UHPLC-MS/MS技术同时测定大鼠灌胃白花蛇舌草水提取液后血浆中3种二萜类成分含量及其药代动力学研究

目的：建立超高效液相色谱-质谱联用（UHPLC-MS/MS）方法，同时测定大鼠灌胃白花蛇舌草水提取物后血浆中去乙酰车叶草苷酸、去乙酰车叶草苷酸甲酯和京尼平苷酸的含量。方法：使用C₁₈色谱柱（100 mm×2.1 mm，1.8 μm），以乙腈-0.1%甲酸水为流动相梯度洗脱，流速为0.3 mL·min^-1；采用质谱检测法，多反应监测（MRM）方式检测。结果：各成分线性良好，r²均≥ 0.993 4。各成分日内及日间精密度均在15%以内，准确度回收率范围为91.8%~99.0%。血浆中各待测物及内参提取回收率均≥ 94.5%。去乙酰车叶草苷酸、去乙酰车叶草苷酸甲酯及京尼平苷酸分别在灌胃后0.5、3和3 h达到最高浓度，AUC_（0-t）分别为（1 281.95±1 079.75）、（1 679.10±681.93）、（129.35±69.23）ng·L^-1·h^-1，AUC_（0-∞）分别为（1 373.21±1 054.31）、（2 018.92±1 102.21）、（152.94±71.34）ng·L^-1·h^-1，t_1/2分别为（6.50±5.95）、（8.54±4.14）、（5.22±2.11）h，MRT_（0-t）分别为（6.08±0.86）、（7.98±2.59）、（8.31±3.35）h。结论：该UHPLC-MS/MS方法成功地应用于大鼠灌胃白花蛇舌草提取物后的3种二萜类成分药物代谢动力学研究。

Objective:To develop and validate a rapid and sensitive ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of deacetylasperulosidic acid, deacetylasperulosidic acid methyl ester and geniposidic acid in rat plasma after oral administration of Oldenlandia diffusa (Willd.) water extract.Methods:The analytes were separated on a C₁₈ column (100 mm×2.1 mm, 1.8 μm) with acetonitrile-0.1% aqueous formic acid solution as the mobile phase.The flow rate was set at 0.3 mL·min^-1.The analytes were monitored by mass spectrometry with multiple reaction monitoring (MRM).Results:The linearity r² was ≥ 0.993 4 for all the analytes.The intra-and inter-day precision were all within 15%, and the accuracies ranged from 91.8% to 99.0%.The mean extraction recoveries of analytes and IS from rat plasma were all ≥ 94.46%.Deacetylasperulosidic acid, deacetylasperulosidic acid methyl ester and geniposidic acid ester reached highest concentrations at 0.5, 3 and 3 h after oral administration.The pharmacokinetic of deacetylasperulosidic acid, deacetylasperulosidic acid methyl ester and geniposidic acid ester were AUC_(0-t):(1 281.95±1 079.75) ng·L^-1·h^-1, (1 679.10±681.93) ng·L^-1·h^-1, (129.35±69.23) ng·L^-1·h^-1, AUC_(0-∞):(1 373.21±1 054.31) ng·L^-1·h^-1, (2 018.92±1 102.21) ng·L^-1·h^-1, (152.94±71.34) ng·L^-1·h^-1, t_1/2:(6.50±5.95) h, (8.54±4.14) h, (5.22±2.11) h, MRT_(0-t):(6.08±0.86) h, (7.98±2.59) h and (8.31±3.35) h.Conclusion:The developed UHPLC-MS/MS method is successfully applied to pharmacokinetic study of these compounds in rats after oral administration of Oldenlandia diffusa (Willd.) extract.

[1] CHEN R,HE J,TONG X,et al.The Hedyotis diffusa Willd.(Rubiaceae):a review on phytochemistry,pharmacology,quality control and pharmacokinetics[J].Molecules,2016,21(6):710
[2] CHEN Y,LIN Y,LI Y,et al.Total flavonoids of Hedyotis diffusa Willd inhibit inflammatory responses in LPS-activated macrophages via suppression of the NF-κB and MAPK signaling pathways[J].Exp Ther Med,2016,11(3):1116
[3] YE J,LIU M,ZHANG X,et al.Chemical profiles and protective effect of Hedyotis diffusa Willd. in lipopolysaccharide-induced renal inflammation mice[J].Int J Mol Sci,2015,16(11):27252
[4] XIN G,CHANG L,TANG YL,et al.Effect of Hedyotis diffusa water extract on protecting human hepatocyte cells(LO2)from H₂O₂-induced cytotoxicity[J].Pharm Biol,2016,54(7):1148
[5] KUO YJ,LIN JP,HSIAO YT,et al.Ethanol extract of Hedyotis diffusa Willd. affects immune responses in normal Balb/c mice in vivo[J].Vivo,2015,29(4):453
[6] LIN J,LI Q,CHEN H,et al.Hedyotis diffusa Willd.extract suppresses proliferation and induces apoptosis via IL-6-inducible STAT3 pathway inactivation in human colorectal cancer cells[J].Oncol Lett,2015,9(4):1962
[7] LI YL,ZHANG J,MIN D,et al.Anticancer effects of 1,3-dihydroxy-2-methylanthraquinone and the ethyl acetate fraction of Hedyotis diffusa Willd. against HepG2 carcinoma cells mediated via apoptosis[J].PLoS One,2016,11(4):e151502
[8] SUN G,WEI L,FENG J,et al.Inhibitory effects of Hedyotis diffusa Willd.on colorectal cancer stem cells[J].Oncol Lett,2016,11(6):3875
[9] ZHANG L,ZHANG J,QI B,et al.The anti-tumor effect and bioactive phytochemicals of Hedyotis diffusa Willd. on ovarian cancer cells[J].J Ethnopharmacol,2016,192:132
[10] LIN J,WEI L,SHEN A,et al.Hedyotis diffusa Willd extract suppresses sonic hedgehog signaling leadingto the inhibition of colorectal cancer angiogenesis[J].Int J Oncol,2013,42(2):651
[11] ZHANG P,ZHANG B,GU J,et al.The study of the effect of Hedyotis diffusa on the proliferation and the apoptosis of the cervical tumor in nude mouse model[J].Cell Biochem Biophys,2015,72(3):783
[12] PARK KS,KIM BH,CHANG IM.Inhibitory potencies of several iridoids on cyclooxygenase-1,cyclooxygnase-2 enzymes activities,tumor necrosis factor-α and nitric oxide production in vitro[J].Evid Based Complement Alternat Med,2010,7(1):41
[13] KIM SJ,KIM KM,PARK J,et al.Geniposidic acid protects against D-galactosamine and lipopolysaccharide-induced hepatic failure in mice[J].J Ethnopharmacol,2013,146(1):271
[14] FERNANDO AS,OLIVEIRA CFD,ZAVERUCHA DVT,et al.Morinda citrifoliaLinn.Reduces parasite load and modulates cytokines and extracellular matrix proteins in C57BL/6 mice infected with leishmania amazonensis[J].Plos Neglect Trop D,2016,10(8):e4900
[15] LIU K,YAN L,YAO G,et al.Estimation of p-coumaric acid as metabolite of E-6-O-p-coumaroyl scandoside methyl ester in rat plasma by HPLC and its application to a pharmacokinetic study[J].J Chromatogr B,2006,831(1-2):303
[16] LI YL,ZHANG J,MIN D,et al.Anticancer effects of 1,3-dihydroxy-2-methylanthraquinone and the ethyl acetate fraction of Hedyotis diffusa Willd.against HepG2 carcinoma cells mediated via apoptosis[J].PLoS One,2016,11(4):e151502
[17] YANG T,YANG YH,YANG JY,et al.Fingerprint of Hedyotis diffusa Willd.by HPLC-MS[J].Phytochem Anal,2008,19(6):487