具有高灵敏度和选择性的分析技术对于临床样本的定量分析至关重要。液相色谱串联质谱法是临床化学的黄金标准。然而，串联质谱仪的资本支出和维护成本较高。我们最近表明，通过执行增强的源内碎片化/注释（EISA）结合相关的离子监测，可以使用更简单的单质谱探测器产生非常相似的结果。在这里，我们提供了一个逐步优化EISA分析条件的方案，以便于任何受过良好液相色谱-质谱法培训的人员都可以遵循并将此技术应用于任何给定的分析物。我们使用2-羟基戊二酸（2-HG）作为范例，该物质是一种临床相关代谢物，其D-对映异构体被认为是与突变异构酸脱氢酶1或2（IDH1 / 2）相关的癌症的“癌代谢物”。我们包括确定定量稳健性的程序，并展示这些程序与分析细胞中的DL-2-羟基戊二酸以及患有IDH1 / 2突变的急性髓性白血病患者的血清样本相关的结果。这种EISA-质谱法方案是一种广泛适用且成本低廉的方法，可用于小分子的定量，并且已经开发用于单四极杆和飞行时间质谱分析仪。© 2023年 Springer Nature Limited。

Analytical techniques with high sensitivity and selectivity are essential to the quantitative analysis of clinical samples. Liquid chromatography coupled to tandem mass spectrometry is the gold standard in clinical chemistry. However, tandem mass spectrometers come at high capital expenditure and maintenance costs. We recently showed that it is possible to generate very similar results using a much simpler single mass spectrometry detector by performing enhanced in-source fragmentation/annotation (EISA) combined with correlated ion monitoring. Here we provide a step-by-step protocol for optimizing the analytical conditions for EISA, so anyone properly trained in liquid chromatography-mass spectrometry can follow and apply this technique for any given analyte. We exemplify the approach by using 2-hydroxyglutarate (2-HG) which is a clinically relevant metabolite whose D-enantiomer is considered an 'oncometabolite', characteristic of cancers associated with mutated isocitrate dehydrogenases 1 or 2 (IDH1/2). We include procedures for determining quantitative robustness, and show results of these relating to the analysis of DL-2-hydroxyglutarate in cells, as well as in serum samples from patients with acute myeloid leukemia that contain the IDH1/2 mutation. This EISA-mass spectrometry protocol is a broadly applicable and low-cost approach for the quantification of small molecules that has been developed to work well for both single-quadrupole and time-of-flight mass analyzers.© 2023. Springer Nature Limited.