液相色谱-串联质谱法测定人血浆中二甲双胍的浓度

目的: 建立LC-MS/MS法测定人血浆中二甲双胍的浓度。 方法: 人血浆样本以乙腈沉淀蛋白后,选用Zorbax SB-C₁₈ Narrow-Bore色谱柱(150 mm ×2.1 mm,5 μm),以甲醇-10 mmol·L^-1乙酸铵(含1%甲酸)(5∶ 95)为流动相,流速为0.3 mL·min^-1;选用API3200型三重四极杆串联质谱仪的多重反应监测(MRM)扫描方式进行监测,电喷雾离子化源,正离子方式,选择监测离子反应分别为m/z 130.1→m/z 71.0(二甲双胍)和m/z 147.1→m/z 58.2(内标米曲肼)。 结果: 二甲双胍和米曲肼的保留时间分别为1.27 min和1.26 min;血浆中二甲双胍的线性范围为0.010~3.000 mg·L^-1(r>0.99),定量下限为0.010 mg·L^-1;日内、日间RSD均小于6%;相对偏差(RE)均在±6%的范围以内;平均提取回收率为(86.6±5.4)%;稳定性试验中,在各种贮存条件下血浆中二甲双胍均较稳定。 结论: 该方法快速、灵敏,专属性强,重现性好,适用于人血浆中二甲双胍浓度的测定,可应用于盐酸二甲双胍肠溶片的人体生物等效性研究。

Objective: To develop an LC-MS/MS method for determination of metformin in human plasma. Methods: After protein precipitation with acetonitrile,the analyte and internal standard,mildronate,were separated on a Zorbax SB-C₁₈(150 mm×2.1 mm,5 μm) Narrow-Bore analytical column using the mobile phase of methanol and 10 mmol·L^-1 ammonium acetate (containing 1% formic acid) (5∶ 95) at a flow rate of 0.3 mL·min^-1.Detection was carried out by electrospray positive ionization mass spectrometry in the multiple reaction monitoring (MRM) mode.The MRM transitions of m/z 130.1→m/z 71.0 and m/z 147.1→m/z 58.2 were used to quantify metformin and internal standard,respectively. Results: Metformin and internal standard were eluted at 1.27 and 1.26 min,respectively.The calibration curve was linear over the concentration range of 0.010-3.000 mg·L^-1 with the lower limit of quantitation (LLOQ) 0.010 mg·L^-1.Intra and inter day RSD were both less than 6%,and the relative errors(RE) were within 6%.The mean extract recoveries were (86.6±5.4)%.In the stability studies,metformin in plasma was found to be stable under various storage conditions. Conclusion: It is a rapid,sensitive,selective and reliable method for the determination of metformin in human plasma.The method is successfully applied to a bioequivalence study of metformin hydrochloride enteric-coated tablets in healthy volunteers.