Phipps William S, Crossley Eric, Boriack Richard, Jones Patricia M, Patel Khushbu
Department of Pathology University of Texas at Southwestern Medical Center Dallas Texas.
Chemistry, Metabolic Disease and Mass Spectrometry Laboratories Children's Health Dallas Texas.
JIMD Rep. 2019 Nov 12;51(1):62-69. doi: 10.1002/jmd2.12080. eCollection 2020 Jan.
Amino acid analysis is central to newborn screening and the investigation of inborn errors of metabolism. Ion-exchange chromatography with ninhydrin derivatization remains the reference method for quantitative amino acid analysis but offers slow chromatography and is susceptible to interference from other co-eluting compounds. Liquid-chromatography tandem mass spectrometry (LC-MS/MS) provides a rapid and highly specific alternative, but sample preparation is frequently laborious and sometimes cost prohibitive. To address these limitations, we validated an LC-MS/MS method using the aTRAQ Reagents Application Kit with a modified protocol consuming only half reagents. Adequate performance for clinical specimen measurement of 26 amino acids with high clinical relevance was achieved. An automated liquid handler and modified calibration and normalization approaches were used to ensure reproducible assay performance. Linear measurement between 5 and 2000 μM was achieved for most analytes despite use of a small, 10 μl sample size. Overall the method achieved near substantially improved throughput and enabled use of smaller samples volumes for batched analyses of clinical samples.
氨基酸分析是新生儿筛查和先天性代谢缺陷病调查的核心。茚三酮衍生化离子交换色谱法仍是定量氨基酸分析的参考方法，但色谱分析速度慢，且易受其他共洗脱化合物的干扰。液相色谱串联质谱法（LC-MS/MS）提供了一种快速且高度特异的替代方法，但样品制备通常很繁琐，有时成本过高。为解决这些局限性，我们使用aTRAQ试剂应用试剂盒并采用仅消耗一半试剂的改良方案，验证了一种LC-MS/MS方法。该方法在测量26种具有高度临床相关性的氨基酸时表现良好。使用自动液体处理器以及改良的校准和归一化方法来确保检测性能的可重复性。尽管使用的样本量较小，仅10 μl，但大多数分析物在5至2000 μM之间实现了线性测量。总体而言，该方法显著提高了通量，并能够使用更小的样本量对临床样本进行批量分析。
