液相色谱-串联质谱法检测干血点中的同型半胱氨酸

建立了一种高通量液相色谱-串联质谱技术检测干血点(DBS)中同型半胱氨酸(homocycteine, Hcy)的方法。以DBS为样本,homocystine-D8为同位素内标,二硫苏糖醇(DTT)为蛋白结合态Hcy的还原剂,使用含0.1%(v/v)甲酸、0.05%(v/v)三氟乙酸的乙腈溶液萃取。整个前处理过程使用自动移液平台及96孔板实现高通量自动化操作。处理后的样本经过Phenomenex CN柱分离,使用多反应监测模式进行LC-MS/MS分析。结果表明:Hcy的检出限为0.12 μ mol/L(S/N=3),定量限为0.46 μ mol/L(S/N=10)。Hcy在1.16~148.00 μ mol/L范围内线性关系良好,R²=0.994。Hcy的平均回收率为(103.0±4.97)%~(112.0±2.13)%,日内相对标准偏差(RSD)为1.9%~4.6%,日间RSD为1.5%~7.1%。DBS样本在不同温度(-4、-20、22和37℃)下储存不同时间(0、1、2、3、4、5、6、14天)后的稳定性试验显示样本总体RSD<15%,经前处理后的样本在48 h内的稳定性试验显示样本总体RSD<5%。该方法与传统生化分析方法的相关性好(R²=0.9818, n=47)。

Determination of homocysteine in dried blood spots using liquid chromatography-tandem mass spectrometry

A high-throughput method to measure homocysteine (Hcy) in dried blood spots (DBS) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established. The DBS sample processing includes adding homocystine-D8 as internal standard, dithiothreitol (DTT) as reducing agent for bound Hcy forms, acetonitrile containing 0.1% (v/v) formic acid and 0.05% (v/v) trifluoroacetic acid for Hcy extraction. This procedure was carried out in a 96-well plate format in an automated liquid handling platform to facilitate high-throughput analysis. The processed samples were separated on a Phenomenex CN column and quantitated by LC-MS/MS in multiple reaction monitoring (MRM) mode. The limit of detection was 0.12 μ mol/L (S/N=3) and the limit of quantification was 0.46 μ mol/L (S/N=10). The linear range was 1.16-148.00 μ mol/L (R²=0.994). The average recoveries were (103.0±4.97)%-(112.0±2.13)%. The intra-day relative standard deviations (RSDs) were 1.9%-4.6% and the inter-day RSDs were 1.5%-7.1%. DBS sample stability was confirmed by measuring same DBS samples stored for 0, 1, 2, 3, 4, 5, 6 and 14 days at -4, -20, 22 and 37℃, and an overall sample RSD<15% was found under each temperature. Processed sample stability within 48 hours was also confirmed with an overall sample RSD<5%. The comparison of this method with conventional biochemistry assay was made by measuring 47 blood samples both in an automated biochemistry analyzer (samples in plasma form) and with the LC-MS/MS method (samples in DBS form), which showed excellent correlation (R²=0.982).