用液相色谱-串联质谱法测定血浆总同型半胱氨酸

Determination of total homocysteine in human plasma by liquid chromatography-mass/mass spectrometry method

目的:建立血浆中总同型半胱氨酸(tHcy)的液相色谱-串联质谱(LC-MS/MS)测定法。方法:血浆tHcy及内标氘代高胱氨酸经巯基乙醇还原后,用10％三氯乙酸沉淀血浆蛋白,经液相色谱分离后,采用电喷雾离子化-串联质谱进行多反应监测(MRM)检测,离子选择通道分别为m/z:136.2/46.9(Hcy),140.2/48.9(Hcy-d4,氘化Hcy)。Hcy、Hcy-d4的保留时间均为6.3 min。结果:Hcy在2.5-640μmol/L范围内线性关系良好。本法的最低检测浓度为0.3 μmol/L(S/N≥5)。批内、批间精密度分别为1.97％-8.79％、2.55％-4.14％。方法回收率为100.3％-109.1％。结论:该方法的预处理快速、简便,检测专一灵敏,试剂成本低廉,可适应开展大规模临床Hcy研究的需要。

Objective: To establish a method for determining total homocysteine (tHcy) in plasma with liquid chromatography-mass/mass spectrometry(LC-MS/MS) method. Methods: After the tHcy in plasma and the D-homocystine (internal standard) were reduced with mercaptoethanol, the proteins were deposited with 10% trichloroacetic acid. The analysis was performed using the multiple reaction monitoring in which tHcy and Hcy-d4were detected through the transition from the precursor to the product ion (m/z 136. 2-46. 9 and m/z 140. 1-48. 9). The retention time of tHcy and Hcy-d4was 6. 3 min. Results: Calibrations of Hcy between 2. 5 and 640 μmol/L exhibited consistent linearity and reproducibility. At a plasma concentration of 0. 3 F11mol/L,the signal-to-noise ratio for tHcy was 5 : 1. Intra- and interassay RSD were 1. 97%-8.79% and 2. 55%-4. 14% , respectively. Method recovery of tHcy was 100. 3%-109. 1%. Conclusion:The pretreatment of this method is simple and quick. Analysis has a high specificity and the reagent cost is also low. So this method is very suitable for the need of wide clinical homocysteine research.