Objective: To establish an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of 22 amino acids contents in 4 kinds of compound amino acid injections. Methods: The gradient elution was achieved on an Agilent Zorbax SB-C18 column (150 mm×3.0 mm, 5 μm) with a binary elution system (phase A, 0.2% formic acid and 0.02% heptafluorobutyric acid in water; phase B, methanol). The column temperature was 50 ℃. The flow rate was 0.4 mL·min-1. The multiple reaction monitoring mode was chosen and detected with an ionization source operating in the positive ionization mode for data acquisition. The run time was 9.5 min. Results: The linear relationship was good for Gly, Ala, Val, Leu, Ile, Lys, Met, His, Phe, and Arg in the concentration ranges of 0.32-8 μg·mL-1, and for Ser, Pro, Thr, Asn, Orn, Asp, Glu, Ace, Trp, and Aty in the concentration ranges of 0.16-4 μg·mL-1, and for Tyr and Cyss in the concentration ranges of 0.032- 0.8 μg·mL-1 (all r >0.99). The intra-day and inter-day precision, the stability, and the repeatability were all good. The average sample recovery rates ranged from 90.0% to 101.3% (all RSD ≤5.9%, n=6). The method was used to simultaneously determine 22 amino acids contents in 4 kinds of compound amino acid injections. The ratios of detection content to identification content of 22 amino acids ranged from 86.67% to 102.86%. Conclusion: The method is simple to operate, rapid in analysis, strong in specificity, high in sensitivity, and accurate in determination results. It can be used to determine each amino acid contents in compound amino acid injections, and systematically and comprehensively carry out quality control and quality evaluation.
FEI Yan, WANG Zhi-peng, LIANG Feng-ying, LIU Wei-xiang, YANG Yang, GAO Shou-hong
. Simultaneous determination of contents of 22 amino acids in 4 kinds of compound amino acid injections by UHPLC-MS/MS[J]. Chinese Journal of Pharmaceutical Analysis, 2023
, 43(8)
: 1291
-1301
.
DOI: 10.16155/j.0254-1793.2023.08.04
[1] CHEN XW, GAO D, LIU F, et al. A novel quantification method for analysis of twenty natural amino acids in human serum based on N-phosphorylation labeling using reversed-phase liquid chromatography-tandem mass spectrometry[J].Anal Chim Acta,2014,836:61
[2] WANG CY, ZHU HB, PI ZF, et al. Classification of type 2 diabetes rats based on urine amino acids metabolic profiling by liquid chromatography coupled with tandem mass spectrometry[J].J Chromatogr B,2013,935:26
[3] 蔡诗贵,熊家林. 我国氨基酸生产现状和发展建议[J].湖北化工,2000,17(6):1
CAI SG, XIONG JL. Production status and development suggestions of amino acid in my country[J].Hubei Chem Ind,2000,17(6):1
[4] 邓红英,张永文,李永贵. 柱前衍生高效液相色谱法测定复方氨基酸注射液中氨基酸的含量[J].药学研究,2020,39(1):27
DENG HY, ZHANG YW, LI YG. Determination of amino acids in Compound amino acids injection by HPLC after pre-column derivatization[J].J Pharm Res,2020,39(1):27
[5] 方方,纪宇. 柱前衍生HPLC法测定复方氨基酸注射液(20AA)中氨基酸含量[J].中国药品标准,2016,17(1):28
FANG F, JI Y. Determination of amino acids in compound amino acid injection (20AA) by HPLC after pre-column derivatization[J].Drug Stand China,2016,17(1):28
[6] 张莉,黄哲甦. HPLC法测定复方氨基酸注射液(18AA-IV)中5-羟甲基糠醛的含量[J].天津药学,2012,24(6):4
ZHANG L, HUANG ZS. Determination of 5-hydroxymethylfurfural in compound anlino acid injection (18AA-IV) by HPLC[J].Tianjin Pharm,2012,24(6):4
[7] 李莉,崔红艳,钟家棋,等. HPLC法同时直接测定复方氨基酸注射液(18AA)中的蛋氨酸亚砜和蛋氨酸砜[J].分析试验室,2018,37(11):1309
LI L, CUI HY, ZHONG JQ, et al. Simultaneous and direct determination of methionine sulfoxide and methionine sulfone in compound amino acid injection (18AA) by HPLC[J].Chin J Anal Lab,2018,37(11):1309
[8] 谢升谷,胡楚楚,黄巧巧,等. HPLC法检测复方氨基酸注射液(18AA)中焦谷氨酸的含量[J].药物分析杂志,2015,35(4):705
XIE SG, HU CC, HUANG QQ, et al. HPLC method for analysis of pyroglutamic acid in compound amino acid injection (18AA)[J].Chin J Pharma Anal,2015,35(4):705
[9] YOSHIDAA H, KONDOA K, YAMAMOTOB H,et al. Validation of an analytical method for human plasma free amino acids by high-performance liquid chromatography ionization mass spectrometry using automated precolumn derivatization[J].J Chromatogr B,2015,998-999:88
[10] WATERVAL WAH, SCHEIJEN JLJM, ORTMANS-PLOEMEN MMJC, et al. Quantitative UPLC-MS/MS analysis of underivatised amino acids in body fluids is a reliable tool for the diagnosis and follow-up of patients with inborn errors of metabolism[J].Clin Chim Acta,2009,407(1-2):36
[11] 王棘,潘雪妍,杨宏伟. HPLC法和氨基酸分析仪(AAA)法测定肠外营养注射液(25)中18种氨基酸的含量的比较[J].药物分析杂志,2012,32(6):1085
WANG J, PAN XY, YANG HW. Comparison of HPLC and amino acid analyzer methods in analyzing 18 different amino acids in total parenteral nutrition injection (25) [J].Chin J Pharm Anal,2012,32(6):1085
[12] GATTI R, GIOIA MG, ANDREATTA P, et al. HPLC-fluorescence determination of amino acids in pharmaceuticals after pre-column derivatization with phanquinone[J].J Pharm Biomed,2004,35(2):339
[13] DEVALL AJ, BLAKE R, LANGMAN N, et al. Monolithic column-based reversed-phase liquid chromatography separation for amino acid assay in microdialysates and cerebral spinal fluid[J].J Chromatogr B,2007,848(2):323
[14] 中华人民共和国药典2020年版. 四部[S].2015:480
ChP 2020. Vol Ⅳ[S].2015:480
[15] 于泓,牟世芬. 氨基酸分析方法的研究进展[J].分析化学,2005,33(3):398
YU H, MOU SF. Method development for amino acid analysis[J].Chin J Anal Chem,2005,33(3):398
[16] 王清华,熊筱娟,张凤,等.血液中氨基酸含量的高效液相色谱及其质谱联用测定方法研究进展[J].宜春学院学报,2015,37(12):76
WANG QH, XIONG XJ, ZHANG F, et al. Research progress of analysis methods for amino acid in blood with high performance liquid chromatography and high performance liquid chromatography coupled with tandem mass spectrometry[J].J Yichun Coll,2015,37(12):76
[17] 安卓玲,史忱,赵瑞,等. 基于超高效液相色谱-质谱的药物性肝损伤患者血清代谢组学研究[J].分析化学,2015,43(9):1408
AN ZL, SHI C, ZHAO R, et al. Ultra high performance liquid chromatography coupled with Q exactive hybrid quadrupole-orbitrap mass spectrometry for serumal metabonomics study of drug-induced liver injury in patients[J].Chin J Anal Chem,2015,43(9):1408
[18] YANG ZL, LI H, WANG B, et al. An optimized method for neurotransmitters and their metabolites analysis in mouse hypothalamus by high performance liquid chromatography-Q exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry[J].J Chromatogr B,2016,1012-1013:79
[19] YANG Y, ZHANG F, GAO SH, et al. Simultaneous determination of 34 amino acids in tumor tissues from colorectal cancer patients based on targeted UHPLC-MS/MS method[J].J Anal Methods Chem, 2020, 2020:4641709
