H. Mascher et al., DIASTEREOMERIC SEPARATION OF FREE AND CONJUGATED SILIBININ IN PLASMA BY REVERSED-PHASE HPLC AFTER SPECIFIC EXTRACTION, Journal of liquid chromatography, 16(13), 1993, pp. 2777-2789
The analytical method outlined herein makes it possible, for the first
time ever, to detect the diastereomers of silibinin separately in hum
an plasma following oral administration of silymarin or silibinin to h
uman subjects in the course of pharmacokinetic investigations, with un
precedentedly low detection limits. The method permits detection of bo
th free (= unconjugated) silibinin diastereomers and of silibinin dias
tereomers following enzymatic cleavage of the silibinin glucuronides a
nd sulphates. The detection limit per diastereomer is 2.5 ng/ml for th
e free silibinin and 5 ng/ml following enzymatic cleavage. A crucial a
spect of this method is its extremely selective extraction and re-extr
action of the silibinin, with recovery rates of around 80 %. The diast
ereomers are separated, without derivatization, on a reversed phase C1
8-column followed by UV detection at 285 nm. The linearity in the rang
e tested (6 - 98 ng for each diastereomer/ml plasma in the case of fre
e 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 a
ccuracy of -1.1% to 6.1%.