Quantitative Determination And Metabolism Studies Of Semen Ziziphi Spinosae By HPLC-MS Technologies And Quality Criteria Establishment Of Lian Qiao Xin

Part one Simultaneous quantification of nine constituents in Semen Ziziphi Spinosae by liquid chromatography tandem mass spectrometryObjective: To develop an HPLC-MS/MS method for the determination of jujuboside A,jujuboside B,spinosin,betulinic acid,betulin,rutin,apigenin,hesperidin,and naringin in Semen Ziziphi Spinosae.Methods: Analysis was performed on a Sapphire C18 column(150 mm× 4.6 mm,5 ?m)eluted with acetonitrile and 0.1% methanoic acid solution containing 1 mmol·L-1 ammonium acetate in a gradient program.The flow rate was 1 mL·min-1,the injection volume was 10 ?L,and the column temperature was 30?.The multiple-reaction monitoring scanning(MRM)was employed for the quantification with switching electrospray ion source polarity in a negative mode.The ion spray voltage was set at-4500 V and the turbo spray temperature was maintained at 650?.Results: The regression equations showing linear relationships between peak areas and contents of each compound were obtained.The average recoveries of the compounds ranged from 98.0% to 101.5% and the precision in terms of RSD was in the range of 0.03%-0.74%.Conclusion: The method is simple,rapid,and sensitive.It could be used as a quantitative determination method for the nine components in Semen Ziziphi Spinosae.Part two Metabolism studies of spinosin in human liver microsomesObjective: A UHPLC-Q-TOF-MS method was established to characterize the metabolites of spinosin in human liver microsomal incubation system.Methods: The chromatography separations were performed on a C18 reversed phase LC column(Poroshell 120 EC-C18,2.1×100 mm,2.7 ?m).The mobile phase consisted of acetonitrile and water-acetic acid(100: 0.1,v/v)at the flow rate of 300 ?L·min-1 and a gradient program was adopted.The mass spectral analysis was performed in a negative electrospray ionization mode,the turbo spray temperature was 550?.The full MS experiments were run with a scan range from m/z 100 to m/z 1000.Results: 8 metabolites were identified in human liver microsomal incubation system and the possible fragmentation pathways of spinosin were speculated in a negative electrospray ionization mode.No glucuronidation metabolites were detected in human liver microsomal incubation system.Conclusion: The UHPLC-Q-TOF-MS method was very convenient and efficient for detecting the spinosin in human liver microsomes.Part three Inhibitory potency of spinosin on cytochrome P450 enzymes from human liver microsomesObjective: To study the inhibitory effect of spinosin on seven cytochrome P450 enzymes(CYP1A1,CYP2B6,CYP2C8,CYP2C9,CYP2C19,CYP2D6 and CYP3A4)from human liver microsome as analyzed by UHPLC-Q-TOF-MS method.Methods: Seven cytochrome P450 probe substrate metabolites(including tacrine 1-hydroxylation,bupropion-hydroxylation,amodiaquine N-deethylation,diclofenac 4'-hydroxylation,s-mephenytoin 4'-hydroxylation,dextromethorphan O-demethylation and midazolam 1-hydroxylation)were measured by UHPLC-Q-TOF-MS in a positive electrospray ionization mode,and the inhibitory effect was evaluated with IC50 values.Results: Spinosin showed no inhibitory potency on the activity of CYP1A1,CYP2B6,CYP2C8,CYP2C9,CYP2C19,CYP2D6 and CYP3A4,the IC50 values were 1714 ?mol·L-1,1158 ?mol·L-1,226.1 ?mol·L-1,2288 ?mol·L-1,80.59 ?mol·L-1,101.1 ?mol·L-1 and 1119 ?mol·L-1,respectively.Conclusion: The analytical method has acceptable accuracy,precision and sensitivity.Spinosin has no inhibitory effect on the activities of the seven cytochrome P450 enzymes.The probability of drug-drug interactions between spinosin and other drugs is very low.Part four The research and the establishment of the Lian Qiao Xin quality standardObjective: To study and build up the quality standard of the Lian Qiao Xin,and to provide the quantity gist for the Lian Qiao Xin that acquire from different resources.Methods: Consulting the standard of Forsythia suspensa and the characteristics of Lian Qiao Xin,the research studied the quality standard of Lian Qiao Xin from the character,identification,impurity test,content determination,etc.Results: The quality standard of Lian Qiao Xin was built up.Conclusion: The establishment of the quality standard is helpful to quality control of Lian Qiao Xin,and provides the gist for establish interfix standard.Part five Simultaneous quantification of eight constituents in Lian Qiao Xin by liquid chromatography tandem mass spectrometryObjective: To develop an HPLC-MS/MS method for the simultaneous determination of forsythoside A,phillyrin,forsythiaside,rutin,quercetin,isoquercitrin,ferulic acid and hesperidin in Lian Qiao Xin.Methods: Chromatographic separation was performed on a Diamonsil C18 column(150 mm×4.6 mm,5 ?m)with linear gradient elution of methanol and 0.1% formic acid(v/v)at a flow rate of 800 ?L·min-1,the injection volume was 10 ?L.Multiple-reaction monitoring(MRM)was employed with switching electrospray ion source polarity in a negative mode.The ion spray voltage was set at-4500 V and the turbo spray temperature was maintained at 650?.Results: All calibration curves showed good linearity within the test ranges.The average recoveries of the compounds ranged from 98.1% to 101.8%,the precisions(RSD)for the investigated components were less than 0.84%.Conclusion: A efficient,rapid and sensitive method was first established for the qualitation and quantification of 8 major components in Lian Qiao Xin.The satisfactory results demonstrated that the method could be applied as a reliable quality control method for Lian Qiao Xin.