LIQUID-CHROMATOGRAPHIC ASSAY OF SILYBIN IN HUMAN PLASMA AND URINE

A specific and sensitive high-performance liquid chromatographic method for the quantitative determination of silybin and its conjugates in human plasma and urine was developed. Silybin glucuronides and sulfates were calculated as silybin after enzymatic hydrolysis with beta-glucuronidase/arylsulfatase. Extraction from the biological fluid was performed at pH 4 on normal-phase solid extraction columns using tert-butylmethylether as eluent. (+)-Catechin was added as internal standard after the extraction procedure. A normal-phase column was used for the HPLC analysis. The mobile phase consisted of n-hexane/ethanol, acidified with 85% phosphoric acid. Detection was performed by a variable-wavelength detector at 214 nm. Detection limits of 5 and 25 ng/ml were respectively achieved for free and total (free and conjugated) silybin in plasma, and 100 ng/ml in urine. The method is suitable for pharmacokinetic studies after oral administration of IdB 1016, a lipophilic silybin-phosphatidylcholine complex.