Mak Justin, Cowan Tina M, Le Anthony
Biochemical Genetics Laboratory, Stanford Health Care, Palo Alto, CA, USA.
Department of Pathology, Stanford University School of Medicine, Palo Alto, CA, USA.
Methods Mol Biol. 2019;2030:85-109. doi: 10.1007/978-1-4939-9639-1_8.
Quantitative amino acid analysis has diverse applications in clinical diagnostics, biomedical research, and agriculture. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables more rapid and specific detection of amino acids in comparison to traditional, gold-standard ninhydrin-based methods. However, triple quadrupole mass spectrometers are unable to definitively differentiate isomers and are susceptible to ion suppression, both of which prevent accurate quantitation. Therefore, appropriate chromatography must be applied before ionization.We have shown that two-dimensional LC enables rapid and specific amino acid quantitation without derivatization by resolving isomers, such as alloisoleucine, isoleucine, and leucine, and reducing matrix effects (Le et al., J Chromatogr B Analyt Technol Biomed Life Sci 944:166-174, 2014). In this clinically validated protocol, we provide an updated description of the chromatographic setup and selected reaction monitoring (SRM) transitions. Then, we describe sample processing for serum, plasma, urine, cerebral spinal fluid, and dried blood spots. Most importantly, we outline a singular quantitative design for efficient data analysis of the listed sample types and quality assurance strategies to ensure test fidelity. Lastly, we share extensive knowledge critical to the success of this method. A liquid sample can be processed and be ready for injection within 5 min, and each sample is analyzed by the MS in 14.5 min.
定量氨基酸分析在临床诊断、生物医学研究和农业领域有多种应用。与传统的基于茚三酮的金标准方法相比，液相色谱 - 串联质谱法（LC-MS/MS）能够更快速、更特异性地检测氨基酸。然而，三重四极杆质谱仪无法明确区分异构体，并且易受离子抑制影响，这两者都会妨碍准确定量。因此，在离子化之前必须应用适当的色谱法。我们已经表明，二维液相色谱法能够通过分离异构体（如别异亮氨酸、异亮氨酸和亮氨酸）并减少基质效应，在不进行衍生化的情况下实现快速、特异性的氨基酸定量（Le等人，《色谱杂志B：分析技术、生物医学与生命科学》944:166 - 174，2014年）。在这个经过临床验证的方案中，我们提供了色谱设置和选择反应监测（SRM）转换的更新描述。然后，我们描述了血清、血浆、尿液、脑脊液和干血斑的样品处理方法。最重要的是，我们概述了一种单一的定量设计，用于对所列样品类型进行高效数据分析以及质量保证策略，以确保检测的准确性。最后，我们分享了对该方法成功至关重要的广泛知识。一份液体样品可以在5分钟内处理好并准备好进样，每个样品由质谱仪在14.5分钟内进行分析。
