Division of Metabolism and Nutritional Medicine, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-University of Munich, Lindwurmstr. 4, D-80337 Munich, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Jan 1;879(1):83-9. doi: 10.1016/j.jchromb.2010.11.016. Epub 2010 Nov 21.
Elevation of plasma homocysteine concentration is recognized as an independent predictor of cardiovascular disease risk. Therefore, quantification of homocysteine and related sulphur amino acids cysteine and methionine from plasma samples is routinely performed in clinical laboratories. Due to the highly hydrophilic character of these amino acids, previously reported LC-MS methods often suffered from very short chromatographic retention resulting in inadequate separation from matrix background and possible co-eluents. In the present method, aqueous normal phase (ANP) chromatography was introduced to improve chromatographic separation for liquid chromatography-electrospray ionization tandem mass spectrometry. Selective qualification of analytes and internal standards was achieved by qualifier ion monitoring. Using this enhanced selectivity, spurious co-eluents were identified and separated from the analyte signal by optimization of chromatographic conditions. Method validation proved high precision and accuracy (intra-assay reproducibility 1.2-4.3% CV, inter-assay reproducibility 3.4-6.1% CV, accuracy 91.3-105.9%). Total cycle time of 7 min and low costs per sample allow high-throughput application in clinical diagnostics and research trials.
血浆同型半胱氨酸浓度升高被认为是心血管疾病风险的独立预测因子。因此，临床实验室通常会定量检测血浆样本中的同型半胱氨酸以及相关的含硫氨基酸半胱氨酸和蛋氨酸。由于这些氨基酸具有很强的亲水性，之前报道的 LC-MS 方法通常存在色谱保留时间很短的问题，导致与基质背景和可能的共洗脱物无法充分分离。在本方法中，引入了水相正相（ANP）色谱法以改善用于液相色谱-电喷雾串联质谱法的色谱分离。通过 qualifier 离子监测实现了对分析物和内标物的选择性定量分析。通过优化色谱条件，使用这种增强的选择性可以从分析物信号中识别和分离出假共洗脱物。方法验证表明，该方法具有很高的精密度和准确度（日内重复性 1.2-4.3%CV，日间重复性 3.4-6.1%CV，准确度 91.3-105.9%）。总分析周期为 7 分钟，每个样本的成本低，可实现高通量应用于临床诊断和研究试验。
