University of Munich Medical Centre, Dr. von Hauner Children's Hospital, Div. Metabolic and Nutritional Medicine, Lindwurmstr. 4, D-80337 Munich, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Mar 1;879(7-8):495-504. doi: 10.1016/j.jchromb.2011.01.010. Epub 2011 Jan 14.
Time efficient and comprehensive quantification of amino acids continues to be a challenge. We developed a sensitive and precise method for quantitative analysis of amino acids from very small plasma and serum volumes. Ion-pair chromatography of amino acid butyl esters proved to provide an optimal combination of selectivity, sensitivity and robustness. 10 μL of plasma or serum are added to precipitation reagent containing stable isotope standards. After protein precipitation, the supernatants is dried and incubated with 3N butanolic HCl for improving chromatographic separation and ionization efficiency. Amino acid butyl esters are separated using ion-pair (heptafluorobutyric acid) reversed-phase chromatography coupled to triple quadrupole mass spectrometry. The established method enables quantitative analysis of 22 amino acids, all 20 proteinogenic amino acids, ornithine and citrulline. Cysteine is measured as cystine. The combination of precipitation, derivatization and chromatographic separation effectively avoids ion suppression and coelution. Simultaneous with quantification, analyte identity is verified in each sample using qualifier ions. The micro-method is very sensitive and accurate. The intra-assay precision for the analysis of plasma was 2.6-10.1%. Absolute accuracy as determined by comparison of external reference samples was 82-117.7%. Excellent linearity of detection response was demonstrated for all compounds in the range representative for clinical samples from infants and adults. Lower limits of quantification were in the range of 1 μmol/L for all analytes. In conclusion, the method is ideally suited for cost-effective high-throughput analysis of large numbers of samples in clinical studies and metabolomics research.
氨基酸的高效、全面定量分析一直是一个挑战。我们开发了一种灵敏、精确的方法，可从小体积的血浆和血清中定量分析氨基酸。氨基酸丁酯的离子对色谱法被证明提供了选择性、灵敏度和稳健性的最佳组合。将 10 μL 的血浆或血清加入含有稳定同位素标准品的沉淀试剂中。在蛋白质沉淀后，将上清液干燥并与 3N 丁醇盐酸孵育，以改善色谱分离和离子化效率。使用离子对（七氟丁酸）反相色谱法与三重四极杆质谱联用分离氨基酸丁酯。所建立的方法能够定量分析 22 种氨基酸、所有 20 种蛋白质氨基酸、鸟氨酸和瓜氨酸。半胱氨酸以胱氨酸的形式进行测量。沉淀、衍生化和色谱分离的结合有效地避免了离子抑制和共洗脱。在定量分析的同时，使用特征离子在每个样品中验证分析物的身份。微量方法非常灵敏和准确。分析血浆的批内精密度为 2.6-10.1%。通过与外部参考样品的比较确定的绝对准确度为 82-117.7%。所有化合物在代表婴儿和成人临床样本范围内的检测响应均表现出优异的线性。所有分析物的定量下限均在 1 μmol/L 范围内。总之，该方法非常适合在临床研究和代谢组学研究中进行具有成本效益的高通量、大量样本分析。
