4 Preparation of Mycotoxin Standards

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The detection and quantitation of mycotoxins requires pure standards or standards for which the purity and identity are known. Methods for identifying and calibrating standards are necessary. Few commercial sources exist for the mycotoxins discussed in this volume, and for most mycotoxins, specifications of purity are not available. If primary standards are not available commercially, other sources may be investigators who have isolated standard materials which they may be willing to share. As a last resort, it may become necessary to isolate mycotoxins from appropriate fungal cultures. Calibration and purity tests have been developed based on physical and chemical properties such as melting points, visible/ultraviolet, nuclear magnetic, and infrared spectros-copy and mass spectrometry, and various methods of chromatography. Standards are quite expensive, and regardless of the source, the purity and authenticity can be variable. Analysts are responsible for calibrating the standards used in analysis. Procedures have been developed for this purpose (1 ). The present protocol outlines the preparation, calibration, purity determination, preparation of solutions, distribution, storage, and uses of quantitative aflatoxin standards, and is intended as a guide for application to other mycotoxins which are used in the protocols in this volume. The amounts needed for most methods of analysis and for fortifying various matrixes for use as laboratory test or control samples, are in the nanogram or microgram ranges. The protocols given are for the aflatoxins B1, B₂ , G₁ , and G₂ . Mycotoxins standards other than the aflatoxins may be prepared in the same way using the information provided in Table 1 . For some mycotoxins, such as the fumonisins and deoxynivalenol, UV spectroscopy cannot be used due to the lack of a suitable chromophore in the molecule. For these molecules, use gravimetric methods combined with gas chromatography, liquid chromatography, and/or mass spectrometry.

Table 1 Calibration of Mycotoxins by UV Spectroscopy

Mycotoxin	Solvent	Conc. μg/mL	Molecular Weight	Molar Absorptivity e	Wavelength,^a nm	Reference
Aflatoxin B_:	Toluene-acetonitrile (9 + 1)	10	312	19,300	350	3
Aflatoxin B₂	Toluene-acetonitrile (9 + 1)	10	314	21,000	350	3
Aflatoxin Gj	Toluene-acetonitrile (9 + 1)	10	328	16,400	350	3
Aflatoxin G₂	Toluene-acetonitrile (9 + 1)	10	330	18,300	350	3
Aflatoxin M ₁	Acetonitrile	10	328	18,900	350	1
Aflatoxin M₂	Acetonitrile	10	330	21,400	350	1
Citrinin	Chloroform	10	250	16,100	332	2
Cyclopiazonic Acid	Methanol	10	336	20,417	284	2
Deoxynivalenol	Ethanol	50	296	4,500	218	2
Moniliformin	Water	50	98	5,600	260	2
Ochratoxin A	Benzene-acetic acid (9 + 1)	40	403	5,600	333	1
Patulin	Ethanol	10	154	14,600	275	1
Sterigmatocystin	Benzene	10	324	15,200	325	1
Zearalenone	Methanol	50	318	6,000	314	1

^a The wavelength is variable depending on the solvent. The absorbance, A, is themaximum as measured