Mycotoxin Analysis

A. Sampling

 Collect the samples at the following quantities for ensuring
meaningful representation of the whole lot of feed / feedstuff
Min. sample size
Small particle type (milk, vegetable oils) 500 g
Intermediate particle type (ground meals, flours,
compounded feed)
3 kg
Small grains (wheat, rice, sorghum, ragi, barley etc.) 5 kg
Intermediate grains (maize, cotton seed / cake) 10 kg
Large grains (groundnuts / cake) 20 kg
 Collect at least 100 subsamples from the whole lot. For eg. from a
truck of 100 bags of maize, collect 100 g maize from each bag to
obtain a total sample size of 10 kg
 Get about 50 - 100 g subsample from the whole sample employing
either coning and quartering method (in a series of steps) or using
sample divider
 The subsample thus collected can be directly subjected for analysis

B. Outline of Mycotoxin analysis
Sampling

Toxin extraction
(using organic solvents)
Clean-up
(To remove fat, impurities etc.)
Work up
Identification & Quantification
(TLC, HPLC, ELISA etc.)

C. Different methods of Mycotoxin analysis

C. 1. Thin layer chromatography (TLC)

 Principle

It is the cheapest and most commonly used method. It makes use of
heterogenous equilibrium established during the flow of a solvent
(mobile phase) through a fixed phase (stationary phase) to separate ≥
2 components from materials carried by solvent (differential
migration).

 Spotting the extract

Place between 5 - 20 μl of sample extract / standard as a small circular
spot (< 5 mm), 1 - 2 cm from the end of the TLC plate. Micropipette /
microcaps may be used for the purpose. Leave at least 1 cm gap
between two adjacent spots.

 Developing the plate

Place about 50 - 100 ml of mobile phase (solvent) in a tank and keep
the plate at a slight angle with the spots little above the upper level of
the solvent. Due to capillary action, solvent moves upward on the
plate. Allow the solvent to travel at least about 8-10 cms.

 Detection

Air dry the developed plate and view in a UV cabinet under either
longwave (365 nm) or short wave (254 nm) range to identify the
fluorescing mycotoxins. In case of mycotoxins which do not
fluoresce, spray the plate with suitable reagent to develop
fluorescence.

 Resolving front value (Rf)

Each mycotoxin has its characteristic color of fluorescence under UV
light and a constant Rf value in a particular developing solvent
(Table 3). Rf value is computed using the formula,
Distance travelled by sample spot from the origin
Rf =
Distance travelled by solvent front from the origin

 Confirmation

The presence of mycotoxin can be confirmed either by spraying the
plate with suitable reagents (like 50 % aqueous H2SO4, Triflouro
Acetic Acid etc.) or placing an internal standard right over the top of
the sample spot (superimposing).

 Detection by Scanner

The fluorescence intensity of sample and standard spots can be
measured by using TLC Scanner / fluorodensitometer to avoid
possible human errors in comparison.

Table 3. TLC characteristics of mycotoxins
Toxin Rf * Color Color (UV) after
(UV) spray * *
Aflatoxin B1 0.31 Blue Pink
Aflatoxin B2 0.26 Blue Pink
Aflatoxin G1 0.23 Green Blue
Aflatoxin G2 0.17 Green Blue
Ochratoxin A 0.55 Green Blue
T-2 toxin 0.36 Yellow Blue
Zearalenone 0.78 Blue Yellow
DAS 0.33 Yellow Variable
Sterigmatocystin 0.85 Red-brown Yellow

*TEF : Toluene : ethyl acetate : formic acid ( 6:3:1 )
* *P - anisaldehyde

C. 2. Spectrophotometry

This is an extension of TLC method. The sample spots on the
developed TLC plate are scraped out alongwith the sorbent (silica gel)
and extracted with methanol for 3 minutes. The extract is filtered and
the absorbance of the filtrate is measured in a spectrophotometer
at 363 nm.

Reference :

Nabney and Nesbitt. 1965. Analyst 90 : 155-160.

C. 3. High Performance Thin Layer Chromatography (HPTLC)

This is an improvised version of TLC, where sample
application and detection of fluorescence intensity are fully automated
and carried out by using automated sample applicator (like Linomat
IV of Camag, Switzerland) and densitometer, respectively.
Mycotoxin levels less than 0.1 ppb can be detected by this method.

C. 4. Minicolumn method

A glass column of 20 cm length, 6 mm internal diameter with
tapering end (2 mm) is packed serially from the bottom with glass
wool, calcium or sodium sulphate (8-10 mm), florisil (8-10 mm),
silica gel (18-20 mm), neutral aluminia (8-10 mm), calcium or
sodium sulphate (8-10 mm) and a cap of glass wool.
2 ml of final chloroform extract (in case of aflatoxin) is placed
in the column and eluted with chloroform : acetone (9 : 1). Aflatoxin,
if present is trapped as a band above florisil layer which can be
viewed under long wave UV light as a blue fluorescent band. This
method can be used as a qualitative test for rapid identification of
mycotoxin.

C. 5. Immuno assays

These assays are developed on the basic principle of
Antigen - Antibody reaction. Antibodies are highly specific to the
Mycotoxin - Protein conjugate (Hapten) used. Hence the results will
be highly specific.

Commonly employed immuno assays

 Radio immuno assay (RIA)
Standard mycotoxin, labelled onto a radioactive compound like
Tritium is used. Mycotoxin levels as low as 2-5 ppb can be
detected. The disadvantages of this method include high cost,
difficulty in labelling, radio active waste disposal problem and
risk of handling.

 Enzyme linked immuno sorbent assay (ELISA)
It has received great attention in recent times and has been the
most popular and widely practiced immuno assay method.
ELISA is rapid, more sensitive, highly specific and simple to
operate. It does not require any extensive extraction or cleanup.

Commercial ELISA kits
Various companies have been marketing commercial kits
which basically work on ELISA principle. These have gained
wider acceptance as considerable amount of time is saved on
antibody production. Sample is extracted with methanol : water
(60 : 40) or acetonitrile : water (50 :50) and the extract is
directly subjected to analysis.
Elisa tests are good for quick identification of
mycotoxins in feed samples, various tests are developed based
on Antigen - Antibody principle. Some companies which
produce ELISA kits are :
1. Neogen Corp,
620, Lesher place,
Lansing, Michigan 48912, U.S.A.
2. Vicam,
313, Pleasant St.,
Watertown, Massachusetts - 02172, U.S.A.

C. 6. High performance liquid Chromatography (HPLC)

It is highly sensitive and can detect upto 5 x 10-6 ppb level of
mycotoxin. Stainless steel columns (< 18) of 15 cm length and 4 mm
internal diameter, packed with silica gel (particle size - 5 microns) are
used. Sample is first extracted with suitable solvent (generally 60 %
aqeous methanol) and the extract is cleaned - up.
This purified extract (20 μl) is injected into the column and the
eluent (generally a mixture of methanol, water and acetonitrile) is
passed at a flow rate of 0.75 ml / min and at a pressure of 3000 psi.
The eluted toxins coming out of the column are detected and
quantified by fluorimeter.
The columns may be either normal phase (polar stationary
phase) or reverse phase (polar mobile phase) type. The latter type is
most commonly used.

C. 7. Bio - assays

Mostly are useful as confirmatory tests. Toxin extract is
injected as a single dose into stomach (day-old duckling bioassay,
guinea pig bioassay), fertile eggs (chick embryo bioassay) or into skin
of rabbits (skin bioassay). Presence of toxin is confirmed by noticing
pathological changes or mortality.
Safety precautions in mycotoxin analysis
 Carryout the mycotoxin analysis in a separate work area in the
laboratory
 Cover the bench top with non absorbent material
 Solvents used are highly inflammable. So avoid using electric stoves,
bunsen burners etc.
 Do not stock the solvents in larger quantities
 Wear protective clothing, gloves and mask to minimise the risk of
inhalation / contact with hazardous mycotoxins
 Some of the solvents (like benzene, chloroform) are toxic. Avoid
direct skin contact with them
 Any spillage should be immediately mopped-up with cotton. Such
cotton should be incinerated
 After completing the work, decontaminate the area with 4 % sodium
hypochlorite solution
 Decontaminate the glassware by soaking for atleast 2 hours in 1 %
sodium hypochlorite solution
 Spray the TLC plate with reagent only in a fume cup-board / spray
cabinet
 At the UV cabinet, always view the TLC plate only through the UV
filter
 Avoid eating, drinking and smoking in the laboratory
 Keep the lab well ventilated using exhaust fans

MYCOTOXIN QUANTIFICATION

1. Mycotoxin standards

Pure mycotoxins of known concentration are used in mycotoxin

assays for either fluorescence intensity comparison or calibration.

Reconstitution of Mycotoxin standards

Mycotoxin standards are often supplied in crystalline form in sealed

glass vials. They need to be suitably dissolved in appropriate solvents for

preparation of stock and working solutions of desired final concentration.

Procedure

 Carefully remove the metallic seal from the central injecting area of

the cap of the vial and inject about 1 ml of appropriate solvent into the

vial

 Shake the vial gently to dissolve the mycotoxin in the solvent

 Recover the dissolved mycotoxin using the same syringe and transfer

into a volumetric flask

 Inject again 1 ml of solvent into the vial, shake gently, recover and

transfer to volumetric flask. Repeat 5-6 times for complete recovery

of Mycotoxin

 Make up the volume of the dissolved toxin to get the desired

concentration in the stock solution

 Tightly stopper the volumetric flask and store in refrigerator in an

opaque container at 4°C

 For preparation of working standards and those used for

spectrophotometric purity evaluation, dilute the stock standard using

the suitable solvent to get the desired mycotoxin concentration

Table 1. Concentrations of the Mycotoxin standards

required (μg/ml)

Stock UV TLC Solvent

Aflatoxins 100 10 1 Benzene : Acetonitrile (98 : 2)

Ochratoxin A 25 25 2 Benzene : Acetic acid (99 : 1)

T-2 toxin 5000 100 50 Ethyl acetate

Zearalenone 100 10 50 Benzene

Citrinin 40 20 40 Chloroform

DON 500 20 20 Ethyl acetate : Methanol (19 : 1)

Sterigmatocystin 100 100 100 Benzene

Checking the purity / concentration

 The standards thus prepared are required to be checked periodically

for assessing any possible alteration in their concentration during

storage

 Prepare 0.4 mM potassium dichromate solution by dissolving 125 mg

potassium dichromate in 1 litre 0.018 N Sulphuric acid

(1 ml H2SO4 in 2 litre distilled water)

 Prepare 0.2 mM and 0.1 mM solutions of potassium dichromate by

making two successive dilutions of 0.4 mM solution with

0.018 N Sulphuric acid

 Read absorbance of these 3 solutions at 350 nm using 0.018 N H2SO4

as blank

Abs x 1000

 Calculate (E) of each solution =

mM

 Calculate the average of the three solutions (0.4, 0.2 and 0.1 mM)

 Calculate the correction factor (CF) for the instrument

3,160

CF = (normal value : 0.95 - 1.05)

E

 Read the absorbance of the mycotoxin standard at wave length of

maximum absorbance

Abs x Mol wt x CF x 1000

Concn. (μg/ml) =

E

Molecular weight, wave length of maximum absorbance and

absorptivity of some Mycotoxins

Mol. wt. Max. abs. (nm) Absorptivity (E)

Aflatoxin B1 312 353 19,800

Ochratoxin A 403 333 5,550

Zearalenone 318 316 6,020

Citrinin 259 322 16,100

Sterigmatocystin 324 325 15,200