vial containing 25 μg
aflatoxin B1 standard (10 ng/μL). Cap vial, mix contents, and
store vial below -20 °C. Stable for six months
2. Working solution: Dispense 250 μL stock solution into vial. Add
2250 μL methanol (5 ng/μL), mix and store solution at 5 °C. May
be stored for one months (1ng/ μL)
3. Buffer solution of standard: Prepare fresh (<2 h before use).
Dispense 5 μL of working solution into test tube. Add 300 μL of
methanol and 700 μL of PBS, mix by agitation.
4. Proceed as below (Steps 5-8)
Method of Analysis Enzyme Immunoassay#:
1. Allow 1 h for all reagents to reach room temperature (23-29 °C).
34 | M o M - M y c o t o x i n s
2. Prepare fresh substrate in a small culture (test) tube by mixing
500 μL substrate solution A with 500 μL substrate solution B for
each cup/well being used. Do not combine substrate solution A
with solution B more than 15 min before use.
3. Run 1 negative control and 1 positive standard control each day
to ensure that all reagents are functional. Threshold-level
standard should be run with each set of new reagents. Negative
control should be run by using 100 μL buffer. For positive
standard control, using working standard
4. Add 100 μL test extract to 200 μL PBS (300 μL total).
5. Thoroughly mix diluted test extract and apply one 100 μL aliquot
to center of cup/well.
6. Using timer, after exactly 60 s add 100 μL enzyme solution to
center of cup/well. Using timer, wait one min.
7. Wash one time with 1.5 mL wash solution added drop wise. If
more than 1 cup is being used, wash successively with 500 μL
per cup 3 times.
8. Add entire contents of substrate solution 1.0 mL from each test
tube to each cup. (Start time as soon as substrate mixture is added
to cup.). Wait one min and immediately observe the disk (center
of cup) for blue color development (negative) or no color
development (positive)
Inference Observe well/cup for blue color or no color development at exactly
(Qualitative Analysis) after 60 s of adding substrate A and B mixture.
1. Negative- If it turns light blue or darker, test sample total
aflatoxin B1, B2 and G1 is < 20 ng/g.
2. Positive- If no color is observed in disk (center of cup/plate) and
disk remains completely colorless (no color change) for at least
60 s, test sample contains total aflatoxin B1, B2 and G1 at 20
ng/g. Positive samples must be confirmed by quantitative
method.
3. Negative control- Negative control cup must develop blue color
in center of cup.
4. Positive control-Positive standard cup must remain completely
white (no color change) for at least 60 s.
5. Threshold-level standard- Cup must remain completely white (no
color change) for 60 s.
#
Note: The ELISA kits are meant for primary screening purposes and results
obtained must be confirmed with other analytical methods. Various
manufacturers have different protocols for using their kits. It would
be the responsibility of the lab to validate these kits prior to use.
Reference AOAC Official Methods of Analysis (2000), Method, 990.16.
Ch.49.2.11
Approved by Scientific Panel on Methods of Sampling and Analysis
35 | M o M - M y c o t o x i n s
Aflatoxin B1 and Total Aflatoxins using Immunoaffinity Column
Cleanup, Post-column Derivatization, and
LiquidChromatography/Fluorescence Detection
36 | M o M - M y c o t o x i n s
have been found to meet the criteria.
Criteria for acceptance of The aflatoxin IACs to contain monoclonal antibodies that are cross reactive
immunoaffinity column with AFB1, B2, G1, and G2. Thecolumns should have capacity of not less
than 100 ng total AFand should give a recovery of not less than 80% for
AFB1, B2, G1,and G2 when 5 ng of each AF is applied in 10 mL
methanol–PBS(10 + 90, v/v). The columns should have a shelf life of 18
monthsat 4°C or 12 months at room temperature.
Materials and Reagents All chemicals should be of analytical grade
Reagents:
37 | M o M - M y c o t o x i n s
stored in dark at room temperature.
7. Toluene–acetonitrile: 9:1(v/v): Toluene-acetonitrile: Mix 90 mL
toluene and 10 mL acetonitrile
Preparation of standards Stock Aflatoxin standards
1. To containers of dry aflatoxins B1, B2, G1, G2 using the label
statement of aflatoxin t as guide add the required volume of toluene–
acetonitrile (9+1), calculated to give a final concentration of 1000 ng
B1, 200 ng B2, 1000 ng G1, and 200 ng G2/mL.
2. Vigorously agitate solution for one min on a vortex shaker and transfer
without rinsing to a convenient sized glass flask.
3. Do not transfer dry Aflatoxins for weighing or other purposes unless
facilities are available to prevent dissemination to the surroundings
because of electrostatic charge on particles.
4. For Aflatoxins received as solutions transfer solution to convenient
sized glass stoppered flask. Dilute if necessary, to adjust the
concentration as above
5. Record the UV-Vis spectrum of the aflatoxin solution from 200-500
nm. Determine the concentration of individual aflatoxin by measuring
the absorbance (A) at wavelength of maximum absorption close to 350
nm and substitute in the following equation
38 | M o M - M y c o t o x i n s
standards can be stored in dark brown bottles covered with
Aluminium foil
Peanut butter and pistachio paste: Weigh, to nearest 0.1 g, 50 g test portion
into 500 mL Erlenmeyer flask, add 5 g NaCl, 300 mL methanol–water
extraction solvent, and 100 mL hexane or cyclohexane. Blend 3 min with
high speed blender. Filter and pipette 10.0 mL clear filtrate into reservoir
containing 60 mL PBS solution placed on conditioned immunoaffinity
column. Mix with plastic spatula and rinse residues with 1–2 mL PBS from
spatula into reservoir. Transfer solution to column as described below.
Chilli, paprika powder and other spice powders: Weigh, to the nearest 0.1
g, 50 g test portion into 500 mL Erlenmeyer flask with screw top or glass
stopper. Add 5 g NaCl and 300 mL methanol–water solvent. Shake
intensively by hand for 15–30 s and then for 30 min on a shaker. Filter
extract using pre-folded paper. Pipette 10.0 mL clear filtrate into reservoir
containing 60 mL PBS solution placed on conditioned immunoaffinity
column. Mix with plastic spatula and rinse residues with 1–2 mL PBS into
reservoir. Apply solution on immune affinity column as described below.
39 | M o M - M y c o t o x i n s
Dried figs and other dried fruits: Weigh, to nearest 0.1 g, 50 g test portion
into 500 mL Erlenmeyer flask, add 5 g NaCl, 300 mL methanol–water
extraction solvent. Blend 3 min with high speed blender. Filter and pipette
10.0 mL clear filtrate into reservoir containing 60 mL PBS placed on
conditioned immunoaffinity column. Mix with plastic spatula and rinse
residues with 1–2 mL PBS from spatula into reservoir. Transfer solution on
column as described below.
Method of Analysis Immunoaffinity Chromatography:
[Note: Methods for loading onto affinity columns, washing the column, and
elution vary slightly between manufacturers. Follow manufacturer’s
instructions supplied with columns. In general, procedures involve
extraction with methanol–water, filtration or centrifugation, possible
dilution with PBS orwater, loading under pressure onto (possibly
prewashed) column, washing of column with distilled water, and elution of
aflatoxinswith methanol or acetonitrile.]
40 | M o M - M y c o t o x i n s
5. Flow rate, 1.00 mL/min (mobile phase B); current, 100 µA.
6. Inject 200 µL working standard mixture (covering the range of 1–4
ng/g for aflatoxin B1) into injector, following manufacturer‘s
instructions to ensure complete filling of the injection loop.
7. Prepare calibration curve using calibration solutions described and
check curve for linearity.
8. Inject 200 µL extract into injector and identify each aflatoxin peak in
chromatogram by comparing retention timeswith those of
corresponding reference standards. Determine quantity of aflatoxin in
eluate injected from standard curve.
Results Aflatoxins elute in the order G2, G1, B2, and B1 with retention times of ca
6, 8, 9, and 11 min, respectively, and should be baseline resolved.
Calculation with units of Calculate concentration of aflatoxin in test sample as follows:
expression
Plot data [concentration of aflatoxin (ng/mL; y-axis) from calibrant
solution experiments against peak area (units; x-axis)]
Where
Elution volume (mL) = final volume collected after elution from IAC;
41 | M o M - M y c o t o x i n s
Reference J. AOAC Int. 83, 320(2000).
AOAC Official Method 999.07 Aflatoxin B1 and Total Aflatoxins in
Peanut Butter,Pistachio Paste, Fig Paste, and Paprika Powder
Immunoaffinity Column Liquid Chromatography with Post-Column
Derivatization.
Approved by Scientific Panel on Methods of Sampling and Analysis
42 | M o M - M y c o t o x i n s
Aflatoxin B1 in Baby Food using ImmunoaffinityColumn Cleanup,
Post-column Derivatization, and Liquid Chromatography/
Fluorescence Detection
Caution Follow all personal safety procedures while handling and disposing
solution and washing glassware as described earlier.
Soak new glassware before use in dilute acid (e.g., sulfuric acid, 2 mol/L)
for several hours; then rinse extensivelywith distilled water to remove all
traces of acid(check using pH paper).
43 | M o M - M y c o t o x i n s
10. Post column derivatization system
a. For pyridinium hydrobromide perbromide reagent: Second LC
pulseless pump, zero-dead volume T-piece, reaction tubing
minimum dimensions 45 cm × 0.5 mm id PTFE.
b. For electrochemically generated bromine: Kobra cell.
11. Disposable filter unit: Cellulose or cellulose nitrate, 0.45 µm.
12. Pipets: 10 mL.
13. Analytical balance: Weighing to 0.1 mg.
14. Laboratory balance: Weighing to 0.1 g.
15. Calibrated microliter syringes or micropipette(s):25 and 500 µL.
16. Calibrated UV spectrophotometer
17. Affinity Columns: Vicam (Watertown, MA) or Rhone-Diagnostics
have been found to meet the criteria.
Criteria for acceptance of The affinity column must contain antibodies raised againstaflatoxin B1
immunoaffinity column with a capacity of not less than 50 ng aflatoxin B1and should give recovery
of not less than 80% when applied asa standard solution in methanol–H2O
containing 5 ng aflatoxin B1.
Materials and Reagents 1. All chemicals should be of analytical grade
2. Water, except where specified, should be produced by single
distillation, deionization, or reverse osmosis
3. Potassium chloride (KCl)
4. Dihygrogen potassium phosphate (KH2PO4)
5. Disodium mono hydrogen phosphate (Na2HPO4)
6. Sodium chloride (NaCl)
7. Hydrochloric acid
8. Pyridinium hydrobromide perbromide (PBPB): CAS-39416-48-3
9. Potassium bromide
10. Acetonitrile: HPLC grade
11. Methanol: HPLC grade
12. Methanol: Technical grade, pure, or distilled
13. Water: HPLC grade; complying with grade 1 of ISO 3696
14. Hexane or cyclohexane
15. Concentrated Nitric acid
16. Toluene
Reagents:
44 | M o M - M y c o t o x i n s
4. Mobile phase A—Water–acetonitrile–methanol solution (6 + 2 + 3,
v/v/v). Mix 600 mL of HPLC grade water, 200 mL of acetonitrile and
300 mL of HPLC grade methanol.
5. Mobile phase B —For use with electrochemically generated Br: water:
acetonitrile: methanol solution (6:2:3 v/v/v). To each liter of mobile
phase, add 350 µL nitric acid [4M] and 120 mg potassium bromide, and
mix to dissolve.
6. Post column reagent: Dissolve 25 mg Pyridinium hydrobromide
perbromide in 500 mL H2O. Solution can be used for up to 4 days if
stored in dark at room temperature.
7. Toluene–acetonitrile: 9:1(v/v): Toluene-acetonitrile: Mix 90 mL
toluene and 10 mL acetonitrile.
Preparation of standards Stock Aflatoxin standards:
Option A For aflatoxin B1 standard received as a dry films or crystal:
45 | M o M - M y c o t o x i n s
Where A350 = the absorbance of the aflatoxinB1 at 350 nm,
Mw = molecular weight of the aflatoxin B1 =312
ε = the molar absorptivity of the aflatoxinB1 in Toluene–acetonitrile
solution= 19300 [from J. AOAC Int. 82, 252(1999)].
4. Return aflatoxin solution to original glass-stoppered flaskand dilute
with toluene–acetonitrile (9 + 1) to obtain a concentration of 5.00
ng/mLWrap flasks tightly with aluminum foil and store at 0 °C. Do
not remove aluminum foil until contents have reached room
temperature. Standard solutions of aflatoxin B1 is stable for more
than one year.
Working Standard Solution Option, A (To be prepared daily)
I. Use this solution (5ng/mL) for pipetting the volumes listed in Table
below into a set of 10 mL calibrated volumetric flasks.
II. Evaporate toluene–acetonitrile solution just to dryness under stream of
N2 at room temperature (22–25 °C).
III. To each flask, add 3.5 mL methanol, and mix; dilute to volume (10 mL)
with water and mix again
Preparation of Working Calibration Solutions Option A
Prepared fresh daily
Working Aliquot taken Final concentration of
standard from stock working calibrant, ng
solution, µL AfB1/mL
1 20 0.01
2 40 0.02
3 60 0.03
4 80 0.04
5 100 0.05
6 120 0.06
7 140 0.07
Working Standard Solution Option, B (To be prepared daily)
1. Pipet from aflatoxin standard solution (5 ng/mL) volumes as listed in
Table below into a set of 10 mL calibrated volumetric flasks.
2. Evaporate the toluene–acetonitrile solution just to dryness under stream
of N2 at room temperature (22–25 °C).
3. Add 3.5 mL methanol, let aflatoxins dissolve, fill to the mark with
methanol, and shake well.
4. Transfer exactly 1 mL of this working calibrant into an acid-washed
vial and evaporate to dryness.
5. Re-dissolve in exactly the same amount of aqueous methanol that will
be used for test solutions.
6. Calculate concentration of aflatoxin B1 in the re-dissolved working
46 | M o M - M y c o t o x i n s
calibrant solution in ng/mL.
7. Use these values for the calculation (the calibration range in ng/g will
remain unchanged).
47 | M o M - M y c o t o x i n s
and elution vary slightly between manufacturers. Follow manufacturer’s
instructions supplied with columns. In general, procedures involve
extraction with methanol–water, filtration or centrifugation, possible
dilution with PBS or water, loading under pressure onto (possibly
prewashed) column, washing of column with distilled water, and elution
of aflatoxins with methanol or acetonitrile.)
5. Pass filtrate of the extractions through column at flow rate of ca 1 drop/s
(ca 3 mL/min by gravity). Do not exceed 5 mL/min.
6. Wash column with 15 mL water in 5 mL portions, and dry by applying
small vacuum for 5–10 s or passing air through by means of syringe for
10 s.
7. Elute aflatoxin B1 in two steps, First, apply 0.5 mL methanol on the
column and let it pass through by gravity. Collect eluate in either 5 mL
volumetric flask (option A below) or LC injection vial (option B below).
48 | M o M - M y c o t o x i n s
When using PBPB, mount mixing T-piece and reaction tubing, then operate
using the following parameters: flow rates,
Flow rate, 1.00 mL/min (mobile phase B); current, 100 µA.
Inject same volumeof working standards and extract into injector and
identifyeach aflatoxin peak in the chromatogram by comparing
retentiontimes with those of corresponding reference standards.
Calculation with units of Plot the data: concentration of aflatoxin (ng/mL) as they-axis against peak
expression area (units) as the x-axis, from thecalibrant solutions.
Where
49 | M o M - M y c o t o x i n s
g = test portion (g; 50);
50 | M o M - M y c o t o x i n s
Determination of Aflatoxins B1, B2, G1, and G2 in Olive Oil, Peanut Oil,
and Sesame Oil using Immunoaffinity Column Cleanup, Post-column
Derivatization, and LiquidChromatography/Fluorescence Detection
51 | M o M - M y c o t o x i n s
visual exposure to the UV light).
c. Kobra cell (electrochemical cell, post column bromination derivatization
cell. (Caution: Set at 100 mA. Do not turn on current until LC pump is
operating to avoid overheating the cell membrane.)
13. Disposable filter unit: Cellulose or cellulose nitrate, 0.45 µm.
14. Graduated measuring cylinders: 25 and 50 mL.
15. Analytical balance: Weighing to 0.1 mg.
16. Laboratory balance: Weighing to 0.1 g.
17. Calibrated microliter syringes or micropipette(s): 25 and 500 µL.
18. Calibrated UV spectrophotometer
19. Immunoaffinity columns—AflaTest WB columns (G1024; VICAM, meet
the criteria below.
Criteria for acceptance The aflatoxin IACs to contain monoclonal antibodies that are cross reactive with
of immunoaffinity AFB1, B2, G1, and G2. Thecolumns should have capacity of not less than 100
column ng total AFand should give a recovery of not less than 80% for AFB1, B2,
G1,and G2 when 5 ng of each AF is applied in 10 mL methanol–PBS(10 + 90,
v/v). The columns should have a shelf life of 18 monthsat 4 °C or 12 months at
room temperature.
52 | M o M - M y c o t o x i n s
3. Washing solution: Methanol–H2O (10 + 90, v/v), mix, equilibrate to room
temperature.
4. 4M Nitric acid: Dilute 28.1 mL concentrated HNO3 (65%) or 26.1 mL 70%
HNO3) in water to final volume of 100 mL.
5. Mobile phase A: For AF post column derivatization with PHRED cell or
UVE device. Methanol–acetonitrile–water (25 + 17 + 60, v/v/v). Mix 600
mL of HPLC grade water, 170 mL of acetonitrile and 250 mL of HPLC
grade methanol.
6. Mobile phase B: For AF post column derivatization with Kobra cell.
Methanol–acetonitrile–water (25 + 17 + 60, v/v/v) + 350 μL of 4 M nitric
acid + 120 mg potassium bromide, and mix.
53 | M o M - M y c o t o x i n s
Working Aliquot of Final aflatoxin concentration of
Standard 400 ng/mL working standard solution, ng/mL
solution AFs second
B1 B2 G1 G2 Sum
stockstandard
solution (μL)
1 0 0 0 0 0 0.0
2 10 0.40 0.10 0.20 0.10 0.80
3 25 1.00 0.25 0.50 0.25 2.00
4 50 2.00 0.50 1.00 0.50 4.00
5 100 4.00 1.00 2.00 1.00 8.00
6 250 10.0 2.50 5.00 2.50 20.0
Sample Preparation Extraction:
1. Weigh 5.0 g test portion in a 50 mL centrifuge tube.
2. Add 1.0 g NaCl and 25 mL extraction solvent.
3. Vortex until oil and extract solvent are well mixed.
4. Shake at 400 rpm for 10 min.
5. Centrifuge at 7000 rpm (g value = 5323 mm/s2) for 10 min.
6. Aspirate and discard the upper oil layer.
7. Pass the lower aqueous methanol layer through folded filter paper. Measure
15 mL filtrate with a 25 mL graduated cylinder and place in a 50 mL
centrifuge tube.
8. Add 30 mL water, mix, and filter through glass microfiber paper.
9. Collect 30 mL filtrate (equivalent to 2 g test portion) into a 50 mL graduate
cylinder and proceed immediately with IAC chromatography.
Performance Standard The affinity column must contain antibodies raised againstaflatoxin B1 with a
for Affinity Column capacity of not less than 50 ng aflatoxin Bland should give recovery of not less
than 80% when applied asa standard solution in methanol–H2O containing 5 ng
aflatoxinB1.
54 | M o M - M y c o t o x i n s
7. Let column run dry and then force 10 mL air through column with a syringe.
8. Place a 2 mL volumetric flask under column.
9. Elute with 0.6 mL LC grade methanoland collect AFs in a 2 mL volumetric
flask; let drip freely. Let column run dry.
10. Elute with additional 0.6 mL methanol and collect into the same volumetric
flask.
11. Let column run dry and force 10 mL air through column.
12. Dilute eluate to volume with water and perform LC analysis.
55 | M o M - M y c o t o x i n s
8. These solutions cover the range of 0.4–10.0 ng/mL for AFB1, 0.1–2.5 ng/mL
for AFB2, 0.2–5.0 ng/mL for AFG1, and 0.1– 2.5 ng/mL for AFG2.
9. Check the curve for linearity.
10. If test portion area response is outside (higher) the calibration range, then the
purified test extract should be diluted with methanol– water (50 + 50, v/v)
and reinjected into the LC column.
Results Aflatoxins elute in the order G2, G1, B2, and B1 and should be base-line
resolved to measure each aflatoxinas a discrete peak.
Calculation with units of Plot peak area (response, Y-axis) of eachAF standard againstconcentration
expression (ng/mL, X-axis) and determineslope (S) and Y-intercept Calculate level of toxin
in testsample with the following equation,
The total AFs is the sum of theAFB1, AFB2, AFG1, and AFG2
56 | M o M - M y c o t o x i n s
Direct analysis of Aflatoxins (AF) peanuts, peanut products and cereal
matrices by Ultra-High-Performance Liquid Chromatography with
fluorescence detection
57 | M o M - M y c o t o x i n s
and NaCl (5 g). Shake for 30 min,200 rpm), and then centrifuge (5000 rpm, 5
min). Take an aliquot (3 mL) and dilute with15 mL PBS and add 50 μL NaOH
(2 M) solution.
IAC cleanup:
Load the diluted sample onto IAC connected to a vacuum manifold and allow to
pass without any vacuum. Wash with 10 mL PBS. Elute with methanol (2 × 0.5
mL). Slowly evaporate the final extract (1 mL) to dryness. Reconstituted in 0.5
mL methanol–water (acidified with 0.1%acetic acid, 1:1), and finally inject 10
µL into the UHPLC-FLD instrument.
Method of Analysis Chromatography conditions:
1. Column: C18 column (2.1 × 50 mm, 1.7 μm).
2. Column temperature: 40 °C,
3. Flow rate: 0.4 mL/min
4. Injection volume: 10 μL.
5. The mobile phase: methanol: acetonitrile: water (18:18:64)
6. Elution: Isocratic
7. Detector:
a. Excitation wavelength 365 nm.
b. Emission wavelength: 456 nm
Results
Calculation with units of Prepare a calibration curve for 0.02–10 ng/g for each AF by injecting 10 µl each
expression working standard. From the equation determine the concentration of the AF in
the extracts prepared.
LOQ LOQ is 0.008 µg/kg for the B1 and G1 and 0.003 µg/kg for the B2 and G2.
58 | M o M - M y c o t o x i n s
Reference High-sensitivity direct analysis of aflatoxins in peanuts and cereal matrices by
ultra-performance liquid chromatography with fluorescence detection involving
a large volume flow cell‘ Oulkar, D., Goon, A., Dhanshetty, M., Khan, Z., Satav,
S., & Banerjee, K (2018):
Journal of Environmental Science and Health, Part B Pesticides, Food
Contaminants, and Agricultural Wastes, 53, 255-260
Approved by Scientific Panel on Methods of Sampling and Analysis
59 | M o M - M y c o t o x i n s
Determination of Aflatoxins M1 and M2 in Fluid Milk
Liquid Chromatographic Method
61 | M o M - M y c o t o x i n s
diluted milk into syringe and gently pull liquid through cartridge at
flow rate ca 30 mL/min (very fast drops). (Caution: Too fast a flow will
not allow sufficient time for aflatoxin to adsorb, resulting in low
recoveries).
5. Add 10 mL water-acetonitrile wash solution to syringe and pull
through.
6. Plug syringe barrel with stopper and pull hard vacuum on cartridge for
ca 30 seconds to remove as much wash solution as possible from
packing.
7. Remove cartridge and dry inside of both stems with cotton swab or
tissue paper to eliminate any remaining wash solution.
8. Re-prime cartridge by adding 150 μL acetonitrile to inlet bed support
disk and let solvent soak into packing for 30 seconds. Attach cartridge
to dry glass or plastic 10 mL Leur tip syringe, retaining same stem as
inlet.
9. Insert silica gel cleanup column into 250 mL vacuum flask fitted with
one-hole rubber stopper. Wash column with five mL ether.
10. Add seven mL ether to syringe cartridge positioned above silica gel
cleanup column. With plunger, slowly force through cartridge (in
portions), collecting eluate in column reservoir.
11. Pull ether slowly through silica cleanup column, using vacuum to
maintain flow rate ca 10 mL/min (fast drops).
12. Rinse silica column with 2 mL additional ether, continuing to use
vacuum. Discard ether.
13. Remove column and stopper from flask and place 16 ×125 mm
collection tube in flask to catch eluate from column.
14. Add 7 mL elution solution (Methylene chloride-alcohol) to column
reservoir. Pull solvent through column with vacuum at ca 10 mL/min
flow rate, collecting eluate in tube.
15. Discontinue vacuum and remove collection tube from assembly.
Evaporate eluate just to dryness under nitrogen stream, using heat to
keep collection tube near room temperature or under vacuum at ≤35 °C.
16. Transfer residue to one-dram vial with Methylene chloride and
evaporate to dryness under nitrogen on steam bath or in heating block
≤50 °C (Do not overheat dry residue).
17. Save sample for derivative preparation.
Derivatization for LC:
1. Prepare derivative of residue from above by adding 200 μL hexane and
200 μL trifluoroacetic acid to dry residue in vial.
2. Shake on vortex mixer ca 5-10 seconds.
3. Let mixture sit for 10 min at 40 °C, in heating block or bath; then
evaporate to dryness under nitrogen on steam bath or heating block
(<50 °C).
4. Add 2 mL water-acetonitrile (75 + 25) to vial to dissolve residue.
62 | M o M - M y c o t o x i n s
5. Mix well using Vortex mixer for LC analysis.
6. Derivatization of standard containing M1 and M2: Add 200 μL hexane
and 50 μL trifluoroacetic acid to silylated vial and mix. Add 50 μL M1-
M2 working standard solution directly into hexane- trifluoroacetic acid
mixture and mix using vortex mixer 5-10 seconds. Treat as described
above (Steps 3-5).
Method of Analysis Liquid chromatography:
1. Attach the C-18 analytical column to instrument.
2. Wash the column at flow rate of 1.0 mL/min with water-isopropanol-
acetonitrile (80 + 12 + 8) for 30 min.
3. Allow the baseline to stabilize.
4. Adjust detector attenuator so that 50-100 μL injection of standard
(0.625-1.25 ng M1, 0.125-0.25 ng M2) gives 50-70% full-scale
recorder pen deflection for aflatoxin M1.
5. Inject LC standard 2-3 times until peak heights are constant.
6. Prepare standard curve from either peak heights or peak areas to ensure
linear relationship. Inject test solutions (typically 50-100μL) with
standard injections interspersed to ensure accurate quantitation.
7. Retention times M1 (as trifluoroacetic acid derivative) and M2 are ca 4-
5 min and ca 7 min, respectively.
Calculation with units of Calculate aflatoxin concentration using the following equation
expression
Where,
H and H‘ = peak height or area of injected test solution and M1/M2
standard, respectively; C‘=concentration of standard (ng/μL);
V1‘ and V1 = volume injected of standard and test solution, respectively;
V = final volume of test solution(μL);
W= volume of milk represented by test solution (typically 20 mL).
Separately calculate concentration for M1and M2
Reference AOAC Official Methods of Analysis (2005), Ch.49.3.06 Method, 986.16
Approved by Scientific Panel on Methods of Sampling and Analysis
63 | M o M - M y c o t o x i n s
Method for Determination of Aflatoxin M1 In Liquid Milk
Immunoaffinity Column Chromatography followed by Liquid
Chromatography
Method No. FSSAI 07.014:2020 Revision No. & Date 0.0
Scope Applicable to determine aflatoxin M1 in raw liquid milk at >0.02 ng/mL.
Caution Follow all personal safety procedures while handling and disposing solution and
washing glassware as described earlier.
Trifluoroacetic acid is a corrosive chemical and contact can severely irritate and
burn the skin and eyes with possible eye damage. Use face shield or eye
protection (safety goggles) in combination with breathing protection.
64 | M o M - M y c o t o x i n s
12. Liquid Chromatography System:
a) With pump delivering a steady flow rate of 0.8 mL/min; loop injection system
of 50-200 μL capacity; equipped with a fluorescent detector with 365 nm
excitation and 435 nm emission; and recorder, integrator, or computer-based
processing system.
b) Reversed-phase LC analytical column: A suitable ODS (C18) column with
particle size 5 µm may be used. Column dimensions can vary (mm): 100
×2.3/4.6/5 i.d. or 125 ×4 i.d. or 200 ×2.1/3/4 i.d.; 250 ×4.6 i.d.; with orwithout
guard columns.
c) Mobile phases- Water –acetonitrile (75+25) or (67+33); water-acetonitrile-
methanol (65+25+10);‘ or water-isopropanol-acetonitrile (80 + 12 + 8). Degas
for 2 min before use.
Materials and Reagents 1. Chloroform-stabilized with 0.5-1.0% ethanol.
2. Nitrogen
3. Aflatoxin M1 standard solutions
a) Stock standard solution (1 μg/mL): Suspend a lyophilized film of reference
standard aflatoxin M1 in acetonitrile to obtain the required concentration.
Determine the concentration of aflatoxin M1 by measuring its absorbance at the
maximum (ca 365 nm) in a calibrated spectrophotometer against acetonitrile as a
blank between 200-400 nm. Check purity by nothing an undistorted shape of the
recorded peak. Calculate the mass concentration (C, μg/mL) from the equation:
65 | M o M - M y c o t o x i n s
Sample Preparation 1. Warm milk before analysis to ca 37 °C in a water bath.
2. Gently stir with magnetic stirrer to disperse the fat layer.
3. Centrifuge liquid milk at 2000 × g to separate the fat and discard thin upper
fat layer.
4. Filter through one or more paper filters, collecting at least 50 mL.
5. Let immuno- affinity column reach room temperature.
6. Attach syringe barrel to the top of immuno-affinity cartridge.
7. Transfer (Vs) of prepared test portion using a volumetric flask or volumetric
pipet into syringe barrel and let it pass through immuno-affinity column at slow
steady flow rate of ca 2-3 mL/min. Gravity or vacuum system can be used to
control flow rate.
8. Remove syringe barrel and replace with a clean one.
9. Wash column with 20 mL water at steady flow rate.
10. After washing completely, blow dry column to dryness with nitrogen steam.
11. Put another dry clean barrel on the cartridge.
12. Slowly elute aflatoxin M1 from column with 4 mL pure acetonitrile.
13. Allow acetonitrile to be in contact with column at least 60 seconds.
14. Keep a steady slow flow rate.
15. Collect eluate in conical tube.
16. Evaporate eluate to dryness using gentle stream of nitrogen.
17. Dilute to volume Vf with mobile phase, i.e., 200 μL (for 50μL injections) or
1000 μL (for 250 μL injections).
Method of Analysis Liquid Chromatography using a fluorescent detector:
1. Connect the C-18 LC column to the LC system.
2. Equilibrate the LC column with the mobile phase at a constant flow rate for at
least 30 min.
3. Set the fluorescent detector at 365 nm excitation and 435 nm emission.
4. Depending on the kind of column, the acetonitrile-water ratio and flow rate of
the mobile phase may be adjusted to ensure optimal separation of aflatoxin M1
from other extract components. As a guideline for conventional C-18 column
(with a length of 250 ×4.6 i. d. mm), a flow rate of ca 0.8 mL/min gives optimal
results.
5. Equilibrate column to obtain a stable baseline.
6. Check optimal conditions with aflatoxin M1 calibrant solution and spiked
milk extract before analyzing test materials.
7. Check linearity of injection calibrant solutions and stability of
chromatographic system.
8. Repeatedly inject a fixed amount of aflatoxin M1 calibrant solution until
stable peak areas or heights are obtained. Peak areas or heights corresponding to
consecutive injections must be within ±5%.
9. Retention times of aflatoxin M1 can vary as a function of temperature and
must be monitored by injecting a fixed amount of aflatoxin M1 calibrant solution
at regular intervals.
10. Calibration curve of aflatoxin M1: Inject in sequence suitable volumes (Vi)
66 | M o M - M y c o t o x i n s
depending on the injection loop, aflatoxin M1 standard solutions containing from
0.05 to1 ng. Prepare a calibration graph by plotting the peak area or peak height
versus the mass of injected aflatoxin M1.
11. Analysis of purified extracts and injections scheme: Inject suitable volume Vi
(equivalent to at least 12.5 mL milk) of eluate into LC apparatus through
injection loop. Using the same conditions as for calibrant solutions, inject
calibrants and test extracts according to stipulated injection scheme.
12. Inject an aflatoxin M1 calibrant with every 10 injections.
13. Determine aflatoxin M1 peak area or height corresponding to the analyte,
and calculate aflatoxin M1 amount Wain test material from the calibration graph,
in ng.
14. If aflatoxin M1 peak area or height corresponding to test material is greater
than the highest calibrant solution, dilute the eluate quantitatively with mobile
phase and re-inject the diluted extract. For best results this area must fall in the
middle of the calibration curve.
Calculation with units of Calculate aflatoxin M1 mass concentration of the test sample, using the
expression following equation
Where
Wm =the numerical value of aflatoxin M1 in the test sample in ng/mL (ppb or
μg/L);
Wa = the numerical value of the amount of aflatoxin M1 corresponding to the
area or height of the aflatoxin M1 peak of the test extract (ng);
Vf = the numerical value of the final volume of re-dissolved eluate (μL);
Vi = the numerical value of the volume of injected eluate (μL)
Vs = the numerical value of volume of prepared test portion passing through the
column (mL).
Express the results to 3 significant figures.
Reference AOAC Official Methods of Analysis (2005), Ch.49.3.07 Method, 2000.08
Approved by Scientific Panel on Methods of Sampling and Analysis
67 | M o M - M y c o t o x i n s
Determination of Aflatoxins B1, B2, G1, And G2 in Foodstuffs other
than described above
68 | M o M - M y c o t o x i n s
Reagents:
Sodium chloride solution (10%)
Toluene–acetonitrile (98:2)
Sample Preparation Sample preparation for Spices
1. Grind or homogenize sample and mix 5.6 g with 100 mL methanol for
3 min.
2. Add 40 mL water, mix for 4 min, leave to stand for 10 min and then
filter.
3. Shake 20 mL of filtrate with 20 mL Sodium chloride solution (10%) and
20 mL petroleum ether for 2 min.
4. Leave to separate for 10 min (extraction of matrix in petroleum ether).
5. Shake aqueous phase with 50 mL dichloromethane for 1 min and leave
to separate (extraction of aflatoxins in dichloromethane).
6. Dry, dichloromethane phase with 5 g sodium sulfate, filter and evaporate
to dryness.
7. Dissolve residue in 0.5 mL toluene–acetonitrile (98:2).
8. Use extract (= 0.8 g sample) for application to the HPTLC layer.
69 | M o M - M y c o t o x i n s
acetonitrile (98:2).
6. Use extract (= 0.8 g sample) for application to the HPTLC layer.
70 | M o M - M y c o t o x i n s
Determination of Deoxynivalenol (DON) in Wheat (Thin Layer
Chromatographic Method)
71 | M o M - M y c o t o x i n s
stoppered volumetric flask, dilute to volume with ethyl acetate –
methanol (19 + 1) and shake to dissolve.
8. Working standard – 20 μg/mL – Pipette 1 mL of DON stock solution
into a 25 mL volumetric flask and dilute to volume with ethyl acetate–
methanol (19 +1) and shake to dissolve.
Sample Preparation Grind large sample (2 – 4 Kg) to pass through a 20-mesh sieve.
Extraction:
1. Weigh 50 g of ground sample into a 500 mL glass stoppered conical
flask.
2. Add 200 mL acetonitrile- water (84 + 16) mixture, secure stopper with
tape and shake vigorously for 30 min on shaker.
3. Filter and collect 20 mL filtrate in a 250 mL graduated cylinder.
Method of Analysis Column chromatography:
1. Secure a chromatographic column to a 125 mL filter flask.
2. Plug the column with glass wool
3. Add about 0.1 g Celite.
4. Weigh 0.7 g charcoal, 0.5 g alumina, and 0.3 g Celite. Add to a 50 mL
beaker and mix with a spatula.
5. Add mixture to chromatographic column. Tap lightly to settle packing.
6. Apply suction and place a ball of glass wool on top.
7. Add 20 mL of the extract (filtrate) to column and apply vacuum.
8. Flow rate should be 2-3 mL/min with 20 cm Hg vacuum.
9. As solution reaches top of packed bed rinse measuring cylinder with 10
mL acetonitrile – water (84 + 16)
10. Add rinse to column and continue aspiration till flow stops.
11. Do not let column go dry between addition of extract and rinse.
12. Cover vacuum nipple with Aluminium foil and evaporate solvent
slowly to dryness on steam bath. Do not contaminate sample with water
from condensing steam.
13. It is essential that no water droplets remain in flask on cooling.
14. Add 3 mL of ethyl acetate to residue and heat to boiling on steam bath
and gently swirl to dissolve extracted DON.
15. Transfer solution to small vial, rinse with three 1.5 mL portions of ethyl
acetate.
16. Evaporate to dryness and retain dry residue for TLC/HPTLC.
17. Final extract, represents 5 g of sample.
Thin Layer Chromatography:
1. Dissolve above residue in vial in 100 μL chloroform – acetonitrile (4 +
1).
2. Apply 5 and 10 μL aliquots side by side 1, 2, 5. 10 and 20 μL working
standard solution (20 μg DON/mL) on TLC plate.
3. Develop plate with chloroform – acetone – Isopropanol (8 + 1 + 1) in
an unequilibrated tank (development time is about 1 h).
4. Remove plate, let solvent evaporate completely at room temperature.
72 | M o M - M y c o t o x i n s
5. Residual solvent can result in fading of DON spots.
6. Heat plate for 7 min in upright position at 120 °C.
7. Place plate on cool surface in dark for 1 min.
8. DON appears as a blue florescent spot under long wave UV light at R f
about 0.6.
9. Quantify DON by comparing fluorescence intensity of test spots with
those of standard DON using a densitometer
Calculation with units of
expression
Where,
S = μL working standard equal to test spot
C = concentration of standard solution (20 μg/mL)
X = μL test solution that has florescence intensity equal to standard
spot
V = Final volume of test solution (μL)
W = amount of test portion represented by final test solution
Reference AOAC 17th edn, 2000 Official Method 986,17 Deoxynivalenol in Wheat–
Thin Layer Chromatographic Method
Approved by Scientific Panel on Methods of Sampling and Analysis
73 | M o M - M y c o t o x i n s
Determination of Ochratoxin (OTA) in Barley
Thin Layer Chromatographic Method
74 | M o M - M y c o t o x i n s
2. Silica gel for thin-layer chromatography: Test adsorbent for resolution
and fading of ochratoxins. Ochratoxins on occasion fade rapidly on
some silica gel plates, especially when exposed to ≥60% humidity.
Protect plate from humidity during spotting by placing in chamber
under nitrogen or under stream of warm air from hair dryer, or by
covering with clean glass plate. After development, dry plate at 50 °C
for 15 min and immediately cover with clean glass plate, using tape on
sides as spacers, for protection during scanning densitometry.
3. Methanolic sodium bicarbonate solution: Dissolve 0.3 g sodium
bicarbonate in 30 mL water and add 70 mL methanol.
4. Alcoholic sodium bicarbonate solution: Dissolve 6.0 g sodium
bicarbonate solution in 100 mL water and add 20mL alcohol.
5. Formic acid–benzene–hexane solution: Shake 100 mL benzene–hexane
(20:80) with 10 mL of water–methanol (30 + 70), let layers separate.
Discard lower layer. Shake upper layer with 5 mL formic acid, let
separate, and discard lower layer.
6. Boron trifluoride 14% (w/v): Bubble gaseous BF3 into chilled alcohol.
7. Developing solvents: (1) Benzene–methanol–acetic acid (18:1:1).
Combine 2 volumes methanol–CH3COOH (1 + I) with 18 volumes
benzene. Adjust benzene (methanol–CH3COOH) ratio, if necessary, to
produce required resolution. Decrease benzene to increase Rf.
8. Hexane–acetone–acetic acid (18: 2: 1): Combine 3 volumes acetone–
CH3COOH (2 + 1) with 18 volumes hexane. Adjust hexane: (acetone–
CH3COOH) ratio, if necessary, to produce required resolution Decrease
hexane to increase Rf.
Purified cotton: Wash 50 g absorbent cotton in beaker with 1 L of
chloroform. Decant solution, evaporate residual solvent, and store cotton
in closed container.
Preparation of OTA Ochratoxin standard solutions
standard Prepare original solutions, each ca 40 μg/mL, in acetic acid-benzene (1:99)
Determine concentration using the table of Molecular weights and molar
absorptivity of ochratoxins given below. Dilute to required concentration
(1-5 μg/mL) using benzene.
Ochratoxin λ max Molecular Molar absorption
(nm) Weight coefficient
A 333 403 5550
B 320 369 6000
A Ethyl ester 333 431 6200
B ethyl ester 320 397 6500
Sample Preparation Extraction:
1. Weigh 50 g of sample into a 500 mL glass-stoppered Erlenmeyer flask
2. Add 25 mL 0.1M Phosphoric acid and 250 mL Chloroform, and secure
stopper with masking tape.
3. Shake for 30 min using a wrist-action shaker
75 | M o M - M y c o t o x i n s
4. Filter through glass fiber paper, covered with ca 10 g diatomaceous
earth, using a 9 cm Büchner funnel.
Separation of Ochratoxins
Ia Removal of esters:
1. Plug a 700 x 17 mm chromatographic tube with the purified cotton.
2. Mix 2.0 g diatomaceous earth with 1mL 1.25% sodium bicarbonate
solution in 50 mL beaker.
3. Add to chromatographic tube and tap firmly.
4. Mix 50 mL filtrate with 40 mL hexane, and add to column. Reserve
remainder of filtrate for confirmation of identity.
5. Elute at maximum flow rate; then elute with 75 mL chloroform.
6. Combine eluates, evaporate to dryness on steam bath, and reserve for
ochratoxin ester separation.
Ib Removal of acids
1. Elute Ochratoxins A and B with 75 mL freshly prepared formic acid-
chloroform (1 + 99), and collect in 250 mL Erlenmeyer.
2. Immediately add 2 boiling chips and evaporate nearly to dryness on
steam bath
3. Quantitatively transfer residue to 15 mL conical centrifuge tube with
chloroform.
4. Evaporate to dryness under gentle stream of nitrogen on steam bath.
(Note: Delay in evaporation of solvent may result in loss of ochratoxins.)
Reserve residue for TLC.
Separation of Ochratoxin Esters:
1. Prepare column as described above using 350 x 25 mm
chromatographic tube with 2.5 mL methanolic sodium bicarbonate
solution, and 4 g diatomaceous earth.
2. Dissolve residue of Ia (after removal of esters) in 50 mL hexane. Add
to column.
3. Rinse extraction vessel with each subsequent eluting solvent in turn and
add rinses to column.
4. Force eluting solvents through column at convenient rate with
compressed gas at 1-2 psi (6.9-13.8 kPa).
5. Do not let liquid fall below top of column.
6. Eluting solvents
a) Elute with 50 mL benzene-hexane (1 + 9) previously equilibrated
with 2.5 mL methanolic sodium bi carbonate solution (discard).
b) Then elute with 100 mL formic acid-benzene-hexane mixture.
7. Immediately evaporate eluate to dryness, quantitatively transfer to
conical centrifuge tube with chloroform, evaporate to dryness under
gentle stream of nitrogen on steam bath, and reserve for TLC
Method of Analysis Thin Layer Chromatography:
1. Use appropriate silica gel and dissolve residue of Ib (from removal of
acids) in 750 μL acetic acid-benzene (1 + 99).
76 | M o M - M y c o t o x i n s
2. Spot 3, 5, 7.5, and 10 μL on same plate.
3. Spot 10 μLextract superimposed with 10 ng each ochratoxin A and B
standard solutions as internal standard.
4. Also spot 5, 7.5, and 10 μL, ochratoxin A and B standard solutions.
5. Develop plate to solvent stop line, but <90 min, with benzene-
methanol-acetic acid (18 + 1 + 1) in an unlined, unequilibrated tank.
6. Remove plate, let solvent evaporate at room temperature, and view in
dark under long- and shortwave UV lamp.
7. The Rf of Ochratoxins A and B should be in the range of 0.4-0.8.
Ochratoxin A is above B, typically at 0.65 and 0.5, respectively.
8. Ochratoxin A fluoresces most intensely under long wave UV, while
ochratoxin B is brightest under shortwave light.
9. Examine the pattern of test solution for fluorescent spots for spots with
Rf close to those of standards and with similar appearance.
10. Compare fluorescence intensities of test solution spots with those of
standard spots, and determine standard and test spot that match most
closely, interpolating, if necessary.
11. If, concentration of test spots is outside range of standards, concentrate
or dilute, solution and re-chromatograph.
12. Calculate concentration ochratoxin A in μg/kg.
13. Spray plate with alcoholic sodium bicarbonate and dry at room
temperature. View spots in dark under long wavelength light.
Fluorescence should have changed from greenish blue to blue and
increased in intensity.
14. In case of disagreement, use estimate obtained before spraying.
Densitometry:
Prepare and develop as described for visual analysis.
In separate channels spot about 4-5 spots with increasing amounts standard
ochratoxin A in range 3-10 ng/spot.
Scan the plate in a densitometer or scanner following the manufacturer‘s
instructions.
Optimum spectral settings for ochratoxin A are excitation at 310-340 nm
and emission, 440-475 nm.
Plot standard curve from instrument response for linearity and system
performance.
Dissolve residue from I(b) in 0.5 mL acetic acid-benzene and spot
replicates of at least two test extracts and standard of ≥ 3μL each. The test
77 | M o M - M y c o t o x i n s
extract must have ochratoxin within the standard concentration range.
78 | M o M - M y c o t o x i n s
Ochratoxin A in Barley
Immunoaffinity by Column HPLC and fluorescence detection
79 | M o M - M y c o t o x i n s
Materials and Reagents 1. Acetonitrile (CH3CN): 99.9%, LC grade
2. Methanol 99.9%, LC grade,
3. Glacial Acetic acid,
4. HPLC grade water (18.2 MΏ cm)
5. Sodium chloride.
6. Disodium hydrogen orthophosphate (Na2HPO4,).
7. Potassium dihydrogen phosphate (KH2PO4)
8. Potassium chloride.
9. Sodium hydroxide.
10. Toluene.
Reference standards: OTA with purity of >98%.
Preparation of Reagents 1. Extraction solvent (v/v): Mix 6 parts acetonitrile with 4 parts water
2. Injection solvent (v/v): Mix 30 parts methanol with 70 parts water and 1-part
glacial acetic acid
3. Sodium hydroxide, 0.2M; Dissolve 8 g NaOH in 1 L water.
4. Phosphate buffered saline (PBS): Dissolve 8 g NaCl, 1.16 g Na2HPO4, 0.2 g
KH2PO4 and 0.2 g KCl (j) in1 L water. Adjust pH to 7.4 with 0.2M NaOH.
5. HPLC mobile phase (Acetonitrile containing 1% acetic acid): Mix (v/v) 102
parts water with 96 parts CH3CN and 2 parts glacial CH3COOH; filter
through0.22 µm filter Band degas.
6. Toluene–glacial acetic acid mixture (v/v): Mix9 parts toluene with 1-part
CH3COOH.
7. Silanizing reagent (v/v): Surface siliconizing fluid, 5% (v + v) solution (such
as SurfaSilÔfrom Pierce ChemicalCo., PO Box 117, Rockford, IL 61105,
USA, No. 42800). Mix1partSurfaSil with 19 parts toluene.
80 | M o M - M y c o t o x i n s
Preparation of Stock standard solution (10 µg/mL): Prepare OTA standard in tolueneacetic
standards acid, (99 + 1,v/v).
Determine concentration of stock as follows:
Record UV spectrumof ochratoxin A solution against Toluene -acetic acid
solution inreference cell.
Determine concentration of ochratoxin A solutionby measuring A at wavelength
of maximum absorptionclose to 333 nm and using following equation:
where A = absorbance,
MW = molecular weight ofochratoxin A (403),
= molar absorptivity (5440 in toluene–acetic acid, 99 + 1,v/v).
Preparation of working standards
Pipet 200 µL 10 µg/mLOTA stock standard into glass vial and dilute to 1
mLwith 800 µL toluene–acetic acid to give 2 µg/mL OTAsolution. Pipet 100 µL
of 2 µg/mL OTA solution into silanizedglass vial Evaporate solvent under
stream of nitrogen.
81 | M o M - M y c o t o x i n s
Immuno-affinity cleanup:
1. Pipet 4 mL filtrate into 100 mL glass beaker (or similar) and dilute with 44
mL PBS.
2. Connect immunoaffinity column) to vacuum manifold and attach reservoir to
immunoaffinity column.
3. Add diluted extract to reservoir and pass through immunoaffinity column at
0.5 mL/min flow rate.
4. The immunoaffinity column must not be allowed to run dry.
5. Wash beaker and column with 10 mL water,
6. Remove from vacuum manifold, and place over silanized vial.
7. Elute OTA into silanized vial with four 1 mL portions methanol.
8. Evaporate eluate to dryness over steam bath, under N.
9. Re-dissolve in 1 mL injection solvent which has been filtered through 0.2 mm
filter.
10. Transfer to LC vial.
Method of Analysis Chromatography conditions:
1. C18 reversed-phase, 5 mm ODS(equivalent to ODS 1 or 2)
2. Column temperature: 45 ± 1 °C,
3. Isocratic elution, Flow rate:1 mL/min
4. Injection volume: 100 μL.
5. Mobile phases: Acetonitrile containing 1% acetic acid
6. Detector:
A. Excitation wavelength 333 nm.
B. Emission wavelength: 460 nm
Connect the HPLC column, set column temperature and detector wavelength
Wash column thoroughly with mobile phase at a flow rate of 1 mL /min. Wash
for 30 mins.
Inject 100 μL of each calibrant solution to construct a calibration curve.
Inject 100 μL sample in triplicate.
Calculation with units of Determine, from calibration graph, masses in ng of OTA in aliquot of test
expression solution injected ontothe LC column.
The regression should be > 0.998
From the equations determine the concentration of the unknown (MA).
Calculate mass fraction,WOTA, of OTA in mg/kg using theequation:
Where
MA = mass of OTA in test solution extract, ng;
V1 = extractionsolvent, mL (100 mL);
V2 = acetonitrile–water filtratepassed through immunoaffinity column, mL(4
mL)
82 | M o M - M y c o t o x i n s
V3 = testsolution (1 mL);
V4 = test solution injected, mL
MS = Mass of test portion
LOD and LOQ The LODs are 0.02 ng/g (S/N > 3) and 0.1 ng/g (S/N > 10) for AFs and OTA,
respectively
Reference J. AOAC Int. 83, 1379–1383 (2000)
AOAC Official Method 2000.03Ochratoxin A in BarleyImmunoaffinity by
Column HPLCFirst Action 2000
Approved by Scientific Panel on Methods of Sampling and Analysis
83 | M o M - M y c o t o x i n s
Direct analysis of Aflatoxins (AF) and Ochratoxin A (OTA) in cereals and
their processed products by Ultra-High-Performance Liquid
Chromatography with fluorescence detection
84 | M o M - M y c o t o x i n s
Calibration standards: Make serial dilutions of the intermediate solutions to
obtain 0.02–10 ng/mL for each AF and 0.1–10 ng/mL for OTA in 1:1 ratio of
methanol: water (plus 0.2% acetic acid, v/v).
Sample Preparation Grinding:
Cereal grains and processed products (are thoroughly milled and allowed to pass
through a No. 20 sieve.
Extraction:
Add 12.5 g of finely ground dry matrix to 12.5 g distilled water to make a slurry.
Mix the slurry with 100 mL of extraction solvent (methanol–water,8+ 2, v/v) and
NaCl (5 g). Shake for 30 min,200 rpm), and then centrifuge (5000 rpm, 5 min).
Take an aliquot (3 mL) and dilute with 15 mL PBS and add 50 μL NaOH (2 M)
solution.
IAC cleanup:
Load the diluted sample onto IAC connected to a vacuum manifold and allow to
pass without any vacuum. Wash with 10 mL PBS. Elute with methanol (2 × 0.5
mL). Slowly evaporate the final extract (1 mL) to dryness. Reconstituted in 0.5
mL methanol–water (acidified with 0.2% acetic acid, 1:1), and finally inject 10
µL into the UHPLC-FLD instrument.
Method of Analysis Chromatography conditions:
1. Column: C18 column (2.1 × 50 mm, 1.7 μm).
2. Column temperature: 40 °C,
3. Flow rate: 0.2 mL/min
4. Injection volume: 10 μL.
5. The mobile phases: (A) 1% acetic acid in water and (B) 1% acetic acid in
methanol.
6. Detector:
7. Excitation wavelength 365 nm up to 8 min and subsequently switched to 333
nm and continued up to 15 min.
8. Emission wavelength: 456 nm t
9. Linear Gradient program
Time %A %B
(mins)
Initial 90 10
0.25 90 10
2.50 58 42
6.00 58 42
7.00 20 80
11.50 20 80
12.00 90 10
15.00 90 10
85 | M o M - M y c o t o x i n s
Results
86 | M o M - M y c o t o x i n s
Determination of Patulin in Apple and Apple Juice
87 | M o M - M y c o t o x i n s
Sample Preparation 1. Take 10 g of homogenized sample of apple or 10 mL juice in a 50 mL
polypropylene centrifuge tube.
2. Extract with 10 mL ethyl acetate in presence of 10 g anhydrous sodium
sulfate.
3. Vortex for 2 min and centrifuge at 2795 ×g for 5 min.
4. Take 1 mL of the supernatant in a 2 mL Eppendorf tube and add 25 mg
of PSA.
5. Centrifuge the Eppendorf tube at 5040 ×g for 5 min.
6. Evaporate the final extract under a gentle stream of nitrogen to dryness
and reconstitute in 1 mL of methanol:water (2:8).
7. Inject 20 µL of the extract into LC-MS/MS.
Method of analysis LC conditions (for reference only*):
a. Column: e.g., Bridged ethylene hybrid C18 column (2.1 × 100 mm,1.7
μm) or equivalent
b. Mobile Phase
A: Water with 0.1% acetic acid
B: Acetonitrile with 0.1% acetic acid
c. Column oven: 35 °C
d. Flow rate: 0.3 mL/min
e. Injection volume: 20 µL
88 | M o M - M y c o t o x i n s
Inference The method provides sensitive analysis of patulin in apple and apple juice (LOQ-
(Qualitative Analysis) 0.005 mg/kg). The detection is confirmed based on the detection of two selective
reaction monitoring transitions and their ion ratio within a deviation of ±30%.
Reference Shinde, R., Dhanshetty, M., Lakade, A., Elliott, C.T. and Banerjee, K., 2021.
Development and validation of a liquid chromatographic tandem mass
spectrometric method for the analysis of patulin in apple and apple juice.
Mycotoxin Research, https://doi.org/10.1007/s12550-021-00422-2
Approved by Scientific Panel on Methods of Sampling and Analysis
89 | M o M - M y c o t o x i n s
RAPID ANALYTICAL FOOD TESTING (RAFT) KIT/ EQUIPMENT
Alternate Rapid kits/equipment may be used to get quick results for screening and surveillance
purposes, provided the kit/equipment is approved by FSSA(I). Details of the rapid food testing kit/
equipment approved by FSSA(I) are available at https://www.fssai.gov.in/cms/raft.php
90 | M o M - M y c o t o x i n s
Food Safety and Standards Authority of India
(Ministry of Health and Family Welfare)
FDA Bhawan, Kotla Road,
New Delhi-110002
www.fssai.gov.in
For any query contact: [email protected]