Development and validation of two liquid chromatography-tandem mass spectrometry methods for the determination of silibinin and silibinin hemisuccinate in human plasma.

Abstract To investigate the pharmacokinetics of silibinin and silibinin hemisuccinate in human plasma, two high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) methods were developed and validated. The methods require a small volume of sample (100 μL), and the recovery of the analytes was complete with a good reproducibility (CV% 1.7–9.5), after a simple protein precipitation. Naringenin was used as internal standard. The chromatographic methods provided a good separation of diastereoisomers A and B of both silibinin and silibinin hemisuccinate onto a Chromolith Performance RP18e 100 mm × 3 mm column, with a resolution of peaks from plasma matrix in less than 6 min. The methods precision values expressed as CV% were always ≤6.2% and the accuracy was always well within the acceptable 15% range. Quantification was performed on a triple-quadrupole tandem mass spectrometer by Selected Reaction Monitoring (SRM) mode, in a negative ion mode, via electrospray ionization (ESI). The lower limit of quantitation was set at 5.0 ng/mL (silibinin) and 25.0 ng/mL (silibinin hemisuccinate), and the linearity was validated up to 1000.0 and 12,500.0 ng/mL, for silibinin and silibinin hemisuccinate, respectively, with correlation coefficients ( R 2 ) of 0.991 or better. The methods were suitable for pharmacokinetic studies and were successfully applied to human plasma samples from subjects treated intravenously with Legalon ® SIL at the dose of 20 mg/kg, expressed as silibinin.