Diastereomeric Separation of Free and Conjugated Silibinin in Plasma by Reversed Phase HPLC After Specific Extraction

Abstract

The analytical method outlined herein makes it possible, for the first time ever, to detect the diastereomers of silibinin separately in human plasma following oral administration of silymarin or silibinin to human subjects in the course of pharmacokinetic investigations, with unprecedentedly low detection limits. The method permits detection of both free (= unconjugated) silibinin diastereomers and of silibinin dia-stereomers following enzymatic cleavage of the silibinin giucuronides and sulphates. The detection limit per diastereomer is 2.5 ng/ml for the free silibinin and 5 ng/ml following enzymatic cleavage. A crucial aspect of this method is its extremely selective extraction and re-extraction of the silibinin, with recovery rates of around 80 %. The diastereomers are separated, without derivatization, on a reversed phase C18-column followed by UV detection at 285 nm. The linearity in the range tested (6—98 ng for each diastereomer / ml plasma in the case of free silibinin and 7– 1829 ng for each diastereomer / ml plasma in the case of total silibinin) is very good indeed. The day to day-variation (3 days, 3 concentrations; each n = 12) is lower than 4.8% (CV) with an accuracy of -1.1% to 6.1%.