Mycotoxins, HPLC analysis

Oshima, Y, Sugino, K., and Yasumoto T., Latest advances in HPLC analysis of paralytic shellfish toxins, in Mycotoxins and Phycotoxins 88, a Collection of Invited Papers Presented at the VII International lUPAC Symposium on Mycotoxins and Phycotoxins, Natori, S., Hashimoto, K., and Ueno, Y. Eds., Elsevier Science Publishers, Amsterdam, 1989, 319. 

As far as the mycotoxin analysis based on HPLC techniques is concerned, most methods include the following steps. 

Determination. The last step in mycotoxin analysis includes an additional separation achieved by HPLC column, followed by detection and quantitative determination, performed on the basis of the physicochemical characteristics of the mycotoxin. 

Owing to the varied structures of various food dyes, they can often be differentiated from one another by their characteristic ultraviolet/visible absorbance spectra. Using HPLC coupled with a diode array detector (HPLC-DAD) it is possible to collect a compound s absorbance spectrum as it elutes from the HPLC column, which greatly assists in identification. At Reading Scientific Services Ltd (RSSL) this type of detector is routinely used in a range of analyses of such substances as patulin, a mycotoxin found in apple juice, and in the analysis of colours and vitamins, which allows a more certain assignment of a particular peak to a specific compound to be made. 

While HPLC does not always produce superior results to those with TLC it allows greater versatility and is more suitable for the analysis of complex organic matrices such as cereals. HPLC coupled to sensitive detection and sophisticated data retrieval has improved the identification of selected mycotoxins at levels much less than achieved by TLC. Additional chromatographic modes such as normal-phase, reverse phase and ion-exchange chromatography have been employed. There are no truly universal detectors available for HPLC. Detectors presently in use include Fourier transform infrared detections (FT-IRD), diode array ultraviolet detection (DAD) and mass selection detectors (MSD) (Coker, 1997). 

For mycotoxin analyses radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISAs) and affinity chromatography are the principal immunochemical methods in commercial application. Immunoaffinity columns or cartridges for specific mycotoxins are now being increasingly used in preliminary clean-up of extracts prior to final analysis by HPLC or GLC methods. 

Chemical analysis of hair samples may also provide a method for examining chronic mycotoxin exposures. In 2003, Sewram et al. (2003) reported that human hair testing could be used to detect fumonisins. After extraction and clean up, high performance liquid chromatography coupled to electrospray ionization-mass spectrometry (HPLC-ESI-MS) was able to detect fumonisin Bi, fumonisin B2, and fumonisin B3 from human hair samples (Sewram et al., 2003). However, these were... 

High-performance LC (HPLC) is the technique used most frequently in food analysis for measuring carbohydrates, vitamins, additives (sweeteners, antioxidants, colorants, preservatives, etc.), mycotoxins, amino acids, proteins, tryglicerides in fats and oils, lipids, chiral compounds and pigments, among others (Table 1). Some of these applications will be discussed in this article. 

The analysis of mycotoxins, toxic substances produced by some fungi, is essential to guaranteeing the safety of foods such as cereals, nuts, oilseeds, and spices since the discovery of their significance to human health. The main mycotoxins, like aflatoxins and ochratoxin A, are determined using HPLC with octyl-or octadecyl-bonded phases and polar solvents such as methanol, acetonitrile, and water. For detection with the required sensitivity and selectivity, spectrofluori-metric detection is the most common method, with increasing use of mass spectrometric interfaces. 

TLC plates used are generally silica gel plates, although C] g re versed-phase TLC plates may occasionaly be used. Elution may be monodimensional or bidunensional. The former is more common. Recent automated gradient techniques appear promising for the simultaneous determination of several mycotoxins in a single sample. Solvents used for elution depend on the type of plate used. Most of the time, organic solvents (ethanol chloroform, acetone) are used. One feature of HPTLC is that it uses much less solvent than HPLC and permits analysis of many samples in a very short time. ... 

Mycotoxins are toxic chemical metabolic products produced by microscopic mold fungi that colonize cereal crops, fruits, and vegetables and some of them exhibit carcinogenic and mutagenic properties. The allowed maximum levels in foodstuffs are very low and exhaustive exU action, preconcentration, and clean-up processes are required to attain the desired sensitivity. The alternative of ED (amperometric detection) combined with HPLC can be useful to simplify the detection of such low concentration levels. However, it has been used rarely for the analysis of these compounds in food because many of these compounds can be elecfrochemically oxidized or reduced at extreme potentials. Visconti et al [144], analyzed... 

In comparison with the usual HPLC/UV method, it has heen shown in the analysis of mycotoxins using patulin (TV in Rg. 9.1) that the detection limit decreases hy a factor of 1(X) if an isotopic dilution assay (cf. 5.2.6) is carried out with [ C2] patulin as the internal standard. After sily-lation and gas chromatographic/mass spectromet-ric measurement of analyte and isotopomer, 5.7-26.0 pg/1 of patulin were found in apple juices. 

HPLC has been used for measuring various compounds, for example, carbohydrates, vitamins, additives, mycotoxins, amino acids, proteins, triglycerides in fats and oils, lipids, chiral compounds, and pigments. Several sensitive and selective detectors such as ultraviolet-visible, FL, electrochemical, and diffractometric are available to utilize with HPLC depending on the compound to be analyzed. Various HPLC methods based on these detectors have been published for the measurement of vitamin E in biological and pharmaceutical samples and food products. Excellent literature reviews of HPLC based on various detectors in the analysis of vitamin E content in various matrices have been reported (Abidi, 2000 Aust et al., 2001 Ruperez et al., 2001 Lai and Franke, 2013). Table 19.5 reports several recent HPLC methods for the analysis of vitamin E and similar compounds in various matrices. 

Minerals are usually analyzed by the traditional method of atomic absorption spectroscopy or with the newer induced coupled plasma analyzers. Most vitamins are analyzed by HPLC systems or colorimetric assays. Many laboratories still quantify folic acid with the use of a microbiological assay. The analysis of dietary and detergent (neutral and acid) fibers are critically important for foods and feeds, respectively. Raw materials and feeds should be examined for rodent contamination, pathogenic bacteria, molds and mycotoxins, and undesirable toxicants such as PCB, insecticides, herbicides, and heavy metals. The proper selection of ingredients will ensure the production of high-quality foods that satisfy sensory properties required for humans, and feeds that are palatable and can meet sensory properties required for domestic animals (Chapter 18). 

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