Assays - Silibinin and Isosilibinin (with diastereomeric separation)

Diasteroisomeric separation of free and conjugated Silibinin in plasma by reversed-phase HPLC after specific extraction.

Author: H.J. Mascher, C. Kikuta, R. Weyhenmeyer
Publisher: J. Liqu. Chromatogr. 16 (1993), 2777-2789

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 diastereomers following enzymatic cleavage of the silibinin glucoronides 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-coluumn 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 %.

Study on dose-linearity of the pharmacokinetics of Silibinin diastereomers using a new stereospecific assay

Author: R. Weyhenmeyer, H.J. Mascher, J. Birkmayer
Publisher: Int. J. Clin. Pharmacol., Ther. and Toxicol. 30 (1992), 134-138

Silibinin in single doses of 102, 153, 203 and 254 mg was applied as silymarin in capsules (Legalon ® 140) to 6 healthy male volunteers. Using a newly developed HPLC method, both diastereomers of silibinin were assayed in plasma as unconjugated compounds as well as total isomers after hydrolysis. In the dose range studies, the areas under the curves correlate linearly with the dose. On average, only 10 % of total silibinin in plasma is in the unconjugated form. The ratio of the silibinin isomers is reversed, if unconjugated and total isomers are compared. For unconjugated silibinin, the half-life is less than one hour, but the terminal half-life has probably not been observed, because already after 4-6 hours the levels fell below the limit of determination of 2.5 ng diastereomer/ml. For total silibinin, an elimination half-life of approximately 6 h is estimated. About 5 % of the dose is excreted into urine as total silibinin, corresponding to a renal clearance of approximately 30 ml/min. No adverse events were noted, showing that silymarin even in high doses, up to 5 capsules of Legalon ® 140, is well tolerated.