Department of Pathology, Stanford University, Palo Alto, CA, United States.
Stanford University Medical Center, Palo Alto, CA, United States.
J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Jan 1;944:166-74. doi: 10.1016/j.jchromb.2013.11.017. Epub 2013 Nov 18.
The quantitation of free amino acids from physiologic samples is essential for diagnosing and monitoring patients with inherited metabolic disorders. Current methods are hindered by long preparative and/or analysis times, expensive reagents, and often suboptimal performance characteristics. To overcome these challenges, a improved method for amino acid analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated. Samples were deproteinized with sulfosalicylic acid and supernatants diluted with tridecafluoroheptanoic acid. Chromatographic separation of amino acids occurred using two columns, with conditions favoring resolution of isobaric compounds and minimizing ion suppression. Eluted compounds were detected by selective reaction monitoring, and quantitated by relating peak areas of amino acids to externally run standards. Validation studies evaluated linearity, within- and between-run imprecision, lower limits of detection and quantification for 33 amino acids, and correlation with the Biochrom 30 Amino Acid Analyzer. Total run time including re-equilibration was 15min per sample. Within-run precision averaged 2.8% for all compounds, with an average linear correlation coefficient of 0.995. The majority of compounds were reliably quantitated at ≤0.1μM. Between-run precision averaged 4.0%. Results showed excellent correlation with the Biochrom 30 amino acid analyzer with an average overall correlation of 0.908. We conclude that our method is extremely sensitive, specific and reproducible and represents an improvement over other currently available technologies.
从生理样本中定量游离氨基酸对于诊断和监测遗传性代谢紊乱患者至关重要。当前的方法受到制备和/或分析时间长、试剂昂贵以及性能特征通常不佳的限制。为了克服这些挑战，已经开发并验证了一种使用液相色谱-串联质谱 (LC-MS/MS) 进行氨基酸分析的改进方法。样品用磺基水杨酸沉淀，上清液用十三氟庚酸稀释。使用两根柱子进行氨基酸的色谱分离，条件有利于分离等摩尔化合物并最小化离子抑制。洗脱化合物通过选择反应监测进行检测，并通过将氨基酸的峰面积与外部运行标准相关联来定量。验证研究评估了 33 种氨基酸的线性、批内和批间精密度、检测限和定量限，以及与 Biochrom 30 氨基酸分析仪的相关性。每个样品的总运行时间包括重新平衡为 15 分钟。所有化合物的批内精密度平均为 2.8%，平均线性相关系数为 0.995。大多数化合物在 ≤0.1μM 时可可靠定量。批间精密度平均为 4.0%。结果表明与 Biochrom 30 氨基酸分析仪具有极好的相关性，平均总相关性为 0.908。我们得出结论，我们的方法非常灵敏、特异和重现，并且代表了对其他现有技术的改进。
