Preparative chromatographic purification of diastereomers of silybin and their quantification in human plasma by liquid chromatography-tandem mass spectrometry.
A novel preparative HPLC method separating silybin has been developed to meet the need for both silybin A and silybin B standard. After the preparation of silybin A and silybin B standard, a simple, sensitive, selective and reproducible liquid chromatography-tandem mass spectrometry (LC-MS-MS) method with negative electrospray ionization (ESI) was developed for the quantification of silybin A and silybin B in human plasma. Following rapid sample preparation, silybin A, silybin B and naringin (internal standard, ISTD) were separated on a Zorbax Eclipse XDB-C18 column, using methanol-water containing 0.1% formic acid (48:52, v/v) as the mobile phase. The mass spectrometer was operated in selected reaction monitoring (SRM) mode using the transition m/z 481.1-->300.9 for both silybin A and silybin B and m/z 579.2-->271.1 for naringin, respectively. Linear calibration curves were obtained in the concentration range of 2-5000ng/ml with a lower limit of quantitation (LLOQ) of 2ng/ml for both silybin A and silybin B, respectively. The intra- and inter-day precision values were below 7.5% and accuracy was within +/-4.9% at all three quality control (QC) levels, for both silybin A and silybin B, respectively. This method was successfully applied to the stereospecific analysis of silybin in plasma samples from a pharmacokinetic study of silybin A and silybin B in 22 healthy male Chinese volunteers after a single oral dose of silybin-phosphatidylcholine complex (equivalent to 280mg silybin, including 133mg silybin A and 147mg silybin B).
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
Wei
W
Li
Jianping
J
Han
Zhiwen
Z
Li
Xinxin
X
Li
Shuiping
S
Zhou
Changxiao
C
Liu
D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D000077385:Silybin; D012838:Silymarin / Q000097:blood; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000379:methods*
D012838:Silymarin; D000077385:Silybin