Aflatoxins B , B2, G , and

Fig. 4 Typical chromatogram of aflatoxins B, B2, G, and G2 naturally contaminated pistachio nuts sample. Aflatoxin B, (AFB1) 0.75 fig/kg, aflatoxin B2 (AFB2) 0.12 fig/kg, aflatoxin G] (AFGl) 0.68 fig/kg, and aflatoxin G2 (AFG2) 0.15 /rg/kg. Mobile phase water methanol acetonitrile 54 29 17. Pump A regulated at 1 ml/min Pump B (postcolumn derivatization with bromine) regulated at 0.4 ml/min.

Two reviews [312, 1097] focus on the separation of aflatoxins B, B2, G, and G2. Often isocratic acetonitrile/water or acetonitrile/methanol/water mobile phases are used in conjunction with Cig columns (A = 360 nm). Normally acetonitrile levels of 25-40% are used. If methanol is used it is approximately at the 5-15% level and directly replaces an equivalent amount of acetonitrile. Most Importantly, TFA is almost invariably added at the 0.1-0.5% level. TFA forms a hemiacetal product with the aflatoxins that increases sensitivity by at least a factor of 3. Complete baseline resolution is usually achieved in <20 min. Fluorescence detection has been used, with 360 nm excitation and 440 nm emission wavelengths chosen. Detection limits of 1—lOpg were commonly reported. Postcolumn derivatization with iodine has also been used in conjunction with fluorescence detection to increase sensitivity. 

Aflatoxins are the most feared of the mycotoxins-toxin-pro-ducing molds. There are four main aflatoxins B B2, G and Gj, of which B, is the most common and the most toxic. A high incidence of cancer, especially liver cancer, is associated with aflatoxin. 

Reduction of aflatoxin B, B2, G and G2 levels in "arepa", a very popular corn product in Venezuela, following cooking in a microwave for 3.5 min, was studied. The results (Table 3) demonstrates a slight reduction of aflatoxin levels (14.6%) following microwave cooking. These results are in agreement with a study reported by Stoloff and Trucksess (1981) who reported a 13% of destruction of aflatoxin B when corn meal was made into corn muffins. 

M Castegnaro, DC Hunt, EB Sansone, PL Schuller, MG Siriwardana, GM Telling, HP van Egmond, EA Walker. IARC Laboratory Decontamination and Destruction of Aflatoxins B, B2, G, G2 in Laboratory Wastes. Scientific Publication N. 37, 1980. 

The aflatoxins are a group of related mycotoxins produced by the mould Aspergillus flavus. There are four toxins, B, B2, G, and G2. The mould typically grows on crops such as grain and peanuts in hot, humid climates. There is evidence from epidemiology of an association between exposure to aflatoxin Bi in the diet and liver cancer in humans. Aflatoxin Bj is metabolized by the enzyme system cytochrome P450 in the liver to a chemically reactive metabolite (see pp. 19-23 and fig. 25), which reacts with molecules such as DNA and protein in liver cells. 

Extensive methodology is available for the production of sterigmatocystin and the aflatoxins on stationary and submerged cultures on chemically defined media (Maggon et al., 1977). The amounts and relative proportions of aflatoxins B, B2, Gj, and G2 depend on the strain, balance of nutrients in the medium, and culture conditions. Lower pH and some trace metals, e.g., Mn Mg, and Va favored production of hydroxylated aflatoxins (Pai et al., 1975). In biosynthetic studies, it is essential to determine the rate of metabolite production, as the precursors must not only penetrate the synthetic site but must do so at a time when the enzyme systems mediating the synthetic reactions are present and active, i.e., the late stages in the... 

Currently 16 natural aflatoxins are known. Formulae 12-85 to 12-88 show the four major naturally occurring representatives of the B and G groups of aflatoxins, which are known as aflatoxins Bp B2, Gj and G2. The designation B (blue) or G (green) is associated with fluorescence of aflatoxins under UV light. While Aspergillus flavus produces mainly aflatoxins Bj and B2, contamination by A. parasiticus leads to aflatoxins Gj and G2 in the contaminated substrate. The basis of skeletons of aU aflatoxins is coumarin condensed with bisdihydrofurofuran (dihydrobisfuran) and cyclopentanone in the first two aflatoxins, while the other two substances contain 5,6-dihydropyran-2-one instead of cyclopentanone. 

Fig. 1. Chemical structure of aflatoxins. (a) The B-type aflatoxins are characterized hy a cyclopeutane E-ring. These compoimds have a hlue fluorescence under long-wavelength ultraviolet hght (h) The G-type aflatoxins have a xanthone ring in place of the cyclopentane, (c) Aflatoxins of the B2 and G2 type have a saturated bis-furanyl ring. Only the bis-firran is shown, (d) Aflatoxins of the Bi and Gi type have a hydrated his-furanyl structure.

While the aflatoxins B and G are major compounds of the fungus Aspergillus flavus, there are also minor aflatoxin constituents from this organism, e.g. hydroxylated derivatives of aflatoxin Bi (1) and B2 (2), the so-called milk-toxins . Ml (5) and M2 (6), which bear a hydroxy group at the junction of the two furan rings 19). They are called mOk toxins , because they are metabolites of aflatoxin Bi (1) and B2 (2), formed when cows get fed with contaminated foodstuffs. The toxins are then contained in the cow s milk. Other aflatoxins have a hydroxy group instead of... 

Mycotoxin analyses for the Pamlico county samples were shown in Table 1. Calculated ZE concentrations in the original (reconstituted) samples averaged 53 ng/g. Zearalenone in the fractions buoyant in the brine averaged 320 ng/g. There was no detectable ZE in the nonbuoyant grain from these nine samples. Deoxynivalenol was detected in only one of the nine samples, but the segregation pattern was similar to that of ZE. Aflatoxins B- and B2 did not segregate into the buoyant fractions (Table 1). Moreover, there was no correlation between AF concentration and either ZE or DON. 

Aflatest. This immunoaffinity column also met the lower detection requirement for the USFDA at a permissible limit of 20 ppb for aflatoxin. Qualitative results can be obtained using a florisil tip, however, the florisil tip was found difficult to interpret. Evaluation showed the quantitative analysis to be as accurate as other HPLC procedures, but much more rapid. Using the Aflatest for sample extract purification, and HPLC with post column derivatization using iodine, a limit of quantitation of 2.3 ppb for B and G is easily achieved. Sensitivity to B2 and G- is lower due to less specificity of the Aflatest antibodies to tnese toxins. 

Scheme 2.5 Syntheses of aflatoxin B2 (2) by Roberts et al. (above) and by Horne et al. (below). Reagents and conditions a) diethyl P-oxoadipate, HCl, ethanol, it, 19% b) KOH, ethanol, reflux, 2 h, 76% c) (COCl)2, CH2CI2 d) AICI3, CH2CI2, -5 C, 3 h, 38% overtwo steps e) Me3BnNia2, MeOH/CH2Cl2 f) NaH, 0°C then n-BuLi, -100°C, 15 min, 70% g) BnBr, K2CO3 h) n-BuLi, —78°C i) lithium 2-thienylcyano cuprate, —78°C to 0°C j) 37, —78°C to it, 60% over three steps k) H2, Pd/C, EtOAc, rt, 9 h, 200 psi 1) TFA, CH2CI2, rt, 60% over two steps m) DDQ, dioxane, rt, quant...

Scheme 2.7 Enantioselective total synthesis of aflatoxin B2 (2). Reagents and conditions a) 51, CH2CI2/CH3CN, -78°C to It, 7 h, 65%, 99% ee b) hexamethylenetetramine, HOAc, 110°C, 48 h, 40% c) DMAP (cat.), pyridine, TfiO, CH2CI2, -20°C to 0°C, 80% d) MeMgBr, THF, -20°C, 2 h e) DMP, CH2CI2, 0°C to rt, 85% over two steps f) TFAA, urea H20, CH2CI2, rt, 63% g) Raney-Ni, H2, MeOH, rt, 3 h, 60% h) NaHCOs, ZnCOs, ethyl 2-bromo-5-oxocyclopent-l-enecarboxylate, CH2CI2, rt, 20 h, 36%...

Scheme 2.15 Enantioselective synthesis of the aflatoxin B2 building block 33. Reagents and conditions a) Lipase AL, vinyl acetate, Et20, rt, 72%, 89% ee b) MsCl, DIPEA, DMAP, CH2CI2, 89% c) KCN, 18-Crown-6, DMSO, 72% d) LiOH, THF/H2O, 83% e) TPAP, NMO, 4 A MS, CH2CI2 f) HCi, HC(OEt)3, EtOH g) BnCI, K2CO3, DMF, 50% over three steps h) KOH, EtOH/ H2O i) BH3 SMe2, THE j) p-TsOH, CH2CI2,43% over three steps k) 1,4-cyclohexandiene, Pd/C, MeOH, quant...

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