柱前衍生/高效液相色谱-串联质谱法测定氨基酸工业品中26种游离手性氨基酸含量  

Determination of 26 Free Chiral Amino Acids in Industrial Products by Pre-column Derivatization and HPLC-MS/MS

作　　者：匡慧颖 李子樱 朱波[3] 张玉鑫 倪一平 肖伟敏[2,5] 叶新玉 杨国武 杨燕[1] KUANG Hui-ying;LI Zi-ying;ZHU Bo;ZHANG Yu-xin;NI Yi-ping;XIAO Wei-min;YE Xin-yu;YANG Guo-wu;YANG Yan(School of Public Health(Shenzhen),Sun Yat-sen University,Shenzhen 518107,China;Shenzhen Academy of Metrology and Quality Inspection,Shenzhen 518109,China;Luohu District Center for Disease Control and Prevention,Shenzhen 518020,China;School of Medicine and Chemical Engineering,Guangdong Pharmaceutical University,Guangzhou 510006,China;Shenzhen SMQ Group Medical Laboratory,Shenzhen 518109,China;School of Basic Medicine,Guangzhou University of Chinese Medicine,Guangzhou 510006,China)

机构地区：[1]中山大学公共卫生学院(深圳),广东深圳518107 [2]深圳市计量质量检测研究院,广东深圳518109 [3]深圳市罗湖区疾病预防控制中心,广东深圳518020 [4]广东药科大学医药化工学院,广东广州510006 [5]深圳深检集团医学检验实验室,广东深圳518109 [6]广州中医药大学基础医学院,广东广州510006

出　　处：《分析测试学报》2024年第5期703-713,共11页Journal of Instrumental Analysis

基　　金：国家重点研发计划科研项目(2021YFF0701903)。

摘　　要：采用柱前衍生/高效液相色谱-串联质谱法(HPLC-MS/MS)测定氨基酸工业品中26种游离手性氨基酸含量。样品经0.1 mol/L HCl溶液溶解提取后,在碱性条件下与Marfey试剂进行衍生化反应,使用Phe⁃nomenex Kinetex F5(250 mm×5 mm,4.6μm)色谱柱,以10 mmol/L乙酸铵溶液-乙腈为流动相进行梯度洗脱,质谱分析采用电喷雾离子源负离子(ESI-)模式和多反应监测(MRM)模式。结果表明,各氨基酸衍生物之间的分离度良好,26种手性氨基酸的质量浓度在一定范围内与对应的峰面积呈线性关系,相关系数(r^(2))不小于0.9940,定量下限为0.5~12.5 ng/mL,加标回收率为80.3%~118%,相对标准偏差为0.47%~9.8%。应用该方法对180个工业样品进行检测,在L-型氨基酸纯品试剂、L-型稳定同位素标记氨基酸内标试剂、单品L-型氨基酸原料及复合氨基酸原料粉中均检出D-氨基酸。该研究首次评估了国内相关工业产品中手性氨基酸的质量状况,发现复合氨基酸原料需加强手性构型的监测管理,并深入分析了利用相关氨基酸纯品试剂作为标准物质或同位素内标时,对食品、环境、化工、医药等领域样品检测结果准确性可能造成的影响。该方法可为科研试剂、生物制药、化工食品和营养健康等领域提供质量管理和健康评测的有效技术手段。A highly sensitive and selective pre-column derivatization coupled with high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)assay was developed for the determination of the D,L configurations of 26 free chiral amino acids in industrial products.The samples were dissolved and extracted in 0.1 mol/L HCl solution,and then derivatized by Marfey reagent under alkaline condition at 37℃for 16 hours.Baseline separation of the derivatives was achieved on a Phenomenex Kinetex F5(250 mm×5 mm,4.6μm)column.The mobile phase consisted of 10 mmol/L ammonium acetate and acetonitrile by gradient elution.Reverse phase chromatographic resolution of the enantiomers was obtained within 27 min.The derived analytes were sensitively detected using an electrospray ionization source in negative ion mode(ESI-)and multiple reaction monitoring(MRM)mode.The results showed that the derivatized products of 26 chiral amino acids could be effectively separated.The mass concentrations of 26 chiral amino acids showed a good linear relationship with their corresponding peak areas in a corresponding range.The limits of quantification were determined to be 0.5-12.5 ng/mL and correlation coefficients(r^(2))not less than 0.9940.The extraction recoveries for all analysts were in the ranges of 80.3%-118%and the relative standard deviations were in the ranges of 0.47%-9.8%.The method was applied to determine chiral amino acids in 180 industrial samples.The results indicated that D-amino acids were detected in L-type pure amino acid reagent,L-type stable isotope-labeled amino acid internal standard reagent,single L-amino acid raw material,and composite amino acid raw material powder.It is the first time to evaluate the quality of chiral amino acids in relevant industrial products in China,and it was found that the monitoring and management of chiral configurations need to be strengthened for amino acid complex powders.It also thoroughly investigates the potential impact on the accuracy of test results in the food,environmental,chem

关 键 词：手性氨基酸 手性衍生化 高效液相色谱-串联质谱 工业品 

分 类 号：O657.7[理学—分析化学] TS207.3[理学—化学]