General Considerations

Choice of Analytical Method. The choice of analytical method used to separate, detect, and quantify mycotoxins depends on the physiochemical properties of the mycotoxin, concentration of the mycotoxin in the food or feed, cost and time constraints, and availability of analytical methodology and instrumentation. Early studies used biological methods, for example, animal toxicity assays, to detect the presence of mycotoxins in food and feed. With the development of sophisticated instrumentation, chemical methods became the method of choice for mycotoxin analysis. For mycotoxins that contain a chromophore having high molar absorptivity and/or high fluorescent properties, such as aflatoxin, zearalenone, patulin and ochratoxin, sample extracts are usually subjected to either thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC). For both methods, the compound of interest is identified by comparing the Rf (a measure of migration distance) or retention time to that of an analytically pure standard of the compound. Quantitation is accomplished by determining the fluorescence intensity or UV absorbance of an elution spot or an HPLC peak. Mycotoxins that do not contain a distinctive chromophore, for example, fumonisin and some of the trichothecenes, can be derivatized and analyzed by HPLC or gas chromatography (GC). Alternatively, these compounds can be analyzed directly by gas chromatography-mass spectrometry (GC/MS) or liquid chromatography-mass spectrometry (LC/MS).

Analytical Standards. Essential to mycotoxin analysis is obtaining pure analytical standards for the compound of interest. While standards for the most common mycotoxins are commercially available, newly discovered or uncommon toxins are often available only from the researchers who originally purified and identified the compounds. Analytical criteria of important mycotoxins were published by the Food Chemistry Commission of the International Union of Pure and Applied Chemistry (63). In addition, several extensive tabulations of chemical, physical and spectra characteristics of mycotoxins have been published by Cole and Cox (64) and Savard and Blackwell (65). Purity of primary standards should be determined by at least two analytical methods. If possible, concentration of standards in solution should be determined spectrophotometrically rather than gravimetrically. Some mycotoxins can be chemically labile, consequently, stability of the pure standard in solvent should be determined. Proper storage of the standard is essential, and periodic check of stability during storage should be determined. Since all mycotoxins are potentially hazardous, care should be taken while working with the pure compounds, and all manipulations should be performed in a hood or glove box (5,32).

Confirmation Methods. Interfering compounds can be present in sample extract even after exhaustive cleanup procedures are employed. Consequently, confirmation methods are needed to establish the true identity of the analyte. There are two main techniques for confirmation of identity: chemical derivitization and mass spectrometric analysis. Pre- and postcolumn derivatization of mycotoxins can be used to confirm the identity of mycotoxins analyzed by HPLC. For example, fumonisin previously analyzed by derivitizing with cr-phthaldialdehyde (OPA) can be analyzed using another derivitizing agent, for example, naphthalene dicarboxaldehyde (NDA). Diode array detectors are commonly available HPLC detectors that can be used to confirm the identity of mycotoxins having characteristic UV spectra. Using several detectors in tandem is another excellent confirmatory method (5). Numerous chemical methods for confirming the identity of aflatoxins and other mycotoxins exist with TLC. Various reagents can be sprayed onto developed chromatograms, and the reactions can be compared to those with pure standards (66,67).

The most powerful method for confirming the identity of mycotoxins is by mass spectrometry (MS), and numerous methods exist for the identification of mycotoxins by GC/MS (68-71) or LC/MS (72-77). The mass spectra of compounds eluting from a GC or HPLC column can be used to aid in the structural determination of unknowns or provide unequivocal identification of peaks (6). Selectivity can be increased by using select ion monitoring (SIM) that is unique to the analyte of interest. The affordability of GC/MS and LC/MS systems allows most modern laboratories to be equipped with this powerful analytical tool.

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