Simple high-performance liquid chromatography-fluorescence detection method for plasma, kidney and liver of rat as a tool for toxicology studies.

A fast and simple HPLC-FLD (high-performance liquid chromatography-fluorescence detection) analytical method has been developed and validated for the determination of ochratoxin A in rat plasma, kidney and liver. The extraction method, calibration curves and chromatographic conditions are common for the three matrices. Plasma and homogenized tissue samples (250 microL) were extracted with ethanol (400 microL) and trichloroacetic acid 20% (w/v) (50 microL). Supernatants were directly injected into the HPLC system, analyzed on a 5-microm (25 cm x 0.4 cm) Tracer Extrasil ODS2 column using FLD (excitation wavelength=225 nm, emission wavelength=461 nm). The mobile phase was 29:29:42 (v/v) methanol-acetonitrile-sodium acetate. The small volume of sample needed which allows the obtaining of ochratoxin A levels in individual tissue samples from small animals and the wide range of concentrations that could be analyzed make this method easy to apply in toxicology and toxicokinetic studies of this mycotoxin, even in low dose carcinogenic studies. This method was linear and selective for all the matrices. Precision and accuracy were always <10% and recovery was very efficient in each case. Limits of detection and quantification were also calculated in plasma (1 and 8.4 microg/L), kidney (14.3 and 55.8 microg/kg) and liver (4.1 and 52.8 microg/kg). Stability of the tissue homogenates was assured for at least 10 months at -80 degrees C. The method has been successfully applied to the analysis of rat samples after 7 days of ochratoxin A (0.5mg/kg b.w. dissolved in an aqueous NaHCO(3) solution) administration by oral gavage.