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目的:建立自动化在线固相萃取HPLC/MS/MS法测定犬血浆中葛根素的含量,研究葛根素制剂在比格犬体内的药代动力学。方法:分析流动相为甲醇-乙腈-0.1%甲酸溶液(25∶25∶50,v/v/v),流速为0.2 ml/min,分析色谱柱为Grace Vydac C8柱。在线固相萃取流动相为乙腈-0.1%甲酸溶液(5∶95,v/v),流速为2.0 ml/min,固相萃取柱的填料为Zobax C18(50μm)。配有电喷雾离子源(ESI)的线性离子阱LTQ质谱仪用于定量分析葛根素,采用选择离子反应监测(SRM)方式,用于定量分析的离子反应分别为m/z 415.2→m/z 195.2(葛根素)。结果:线性范围0.39~400 ng/ml(r2>0.999),最低定量限为0.39 ng/ml。日内、日间精密度(RSD%)分别小于7.6%和6.4%,低、中、高3个浓度的QC样品准确度分别平均为-0.51%,1.20%,-2.31%。单样品分析时间为6.5 min。结论:所建立的在线固相萃取HPLC/MS/MS法样品处理简便、分析测试速度快、灵敏度高、线性范围宽、精密度和准确度满足药代动力学研究的需要,可用于非临床和临床药代动力学研究。
OBJECTIVE: To establish an automated on-line HPLC-MS / MS method for the determination of puerarin in canine plasma and to study the pharmacokinetics of puerarin in beagle dogs. METHODS: The mobile phase consisted of methanol-acetonitrile-0.1% formic acid solution (25:25:50, v / v / v) at a flow rate of 0.2 ml / min and the analytical column was a Grace Vydac C8 column. The mobile phase consisted of acetonitrile-0.1% formic acid solution (5:95, v / v) at a flow rate of 2.0 ml / min. The solid phase extraction column was packed with Zobax C18 (50 μm). The linear ion trap LTQ mass spectrometer with electrospray ionization source (ESI) was used for the quantitative analysis of puerarin and the selective ion reaction monitoring (SRM) was used. The ion reactions for quantitative analysis were m / z 415.2 → m / z 195.2 (Puerarin). Results: The linear range was 0.39-400 ng / ml (r2> 0.999). The lowest limit of quantification was 0.39 ng / ml. The intra-day and inter-day precision (RSD%) were less than 7.6% and 6.4%, respectively. The accuracy of QC samples with low, middle and high concentrations were -0.51%, 1.20% and -2.31% respectively. Single sample analysis time was 6.5 min. CONCLUSION: The established HPLC-MS / MS method is simple, rapid and sensitive to analysis, with wide linear range, precision and accuracy to meet the needs of pharmacokinetic studies. It can be used in non-clinical and Clinical Pharmacokinetic Studies.