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