Extraction mycotoxins

Many natural products are charged substances, and can be isolated by IEC methods. Dufresne has published a comprehensive review describing various resins and column operating conditions applicable to purification of natural products from fermentation broths or crude extracts.168 Among natural products, antibiotics are of special interest due to their widespread use in humans and animals. Sample cleanup by IEC prior to analysis by other LC methods for quantitative determination of antibiotics in biological fluids is frequent.I69171 Also, IEC followed by TLC appears useful for the quantitation of fumonisin Bl, a mycotoxin found in agricultural products.172... 

Because estrogenic mycotoxins usually occur at microgram per kilogram (pg/kg) levels there is special interest in analytical procedures for reliable detection of zearalenone and its metabolites between 10 and 100 pg/kg. In response to the risk of a great economic loss to the industry and the threat to human health as a result of exposure to zearalenone, several methods have been developed for the quantification of zearalenone and its metabolites in different foods, feeds, animal tissues, blood and urine. Detailed reviews have been given by Steyn et al. 1991 Betina 1993 Frisvad and Thrane 1993 Scott 1993 Steyn 1995 and Lawrence and Scott 2000. The determination of zearalenone in cereals can be divided into five steps grinding of the sample, extraction of the sample, clean-up, separation and detection. 

The statins, lovastatin (L), simvastatin (S), pravastatin (P), fluvastatin (F), cerivastatin, and atorvastatin, inhibit HMG CoA reductase. The active group of L, S, P, and F (or their metabolites) resembles that of the physiological substrate of the enzyme (A). L and S are lactones that are rapidly absorbed by the enteral route, subjected to extensive first-pass extraction in the liver, and there hydrolyzed into active metabolites. P and F represent the active form and, as acids, are actively transported by a specific anion carrier that moves bile acids from blood into liver and also mediates the selective hepatic uptake of the mycotoxin, amanitin (A), Atorvastatin has the longest duration of action. 

SPR systems also showed encouraging results with their ability to detect mycotoxins. The BIACORE was used to detect a mycotoxin, DON, produced by Fusarium species, from spiked wheat sample in a competitive inhibition assay (Schnerr et ah, 2002). Biotinylated DON was immobilized on the sensor chip which was previously coated with strep-tavidin. Mycotoxin extracts from wheat samples were first allowed to react with the antibody and then injected into the BIACORE. The detection range was established to be 0.13-10 pg/ml. In a slightly modified format, DON was also detected by SPR at a range of 2.5-30 ng/ml (Tudos et ah, 2003). 

The working group CEN TC 275/WG 5 searched for performance criteria to be used in mycotoxin analysis and came up with a document reporting the criteria for the selection of methods (26). The criteria deal with limits of detection, minimum performance characteristics, extraction solvents, and applicability. Criteria for analytical methods in mycotoxin analysis are also included in Directive 98/53/EC (18). 

Cleanup An additional separation of the mycotoxin from lipids and other components of the matrix is accomplished through the cleanup step. Most procedures include solid-phase extraction on stationary phases such as silica, C,8, florisil, and phenyl. Prepacked columns are largely used, with the variations between lots being recently ameliorated. Alternatively, the use of cleanup by immunoaffinity, based on the formation of mycotoxin-protein conjugate, is on the increase, since this is very rapid, selective, and usefully employed in various food matrices. One disadvantage is that the cost is still rather high, and cross-contamination phenomena (false-positive) can occur (30). 

This paper shows a nice example for solving an important analytical problem using MISPE. Mycotoxins and particularly zearalenone (ZON) and /nmv-a-zearalenol (a-ZOL) present an everyday problem in food analysis. Existing sample clean-up techniques have different drawbacks. Liquid-liquid extraction is characterized by... 

Kalinoski et al. [32] has applied this method to the determination of tri-chothecene mycotoxins in wheat. The methods were based on chemical ionisation MS and collision-induced dissociation tandem MS and enabled the rapid identification of ppm levels of several trichothecene mycotoxins. Supercritical carbon dioxide is shown to allow identification of mycotoxins with minimum sample handling in complex natural matrices such as wheat. Tandem MS techniques are employed for unambiguous identification of compounds of varying polarity, and false positives from isobaric compounds are avoided. Capillary column SCFC-MS of a SCF extract of the same sample was also performed, and detection limits in the ppb range appear feasible. 

Analytical methods for mycotoxins have historically utilised chemical detection methods which are easily quantifiable, highly sensitive, less subject to interference by co-extractives but can be quite time demanding. Most quantitative methods use a final chromatographic separator followed by a suitable detection... 

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. 

The third mechanism is peritubular extraction of peptides and proteins from postglomerular capillaries and intracellular metabolism. Experiments using iodi-nated growth hormone (125I-rGH) have demonstrated that, while reabsorption into endocytic vesicles at the proximal tubule is still the dominant route of disposition, a small percentage of the hormone may be extracted from the peritubular capillaries [79, 86]. Peritubular transport of proteins and peptides from the baso-lateral membrane has also been shown for insulin [87] and the mycotoxin ochra-toxin A [88]. 

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... 

R.P. Huopalahti, J. Ebel and J.D. Henion, Supercritical fluid extraction of mycotoxins from feeds with analysis by LC/UV and LC/MS, 7. Liquid Chromatogr. Relat. Technol., 20, 537-551 (1997). 

Removal or elimination of mycotoxins. Since most of the mycotoxin burden in contaminated commodities is localized to a relatively small number or seeds or kernels [reviewed in Dickens, 200], removal of these contaminated seeds/kemels is effective in detoxifying the commodity. Methods currently used include (a) physical separation by identification and removal of damaged seeds, mechanical or electronic sorting, flotation and density separation of damaged or contaminated seeds (b) removal by filtration and adsorption onto filter pads, clays, activated charcoal (c) removal of the toxin by milling processes and (d) removal of the mycotoxin by solvent extraction. 

Lethality due to ingestion of food contaminated by trichothecenes has been reported in horses (Rodricks and Eppley, 1974), cattle (Hsu et al, 1972), and humans (Joffe, 1974). General clinical signs include emesis, food refusal and weight loss, dermal effects, and immune suppression with secondary infection. Clinical signs are dependent on the specific trichothecene involved, the dose, species, route of exposure, as well as the nature of the exposure. Spontaneous and experimental exposures may give somewhat different results, as can exposure to field contaminated materials, when compared to purified toxin. In the case of field contamination or experimental use of crude extracts, multiple mycotoxins, both known and unknown, may be present at the same time. 

Trichothecene mycotoxins are highly irritating to the skin and mucous membranes in all species. Skin irritation was experienced by laboratory workers accidentally exposed to trichothecene producing fungal cultures or crude extracts of T-2 toxin. Skin irritation has also been used as a basis for a semiquantitative bioassay to detect these toxins (Bamburg... 

Mass spectrometry has proved highly suitable for the elucidation of chemical structures and sensitivity. It is thus an invaluable tool for resolving extremely complex natural mixtures. Thus, on-line coupled SFE-MS constitutes a rapid, reliable choice for the determination of mycotoxins in wheat, which possess a high toxicological interest. For example, an SF extractor coupled to an MS chemical ionization source provided limits of detection in the picogram range for diacetoxyscirpenol and toxin T-2 [122]. Used in tandem with chemical ionization-collision induced dissociation MS-MS, SFE has also enabled the rapid detection and identification of species without the need for complete extraction [131],... 

Zearalenone (ZON) is another mycotoxin produced by Fusarium species. It can infect wheat and maize. The analysis of ZON was reported by several groups [106-108]. Rosenberg et al. [107] reported the analysis of ZON in food and feed, using LC separation on a Cig colunrn with 40% acetonitrile in water and positive-ion APCI-MS. The LOQ was 0.12 pg/kg in maize, which is a 50-fold improvement over fluorescence detection. They also compared 100 and 20 nun long columns. Sample clean-up was only required when using the 20 nun colunrn. Pressurized liquid extraction was applied for the isolation of ZON from wheat and com prior to LC-ESI-MS in the negative-ion mode [108]. The LOQ was 12-15 pg/kg. 

Most methods for mycotoxins detection are based on HPLC using reversed phase colnmns followed by flnorescent detection. The methods used for purifying the sample are solid-phase extraction column (SPE) with normal phase or reversed phase absorbents. Recently, immune affinity colnmns have been used more often. 

The ELISA tests are preferred because they require low volume of the sample and fewer clean-up procedures of the extracted sample compared to the conventional methods like TLC (thin layer chromatography) and HPLC (high-performance liquid chromatography). ELISA tests are rapid, simple, specific, sensitive and portable for use in the field for the detection of mycotoxins in foods and fodder (Zheng et al. 2005). 

The analysis of food contaminants, in particular any toxic or biologically active residue, is important for public health or quality control reasons.19 Examples are mycotoxins (aflatoxins) and pesticide and drug residues. Sample preparation is typically elaborate and might involve deproteinization, solvent extraction, and clean-up via solid-phase extraction (SPE).The use of highly sensitive and specific LC/MS/MS is increasing and has simplified some of the sample preparation procedures. 

---