Two high-performance liquid chromatographic assays for the determination of free and total silibinin diastereomers in plasma using column switching with electrochemical detection and reversed-phase chromatography with ultraviolet detection.

A combination of two stereoselective assays was developed using column-switching HPLC with electrochemical detection for the determination of free (unconjugated) silibinin and RP-HPLC with UV detection for the measurement of total (free and conjugated) silibinin in human plasma. After extraction of free silibinin and the internal standard hesperetin with diethyl ether the compounds were pre-separated on a RP-CN column. A cut fraction of eluate containing the analytes was then transferred to the RP-18 main column by means of a switching valve for final separation of the compounds. The limit of quantification with electrochemical detection for free silibinin was 0.25 ng/ml per diastereomer. For the determination of total silibinin diastereomers all conjugates were cleaved enzymatically using beta-glucuronidase/arylsulfatase at pH 5.6 followed by extraction with diethyl ether of the pH 8.5 alkalized solution. Separation of the diastereomers and of the internal standard naringenin was achieved on a RP-18 column. The limit of quantification with UV detection at 288 nm for total silibinin was 5 ng/ml per diastereomer. Both assays were successfully applied to the stereospecific analysis of silibinin in plasma samples from a pharmacokinetic study of silymarin in human volunteers.

Saponins in the leaves of birch? Hemolytic dammarane triterpenoid esters of Betula pendula.

Three hemolytic dammarane triterpenes, esterified with malonate at C-3 and acetate at C-12, were isolated from the leaves of Betula pendula. Their structures were elucidated by chemical and spectroscopic means. Former reports on the presence of saponins in birch leaf extracts could not be confirmed. The hemolytic activity of the extracts, which was ascribed to saponins, is caused by the dammarane esters instead. A fraction containing a mixture of the dammarane esters did not exhibit diuretic activity when tested p.o. in male Wistar rats.