Identification Of Chemical Constituents In Silymarin Capsules And The Effects Of Silymarin On The Pharmacokinetics Of Simvastatin

Part one Identification of chemical constituents in silymarin capsule by UPLC-Q-TOF-MSObjective:Identification of chemical constituents in silymarin capsules by UPLC-Q-TOF-MS.Methods:Silymarin capsule was analyzed by UPLC-Q-TOF-MS in the scanning mode of TOF-MS-IDA-MS/MS under the negative ion mode.Then,the exact mass,fragment ions and retention time obtained by Peak View 2.2data processing system were compared with the standards or the compounds summarized from references to identify the constituents.The separation of was performed on an Waters XBridge BEH C18 column（100 mm×2.1mm,2.5μm）using the mobile phase consisted of methanol（B）and 0.1%formic acid（A）with gradient elution.The flow rate was 0.3 mL/min,the injection volume was 3μL.Results:Based on the references and MS/MS of standards,fourteen compounds from silymarin capsules had been identified.The fragmentation rules of flavonolignans were summarized.The fragment m/z 301、325 were quite different among these compounds and could be used to distinguish between different isomers.Conclusion:UPLC-Q-TOF-MS was high sensitive,accurate and rapid for silymarin capsule identification.And it provided help for the research of the pharmacodynamic material basis.Part two Effects of silymarin on the in vivo pharmacokinetics of simvastatin and its active metabolite in ratsObjective:To develop a sensitive UPLC–MS/MS method to determine the concentration of simvastatin（SV）and simvastatin acid（SVA）and to apply this method for investigating the possible interaction of silymarin with SV through comparing the pharmacokinetic profiles between groups after oral administration in rats.Methods:All rats were administered 0.5%CMC-Na（control group for different corresponding SV doses）or silymarin（50 mg/kg,co-administration group）daily by gastrogavage for six days.Thirty minutes after the seventh treatment,20,40 and 80 mg/kg SV doses were respectively administered to the corresponding control or co-administration rats by gastrogavage.The blood samples were collected from each rat after simvastatin administration by gastrogavage.Then,the plasma samples were analyzed by liquid–liquid extraction with tert-butyl ether.Both lovastatin（LV）and lovastatin acid（LVA）were chosen as the internal standards for SV and SVA,respectively.The chromatographic separation was carried out on a Waters XBridge BEH C18column（2.1 mm×100 mm,2.5mm,Waters,Ireland）at 40°C within 3 minutes.For the detection of simvastatin and lovastatin,the mass spectrometer was set to positive ionization mode,whereas the detection of simvastatin acid and lovastatin acid was performed under the negative ionization mode.The multiple reaction monitoring（MRM）mode was used to quantify the analytes and IS.Non-compartmental analysis was performed to calculate the pharmacokinetic parameters using Phoenix WinNonlin?7.0.Results:The calibration curve for SV and SVA in rat plasma samples showed good linearity within the test concentration range for SV and SVA.The pharmacokinetic parameters of SVA were significantly different between groups when a single dose of SV was given to silymarin pre-treated rats.The pretreatment of rats with silymarin for seven days increased the AUC_0-∞of SVA by 1.3-fold（P<0.05）in the low dose（20 mg/kg SV）group,1.5-fold（P<0.05）in the middle dose（40 mg/kg SV）group,and 1.9-fold（P<0.05）in the high dose（80 mg/kg）.The peak plasma concentrations(C_max)were also found to increase in various dosage groups.No significant differences were observed between groups concerning the pharmacokinetic parameters of SV.Conclusion:In this study,a sensitive and selective UPLC-MS/MS method to quantify SV and SVA in rat plasma samples was developed,validated and subsequently applied for assessing the effect of silymarin on the pharmacokinetic effect of SV at different dosage levels in rats.According to the results of the present study,as the plasma concentration of SVA was found to increase when SV was co-administered with silymarin,the possibility of pharmacokinetic interactions between silymarin and SV should be considered to avoid potential adverse effects.