Chromatography

Introduction. In the past ten years, chromatography has been the most studied technique for detection, analysis, and characterization of mycotoxins. Chromatographic methods employed for these purposes include TLC, GC, HPLC, and supercritical fluid chromatography (SFC). A book by Betina (16) and reviews by Shephard (78) and Dorner (6) are excellent references on the use of chromatography for mycotoxin analysis.

Thin-Layer Chromatography. Despite the fact that many other techniques have been increasingly applied in the determination of mycotoxins in food and feed, TLC is still one of the most popular because of its relative simplicity and low operating cost. Other advantages are that it allows simultaneous analysis of many extracts, offers great selectivity, and makes multitoxin analysis possible (6). As with other chromatographic techniques, TLC requires extensive sample cleanup to remove interferences. Several excellent reviews have been published on TLC methods used for my-cotoxin screening and quantitation (67,79-83).

The choice of type of TLC plate adsorbent and mobile phase depends on the chemical characteristics of the analyte as well the matrix from which it was extracted. Adsorbents used for normal-phase TLC include silica gel, aluminum oxide, and cellulose, whereas reverse-phase TLC plates have a layer of silica gel bonded to various nonpolar functional groups including ethyl, octyl, octadecyl, and phenyl. The majority of TLC plates used for mycotoxin analyses are glass or plastic supports coated with silica gel. Other substances, such as oxalic acid, sulfuric acid, or glycolic acid, can be incorporated into the silica gel layer to aid in the separation of acidic mycotoxins (citrinin, cyclopiazonic acid) (8). TLC plates can be prepared in the laboratory, but most frequently, they are purchased as pre-coated plates since they are more uniform and reproducible.

Sample extracts are applied to the TLC plate with a micropipet or syringe, and the plates are then placed in a mobile phase for chromatographic development. During development, components of the extract partition between the stationary and mobile phases. Mobile phases depend on the mycotoxins to be separated, the type of adsorbent, and the presence of compounds in the extracts that interfere with the analyte (6). In normal-phase TLC, the major component of the mobile phase is usually a relatively nonpolar solvent (chloroform, ethyl ether, or ethyl acetate). Polar solvents (acetone, alcohols, water, or organic acids) are usually added to achieve the desired separation of the an-alytes. With reverse-phase TLC, the mobile phase is usually composed of water in combination with methanol or acetonitrile. Development of plates is usually carried out with one mobile phase. However, use of more than one solvent can be used to improve resolving power (6).

When sample extracts contain few interfering substances, one-dimensional TLC is sufficient for separation and quantitation of analytes. Using this method, many sample extracts can be simultaneously analyzed (81). When cleanup is insufficient to remove interferences, the use of two-dimensional TLC (2D-TLC) can improve resolving power. In 2D-TLC, the plate is dried after developing in the first mobile phase, then it is rotated 90° and developed in a different solvent. 2D-TLC has been applied to the analysis of aflatoxins, ochratoxin A, and citrinin (81).

To visualize the mycotoxin spots on TLC plates, the plates are either examined under UV light for fluorescent mycotoxins such as aflatoxins, zearalenone, sterigmato-cystin, penicillic acid, citrinin, and ochratoxin A, or they are sprayed with a chemical reagent that reacts with the mycotoxins to produce a colored or fluorescent derivative (83). Under long-wave UV light (365 nm), aflatoxins appear as blue, while zearalenone, citrinin, sterigmatocystin, and penicillic acid appear as blue-green, yellow, red, and purple spots, respectively (81). Aluminum chloride has been used to make fluorescent derivatives of deoxyniva-lenol (84) and sterigmatocystin (85), while ammonia was used to increase the fluorescence of ochratoxins (86) and patulin (87). Nonfluorescent mycotoxins are detected by chemical derivatization. For example, T-2 toxin can be detected by spraying plates with a mixture of sulfuric acid and methanol, then heating the plate for several minutes at 110°C (81). Mycotoxins are quantified by comparing the intensity of the fluorescence of the sample spot with that of a series of standards. Visual comparison has been widely employed for mycotoxin screening. More accurate quantitation is achieved instrumentally with a densitometer. Detection limits for TLC are in the range of pg/g to ng/g depending on the analyte, source of contamination, and cleanup method (83).

The development of high-performance TLC (HPTLC) in recent years has kept TLC competitive with other more sophisticated techniques. Plates are smaller than conventional TLC plates, and they are uniformly coated with a 0.1- to 0.3-mm layer of small particle size (2-10 //m) adsorbents (82). The small particle size results in rapid separation of analytes. HPTLC uses automatic sample application equipment for spotting small volumes of test sample on plates and a densitometer to quantify analytes. Detection limits for aflatoxins using HPTLC have been reported in the low picogram range (82).

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