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

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(50 μm).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.     

Simultaneous quantification of urea,uric acid,and creatinine in human urine by liquid chromatography/mass spectrometry

In forensic analysis, the identification of urine or human urine among unknown liquids plays an important role. Urea, uric acid, and creatinine are major organic compounds found in human urine. Previous studies have reported that the concentration quotients of these three compounds can be used as an index for the identification of human urine. Here we describe a method for the simultaneous quantification of urea, uric acid, and creatinine in human urine by liquid chromatography/mass spectrometry (LC/MS), with the aim of forensic identification of human urine. Separation of the three analytes was achieved by hydrophilic interaction chromatography, using a TSK gel Amide-80 column with a mobile phase composed of acetonitrile and ammonium acetate aqueous solution, coupled with detection using a mass spectrometer. For quantification, melamine and violuric acid were used as internal standards. Human urine samples were pretreated for LC/MS analysis by dilution with LC mobile phase, followed by centrifugation and filtration. The analytes and internal standards were separated within 9 min. The linear ranges were 2.0–40.0, 0.10–1.60, and 0.13–2.00 mg/mL for urea, uric acid, and creatinine, respectively, with correlation coefficients > 0.99. The intra- and inter-day accuracies of the analytes were − 10.6% to 7.4%, and the precision was within 7.6%. For all analytes, no significant matrix effects were observed and recoveries ranged from 95.4% to 104.6%. Quantitative results of 3 analytes were obtained within their linear range from 10 human urine samples and the quotients, UA/UN × 20 and UA/Cre, were calculated based on previous reports.