Resolution of Penicillium roqueforti Toxin and Eremofortins A, B, and C by High-Performance Liquid Chromatography
Applied and Environmental Microbiology
A method for the quantitative analysis of Penicillium roqueforti toxin using a high-performance liquid chromatography system is proposed. Penicillium roqueforti toxin (PRT) is a mycotoxin isolated from cultures of P. roqueforti (6), a fungal species used in the ripening of French Roquefort cheeses and also commonly found in fermenting silage (5). The toxin is lethal for rats and mice with a 50% lethal dose of about 7 mg/kg for male rats and mice after intraperitoneal injection (7) . PRT also
... n (7) . PRT also induces biochemical alterations in rat liver, including the inhibition of protein and RNA syntheses (3, 7). The aldehyde group of the molecule has been shown to be directly involved in the biochemical activity of this compound (4). This report describes a method for the quantitative determination of PRT and other structurally related P. roqueforti metabolites by highperformance liquid chromatography (HPLC) (1, 2). MATERIALS AND METHODS Standards. Crystalline eremofortin A (EA), eremofortin B (EB), eremofortin C (EC), and PRT were prepared as described previously (1, 2). A concentrated standard solution of a mixture of EA, EB, and PRT was prepared containing 0.5 mg of each compound per ml in chloroform. A standard solution of EC was made by dissolving EC in chloroform at 0.5 mg/ml. Solvents. Two solvent systems were used: solvent A contained n-hexane and tetrahydrofuran at 75/25, vol/vol (Solvant, Documentation Synthese, Valdonne, France); solvent B contained only chloroform (Merck). AU solvents were of analytical grade. HPLC. HPLC separations were carried out with a Waters Associates ALC 204 instrument equipped with an M-6000 A pump, a U6 K injector, a model 440 absorbance detector (0.005 to 2 absorbance units full scale [AUFS]). Chromatograms were recorded on an omniscribe 10-inch (25.4-cm) recorder (Houston Instruments). A micro Porasil 10-pm silica gel column (4 mm inner diameter by 30 cm long) was used. EA, EB, and PRT were analyzed with solvent A at a flow rate of 1.5 ml/min, and EC was analyzed with solvent B at a flow rate of 2 ml/min. Detection of these compounds after chromatographic separation was made at a wavelength of 254 nm. Sensitivities of 0.005 to 0.5 AUFS were used depending on the amount of compounds injected. Peak heights (in millimeters) were corrected by the expression H = peak height x AUFS x 10 and drawn as a function of the amount of the compounds injected in nanograms. RESULTS AND DISCUSSION The validity of the method depends on the complete separation of the compounds produced by various P. roqueforti strains: EA, EB, EC, and PRT. Since these four compounds have ultraviolet absorption maxima in the region of 250 nm with molar extinction coefficients of about 15,000, a 254-nm filter was chosen for the detection of these compounds after chromatographic separation. A small-particle (10-,um) porous silica gel column was used for chromatographic separation. Among numerous elution solvents tried, optimum resolution of the four compounds was obtained with n-hexane-tetrahydrofuran (75/25, vol/vol) at a flow rate of 1.5 ml/min. A typical chromatogram of the four substances is shown in Fig.