衍生化LC-MS/MS法测定人血浆中氨基葡萄糖的浓度及药代动力学研究

Determination of Glucosamine in Human Plasma by Pre-column Derivatization LC-MS/MS Method and Its Pharmacokinetics in Healthy Volunteers

目的:建立柱前衍生化高效液相色谱-串联质谱(LC-MS/MS)法测定人血浆中氨基葡萄糖的浓度,并研究该药在健康受试者体内的药代动力学特征。方法:采用乙腈沉淀蛋白和邻苯二甲醛/3-巯基丙酸(OPA/3-MPA)衍生化法对血浆样品进行前处理,LC-MS/MS法测定受试者血浆中氨基葡萄糖的浓度,并用BAPP2.0软件计算药动学参数。色谱分离采用Phenomenex ODS柱(150mm×4.6mm,5mm),流动相为甲醇-0.2%醋酸铵溶液(含0.1%甲酸),梯度洗脱;待测样品通过电喷雾正离子化以选择性反应离子监测方式进行检测,用于定量分析的离子反应分别为m/z384→118(氨基葡萄糖衍生物)和m/z326→147(内标酒石酸托特罗定)。结果:血浆中氨基葡萄糖在0.012～8.272mg.L-1范围内线性关系良好(r>0.9980),绝对回收率为87.9%～106.5%,批内、批间精密度RSD分别为4.4%～5.4%和9.8%～19.8%,定量限为0.012mg.L-1。其主要药动学参数Cmax为(1.03±0.47)mg.L-1,AUC为(4.32±1.41)h.mg.L-1,AUC0-∞为(4.34±1.41)h.mg.L-1,Tmax为(2.80±1.37)h,t1/2为(1.20±0.32)h,MRT为(3.74±0.63)h。结论:衍生化LC-MS/MS法可增强氨基葡萄糖在反相色谱系统中的保留,改善与内源性干扰物的分离,有效降低电喷雾质谱检测的基质效应,提高检测灵敏度和准确度,适用于氨基葡萄糖的临床药动学研究。

Objective：To establish a rapid and sensitive pre-column derivatization liquid chromatography-tandem mass spectrometric （LC-MS/MS） method for the determination of glucosamine in human plasma and the pharmacokinetics study on Chinese healthy volunteers.Methods：The sample was protein precipitated with acetonitrile,derivatized rapidly with o-phthalaldehyde（OPA）/3-mercaptopropinonic acid（3-MPA） and then determined by LC-MS/MS.The pharmacokinetic parameters were calculated by using the BAPP 2.0 program.Chromatographic separation was performed on a Phenomenex ODS column （150 mm×4.6 mm,5 μm） using gradient elution by a mobile phase consisting of methanol and an aqueous solution containing 0.2% ammonium acetate and 0.1% formic acid .The MS/MS detections were performed with positive ion ESI and selected reaction monitoring.The ion transitions were selected for the quantitation of glucosamine with m/z 384→118 for the glucosamine-OPA/3-MPA derivatives and m/z 326→147 for tolterodine tartrate.Results：The linear range of glucosamine was 0.012-8.272 mg ·L^-1,the correlation coefficient was above 0.998 0,the absolute recovery was 87.9%-106.5%,within- and between-batch precision （RSD） was 4.4%-5.4% and 9.8%-19.8%,respectively,and the LOQ was as low as 0.012 mg ·L^-1.The main pharmacokinetic parameters were as follows：Cmax was （1.03±0.47） mg ·L^-1,AUC was （4.32±1.41） h ·mg ·L^-1,AUC0-∞was （4.34±1.41） h ·mg ·L^-1,Tmax was （2.80±1.37） h,t1/2 was （1.20±0.32） h,and MRT was （3.74±0.63） h.Conclusion：The established pre-column derivatization LC-MS/MS method enhanced the chromatographic retention of glucosamine,improved the resolution with the endogenous substances,reduced the matrix effects significantly and guaranteed the sensitive,specific and accurate determination of glucosamine in plasma,which was suitable for the clinical investigation of glucosamine.