Reverse Phase Analytical and Preparative High Pressure Liquid Chromatography of Aflatoxins

Abstract

Abstract Reverse phase high pressure liquid chromatography (HPLC) was investigated and found applicable for determining 6 aflatoxins. Aflatoxins M2, M1, G2, G1, and B1 were completely resolved and B2 was satisfactorily separated on a C18 (10 μm, 4 mm id × 30 cm) column with an eluting solvent of acetonitrile-water (35 + 65) and flow rate of 1.5 ml/min. Compounds were detected by ultraviolet absorbance at 350 nm. Peak height and retention time reproducibility of multiple injections was excellent with coefficients of variation of 1.0% (M1) and 1.9% (B1) when laboratory temperatures were relatively constant throughout a day ; however, coefficients of variation increased significantly when temperatures varied by 10°F. HPLC determinations of aflatoxin B1 added to uncontaminated corn extracts at 20 and 50 ppb levels were 83±14 and 92.5±8%, respectively, of those expected. Comparison of com extract assays (6–98 ppb B1) by thin layer chromatography (TLC) and HPLC revealed that HPLC values averaged 25% less than TLC values. Numerous peaks and background interferences were present in corn extracts which made interpreting chromatograms difficult. None of the cleanup procedures tried was successful in removing these interferences. Preparative HPLC was used to isolate and purify quantities of aflatoxins M1, B1, and G1 from silicic acid column mixtures of M1-M2, B1-B2, and G1-G2. Separations were achieved on C18/Porasil B (35–75 μm) columns (⅜″ od × 8′) developed with acetonitrile-water mixtures (M1, 20+80; B1, 35+65; G1, 25+75) at 9.0 ml/min. These columns permitted isolation of 40 mg aflatoxin B1 or G1 in less than 3 hr. Aflatoxin M1 required 2 HPLC steps.