Li Shuijun, Jia Jingying, Liu Gangyi, Wang Wei, Cai Yongbao, Wang Yiping, Yu Chen
Central Laboratory, Shanghai Xuhui Central Hospital, Shanghai 200031, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Jul 1;870(1):63-7. doi: 10.1016/j.jchromb.2008.06.003. Epub 2008 Jun 6.
A liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for the determination of human plasma homocysteine (Hcy), an important independent risk factor for cardiovascular disease, with a simplified sample pretreatment procedure and a zero blank free of endogenous Hcy for calibrator/QC preparation. Following protein precipitation, chromatographic separation was performed on Hypersil Aquasil C18 column (50 mm x 2.1mm, 5 microm, Thermo) using mobile phase of aqueous 10% methanol containing 0.02% formic acid at 0.25 mL/min. Hcy and deuterated internal standard were detected in the multiple reaction monitoring mode with precursor to product ion transitions of m/z 136.1/90.0 and 140.1/94.0, respectively. The retention time was 1.2 min, and the total run time was 2 min per injection. A streamlined three-point calibration curve and one-point QC was used. Excellent linearity was observed with correlation coefficient (r)>0.99. The intra- and inter-batch were < or =3.24% and < or =4.04%, and accuracy was within +/-10%. Method comparison between the proposed method (y) and FPIA assay (x) demonstrated a correlation equation of y=1.003x + 0.4318 (r=0.9589). The developed method, improved for automation with cost-effective reagents, was proven to be suitable for high-throughput quantitative determination of Hcy in clinical practice by successfully applying it to the cardiovascular disease study.
建立并验证了一种液相色谱 - 电喷雾电离串联质谱法（LC - ESI - MS/MS），用于测定人血浆同型半胱氨酸（Hcy），这是心血管疾病的一个重要独立危险因素。该方法具有简化的样品预处理程序，且用于校准品/质量控制（QC）制备的零空白不含内源性Hcy。蛋白质沉淀后，在Hypersil Aquasil C18柱（50 mm×2.1mm，5μm，赛默飞世尔科技公司）上进行色谱分离，流动相为含0.02%甲酸的10%甲醇水溶液，流速为0.25 mL/min。在多反应监测模式下检测Hcy和氘代内标，其母离子到子离子的跃迁分别为m/z 136.1/90.0和140.1/94.0。保留时间为1.2分钟，每次进样总运行时间为2分钟。采用简化的三点校准曲线和一点QC。观察到良好的线性，相关系数（r）>0.99。批内和批间精密度分别≤3.24%和≤4.04%，准确度在±10%以内。所提出的方法（y）与荧光偏振免疫分析（FPIA）法（x）的方法比较显示相关方程为y = 1.003x + 0.4318（r = 0.9589）。所开发的方法通过使用具有成本效益的试剂改进了自动化，通过成功应用于心血管疾病研究，被证明适用于临床实践中Hcy的高通量定量测定。
