基于多步衍生化的GC-MS法测定胸腺法新中D型氨基酸  

Determination of D-amino acids in thymalfasin by GC-MS based on multi-step derivatization

作　　者：林洁虹 尹红锐 邵泓[2] 陈钢[2] 林梅[2] LIN Jie-hong;YIN Hong-rui;SHAO Hong;CHEN Gang;LIN Mei(Shanghai Institute of Pharmaceutical Industry,China State Institute of Pharmaceutical Industry,Shanghai 201203,China;Shanghai Institute for Food and Drug Control,NMPA Key Laboratory for Quality Control of Therapeutic Monoclonal Antibodies,Shanghai 201203,China)

机构地区：[1]中国医药工业研究总院,上海医药工业研究院,上海201203 [2]上海市食品药品检验所,国家药品监督管理局治疗类单抗质量控制重点实验室,上海201203

出　　处：《药学学报》2020年第10期2435-2441,共7页Acta Pharmaceutica Sinica

摘　　要：建立多步衍生化气相色谱-质谱(GC-MS)测定手性氨基酸的方法,并结合氘代盐酸水解应用于胸腺法新中9种D型氨基酸的含量测定。以异丙醇和三氟乙酸酐为衍生化试剂,对多步衍生化的反应条件、供试品复溶试剂的体积、色谱及质谱条件进行优化,建立胸腺法新中9种氨基酸测定方法,考察灵敏度、线性范围、精密度、准确度和加标回收率等。结果表明,16对D/L型氨基酸和Gly衍生物在29 min内实现基线分离;胸腺法新中的9种氨基酸衍生产物在一定浓度范围内线性关系良好(r2>0.9923);定量限低至0.09~2.79μmol·L-1;精密度实验结果表明RSD<10.90%;对照品准确性实验的平均回收率为76.69%~128.18%;样品平均加标回收率在70.41%~125.39%之间。6批胸腺法新原料药中D-Asp和D-Glu含量较高,分别为0.41%~0.49%和0.25%~0.33%,其他D-氨基酸含量均小于0.25%。本方法高效、准确、专属性好,可同时监控胸腺法新中9种D型氨基酸含量,有望为合成多肽药物全面控制杂质提供新的研究思路。We utilized a multi-step derivatization gas chromatography-mass spectrometry method for the deter‐mination of common amino acid enantiomers,combined with deuterated hydrochloric acid hydrolysis,to identify nine trace D-amino acids in thymalfasin.We optimized the conditions for multi-step derivatization,the volume of reagent for redissolving samples,and the conditions for chromatography and mass spectrometry with isopropanol and trifluoroacetic anhydride as derivatization reagents,and validated the procedure,including sensitivity,linear range,precision,accuracy and recovery.Sixteen pairs of D/L-amino acids and glycine derivatives were separated within 29 min,with the limit of quantification as low as 0.09-2.79μmol·L-1.Nine amino acid derivatives of thymal‐fasin showed a good linear relationship within the concentration range examined(r2>0.9923).The precision results showed that RSD values were less than 10.90%.Accuracy test results of a reference substance ranged from 76.69%to 128.18%.Average recoveries of spiked samples ranged from 70.41%to 125.39%.For the nine D-amino acids assayed,D-Asp and D-Glu content in six batches of thymalfasin were highest,ranging 0.41%-0.49%and 0.25%-0.33%,respectively,with others less than 0.25%.The method is sensitive,efficient and reliable,available for seventeen common amino acids and their enantiomers,and works well with simultaneous determination of nine trace D-amino acids in thymalfasin,providing a reference for the comprehensive control of racemic peptide impurities in this synthetic polypeptide drug.

关 键 词：多步衍生化 气相色谱-质谱 胸腺法新 D型氨基酸 消旋肽 

分 类 号：R917[医药卫生—药物分析学]