High Performance Liquid Chromatography One of the

first publications describing the use of HPLC to analyze mycotoxins in food was written by Seiber and Hseih (88). Since then HPLC has become the method of choice for quantifying mycotoxins in food and feed. HPLC is similar to TLC in principle, but it offers the analyst greater resolution, sensitivity, accuracy, and precision (25). HPLC allows ultratrace analysis of mycotoxins; some analytes can be determined at the nanogram or even picogram level. Other advantages of HPLC are its ability to analyze thermally labile, poorly volatile, polar, and ionic compounds. The introduction of autosamplers, computerized data retrieval systems and a variety of sensitive detectors make HPLC very useful for large-scale analyses. Important limitations of HPLC include the high cost of instrumentation and the lack of a sensitive universal detector. Like most of the other chromatographic methods, rigorous cleanup is usually required to achieve accurate, reproducible results. Reviews on the use of HPLC to analyze mycotoxins have been written by Shephard (78), Coker and Jones (89), Fris-vad and Thrane (90), Shepherd et al. (91) and Kuronen (92).

The versatility of HPLC makes it ideal to analyze chemically diverse compounds such as the mycotoxins. This is mainly due to the advances made in the past decade in the chemistry of adsorption materials for column packings. Although use of normal-phase silica columns was initially favored for analysis of some of the mycotoxins, reversephase chromatography is currently being used most often for aflatoxins (93-97), zearalenone (41,98,99), nivalenol and deoxynivalenol (100), citrinin (101), ochratoxin (102), fumonisins (52,53), Alternaria toxins (103-105), patulin (106,107), and sterigmatocystins (108). There have been some reported use of ion-exchange chromatography (IEC) in the determination of moniliformin (109).

Several detectors are available for use with HPLC, which provides for great selectivity and sensitivity. Unlike GC, there is no truly effective universal detector for HPLC. Refractive index (RI) detectors lack sensitivity, which limits their use in mycotoxin analysis. An evaporative light-scattering detector (ELSD) is another universal detector that can be used for the detection of low-volatility components in purified extracts. As with RI detectors, ELSDs lack the sensitivity needed for detection of most mycotoxins. A literature search found only one published method for fumonisins (110).

As most mycotoxins absorb UV light, UV detectors are commonly used detectors for mycotoxin analysis. The photodiode array (PDA) detector has proven to be a powerful HPLC detector for detecting and quantifying mycotoxins. The advantage of PDA detectors is that they provide both multiwavelength and spectra information in a single chromatographic run (42). In combination with computer searching capabilities, it is possible to monitor the spectra of eluting components, allowing further identification of mycotoxins.

The fluorescence detector is commonly used to detect mycotoxins that fluoresce or fluoresce when derivitized. Fumonisins, a recently discovered group of mycotoxins, contain an amino group and several carboxylic acid groups that can be derivitized with fluorogenic reagents such as (T-phthaldialdehyde (OPA) (52), naphthalene dicarboxal-dehyde (111), or iV-hydroxysuccinimidylcarbamate (112). The trichothecenes and sterigmatocystin are other examples of mycotoxins that are derivatized with fluorescent compounds (113,114). Fluorescence detectors, which are highly selective and more sensitive than UV detectors, are the detectors of choice for aflatoxin and fumonisin analyses. In addition to UV and fluorometry, electrochemical detectors have been used for the detection of several mycotoxins (115-117).

Development of LC/MS techniques has been the subject of considerable study since its development in the late 1960s (118). Since that initial work LC/MS interfaces have undergone considerable development and improvement. First (direct liquid introduction, moving belt, particle beam) and second generation (thermospray, electrospray) interfaces have led the way to the generation of so-called atmospheric-pressure ionization (API) interfaces. The API techniques that comprise chemical ionization and other ionization techniques (electrospray and ionspray), have opened a new window in LC/MS, allowing analysis of either slightly polar analytes of low to medium molecular mass or more polar analytes and ions in solutions of medium to high molecular mass (75). Instruments have become smaller (benchtop size), less expensive, more rugged, and compatible with commonly used HPLC mobile phases and flow rates (75). Reports exist on the use of LC/MS for determination of such mycotoxins as aflatoxins (72), trichothecenes (73,74), zearalenone (75), ochratoxin (76), and fumonisins (77,119).

Gas Chromatography. GC is the method of choice for some mycotoxins that exhibit little or no UV absorption or fluorescence. GC methods have been published for virtually all mycotoxins, although their greatest utility lies in the analysis of the trichothecenes (6,119). With GC, extracts containing the analytes are first subjected to vigorous cleanup procedures. The next phase involves volatilizing the analytes. Most mycotoxins are not volatile at GC temperatures (30-350°C), and must be derivatized to a volatile form. The functional group in mycotoxins allowing derivatization is in most cases a hydroxyl group, and the derivatives formed are usually trimethylsilyl (TMS) ethers or heptafluorobutyryl (HFB), pentafluoropropionyl (PFP), or trifluoroacetyl (TFA) esters (120). Onji et al. (121) have described a method for analyzing several Fusarium mycotoxins including deoxynivalenol, 3-acetyldeoxynivalenol, fusarenon-X, diacetoxyscirpenol, 15-monoacetylscirpenol, T-2 toxin, scirpentriol and zearalenone without derivatization.

Packed and capillary GC columns are used for the separation of analytes. Packed columns are usually borosili-cate glass tubes that are packed with an inert support that is coated with the polymer to be used as the stationary phase. With capillary columns, fused silica tubing is coated with thin films of stationary phase. Capillary columns offer great efficiency, which explains why the majority of methods published for the determination of trichothecenes used capillary columns (6).

Several types of detectors are available for GC, but for mycotoxin analysis, the flame ionization detector (FID), the electron capture detector (ECD), and the mass spectrometer (MS) are the most common. The FID detector is considered the universal GC detector because it detects all organic compounds. Although rugged and sensitive, FID detectors are not at all selective and require rigorous cleanup of extracts to obtain reliable results (6). The ECD is a more selective detector than the FID, but it is not necessarily more sensitive. ECD gives strong signals for halogenated compounds but not for hydrocarbons (6). MS is the ultimate detector for use with GC; it is sensitive, selective, and enables confirmation of the identity of elut-ing compounds. Selectivity can be enhanced by selecting SIM mode to monitor a single ion mass. GC/MS is a powerful tool especially for the analysis of trichothecene mycotoxins (69-71). Several recent publications (70,122) describe the use of GC/fourier transform infrared (FTIR) spectroscopy to aid in the characterization and identification of Fusarium mycotoxins. The use of GC/FTIR together with GC/MS provides a very powerful combination for the identification of a variety of mycotoxins that represent diverse structure types in food or feed extracts.

Supercritical Fluid Chromatography. Supercritical fluid chromatography (SFC) uses a mobile phase with the sol-vating power of a liquid and the diffusivity and viscosity of a gas to separate nonvolatile or thermally labile compounds (123). The combination of SFC with MS allows the analysis of compounds which require derivatization prior to GC/MS. Young and Games (124) used SFC on packed HPLC columns combined with UV and a moving belt MS to separate and identify some Fusarium mycotoxins (deoxynivalenol, isodeoxynivalenol, 3-acetyldeoxynivalenol, 3,15-diacetyldeoxynivalenol, calonectrin) in liquid culture extracts. The SFC separations were rapid, and detection limits for a variety of mycotoxin standards analyzed by MS

were from 10 to 250 mg. Roach et al. (123) described a method for analyzing trichothecenes by SFC-negative ion chemical ionization MS. A disadvantage of SFC/MS is that the sensitivity is lower than GC/MS or LC/MS. In all, SFC is in its infancy due to high instrument costs compared with other analytical equipment.

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