自动化在线固相萃取HPLC/MS/MS法分析比格犬血浆中的葛根素含量

An on-line solid phase extraction column switching liquid chromatography tandem mass spectrometry system to quantification of puerarin in beagle plasma

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。

Objectives：To develop a method to quantitate puerarin in beagle plasma samples by on-line solid phase extraction column switching liquid chromatography tandem mass spectrometry, and to study the pharmacokinetics（PK） of puerarin preparationsa in beagles. Methods：The mobile phase of analytical column consisted of acetonitrile/methanoL/0.1% formic acid （25/25/50） at a flow-rate of 0.2 ml,/min. The analytical column was Grace Vydac C8 column. The mobile phase of SPE extraction column consisted of acetonitrile/0.1% formic acid （ 5/95 ） at a flow-rate of 2.0 ml/min. SPE extraction column was packed with Zorbax C18, with average particle size 50 μm. Puerarin was analyzed by a linear ion trap mass spectrometer, LTQ-MS, operating in the negative ion and selective reaction monitoring （SRM） acquisition mode. The SRM detection setup for the analytic peurarin was m/^2 415.2→195.2. Results：Validation of the methodology demonstrated that the linear calibration curves covered the range of 0.39-400 ng/ml, the determinate coefficients（ r^2 ） were above 0.999. The lower limit of detection （LLOD） was 0.39 ng/ml. The intra- and inter-batch precisions were less than 7.6% and 6. 4%, respectively. The accuracy of beagle plasma samples containing puerarin at the QC levels was -0.51% , 1.20% , and - 2.31%, respectively. The single run time of an individual sample was 6.5 min. Conclusion： The established on-line SPE-LC/MS/MS system needs no complicated pretreatment procedures of samples, the analytical speed is rapid, and the specificity, linearity, sensitivity, and precision of the methodology fulfil the requirements of PK study. The results showed that the developed method is feasible and reliable to be applied in preclinical or clinical PK studies of isoflavone drugs.