High-performance liquid chromatographic analysis of the mycotoxin secalonic acid D and its application to biological fluids.

Secalonic acid D (SAD) is an acutely toxic, teratogenic and possibly mutagenic fungal metabolite produced in corn by Penicillium oxalicum. Using ultraviolet absorbance at 340 nm as a means of detection, SAD was resolved as a sharp peak by reversed-phase high-performance liquid chromatography (HPLC) on a small particle (10 mu m) mu Bondapak C18 column in 4 min by an acetonitrile--water--glacial acetic acid-tetrahydrofuran (5:3:0.5:0.5 for solvent system A and 4:3:0.5:0.5 for solvent system B) elution solvent system. The flow-rates for the two solvent systems (A and B) were 1.5 and 1.7 ml/min; column operating pressures of 1500 and 1800 p.s.i., respectively, resulted. The relationship between peak height or peak area and the quantity of SAD injected was linear over a range of 1--50 ng. Detection was very sensitive with lower limits of detection of 0.6 and 0.7 ng of SAD in solvent systems A and B, respectively. Retention times, peak heights and peak areas were highly reproducible in both solvent systems. Detection of SAD in spiked (0.2--10 mu g/ml) urine and bile samples injected without cleanup onto the HPLC column and quantitative extraction of SAD by ethyl acetate from spiked, acidified plasma samples (0.1--5 mu g/ml) followed by HPLC analysis were obtained. Solvent system B gave better resolution of SAD from the interfering substances in bile and urine than solvent system A. However, sensitivity was slightly greater in solvent system A.