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

Rickling, B.; Hans, B.; Kramarczyk, R.; Krumbiegel, G.; Weyhenmeyer, R.

Journal of Chromatography. B, Biomedical Applications 670(2): 267-277


ISSN/ISBN: 1572-6495
PMID: 8548017
DOI: 10.1016/0378-4347(95)00168-9
Accession: 009683953

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A combination of two stereoselective assays was developed using column-switching HPLC with electrochemical detection for the determination of free (unconjugated) silibinin and R.P-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 silvmarin in human volunteers.