Jeong Hyeon-Cheol, Kim Therasa, Yang Deok-Hwan, Shin Kwang-Hee
College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, Korea.
Department of Hematology, Chonnam National University Hwasun Hospital, Hwasun 58128, Korea.
Transl Clin Pharmacol. 2018 Sep;26(3):134-140. doi: 10.12793/tcp.2018.26.3.134. Epub 2018 Sep 14.
This study aimed to develop a UPLC-MS/MS method for determining plasma levels of L-aspartic acid and L-asparagine and the activity of L-asparaginase. L-aspartic acid, L-asparagine, and L-aspartic acid-2,3,3-d were extracted from human plasma by protein precipitation with sulfosalicylic acid (30%, v/v). The plasma samples were analyzed using an Imtakt Intrada amino acid analysis column with 25 mM ammonium formate and 0.5% formic acid in acetonitrile as the mobile phase with step gradient method at a flow rate of 0.5 mL/min. The injection volume was 5 µL, and the total run time was 15 min. Inter- and intra-batch accuracies (%) ranged from 96.62-106.0% for L-aspartic acid and 89.85-104.8%, for L-asparagine, and the coefficient of variation (CV%) did not exceed 7%. The validation results for L-aspartic acid and L-asparagine satisfied the specified criterion, however, the results for L-asparaginase activity assay showed a borderline validity. This study could be a foundation for further development of therapeutic drug monitoring systems using UPLC-MS/MS.
本研究旨在开发一种超高效液相色谱-串联质谱法，用于测定血浆中L-天冬氨酸和L-天冬酰胺的水平以及天冬酰胺酶的活性。采用30%（v/v）磺基水杨酸通过蛋白质沉淀法从人血浆中提取L-天冬氨酸、L-天冬酰胺和L-天冬氨酸-2,3,3-d。血浆样品采用Imtakt Intrada氨基酸分析柱进行分析，以25 mM甲酸铵和0.5%甲酸的乙腈溶液为流动相，采用梯度洗脱法，流速为0.5 mL/min。进样量为5 μL，总运行时间为15 min。L-天冬氨酸的批间和批内准确度（%）范围为96.62 - 106.0%，L-天冬酰胺为89.85 - 104.8%，变异系数（CV%）不超过7%。L-天冬氨酸和L-天冬酰胺的验证结果符合规定标准，然而，天冬酰胺酶活性测定结果显示有效性处于临界状态。本研究可为进一步开发基于超高效液相色谱-串联质谱的治疗药物监测系统奠定基础。
