Quantification of 22 plasma amino acids combining derivatization and ion-pair LC-MS/MS
Quantification of 22 plasma amino acids combining derivatization and ion-pair LC-MS/MS
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DOI：
10.1016/j.jchromb.2011.01.010
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发表时间：
2011-03-01
影响因子：
3
通讯作者：
Peissner, Wolfgang
中科院分区：
文献类型：
--
作者：
Harder, Ulrike;Koletzko, Berthold;Peissner, Wolfgang
对氨基酸进行省时且全面的定量仍然是一个挑战。我们开发了一种灵敏而精确的方法，用于从非常小的血浆和血清体积中定量分析氨基酸。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 ILL 血浆或血清添加到含有稳定同位素标准品的沉淀试剂中。蛋白质沉淀后，将上清液干燥并与 3 N HCl 丁醇一起孵育，以提高色谱分离和电离效率。使用离子对（七氟丁酸）反相色谱法与三重四极杆质谱联用来分离氨基酸丁酯。所建立的方法能够定量分析 22 种氨基酸、所有 20 种蛋白氨基酸、鸟氨酸和瓜氨酸。半胱氨酸以胱氨酸测量。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。在定量的同时，使用定性离子验证每个样品中的分析物身份。显微方法非常灵敏且准确。血浆分析的测定内精度为2.6-10.1%。通过比较外部参考样品确定的绝对准确度为 82-117.7%。对于婴儿和成人临床样品的代表性范围内的所有化合物，都证明了检测响应具有出色的线性。所有分析物的定量下限均在 1 μmol/L 范围内。总之，该方法非常适合临床研究和代谢组学研究中对大量样本进行经济有效的高通量分析。 (C) 2011 Elsevier B.V. 保留所有权利。
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 ILL of plasma or serum are added to precipitation reagent containing stable isotope standards. After protein precipitation, the supernatants is dried and incubated with 3 N 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 mu mol/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. (C) 2011 Elsevier B.V. All rights reserved.