HPLC法对胸腺法新及其制剂中杂质检查

目的: 测定胸腺法新及其制剂中的杂质并对含量超过0.1%的杂质进行结构鉴定。方法: 建立一种高效液相色谱法测定胸腺法新及其制剂中的杂质。含量超过ICH规定质控限度(0.1%)的杂质分别接取流分，脱盐富集之后使用MALDI-TOF/TOF质谱仪分析，推测其结构。合成杂质对照品后确证其结构，并进行杂质测定的方法学研究，测得各杂质相对于胸腺法新的校正因子。结果: 通过对上市的多批胸腺法新及其制剂的分析可知，含量高于0.1% 的杂质主要有4种。杂质1为胸腺法新肽序脱除第3个丙氨酸的多肽；杂质2为第1个丝氨酸右消旋化的多肽；杂质3为最后1个天冬酰胺右消旋化的多肽；杂质4为最后1个天冬酰胺水解为天冬氨酸的多肽。本法精密度、重复性、稳定性、耐用性好。杂质1、2、3、4在各自的线性范围内线性关系均良好(r≥0.9997)，相对于胸腺法新的校正因子分别为0.98、1.06、1.24、1.30。杂质1、2、3、4的检测限分别为0.083、0.077、0.080、0.080 μg·mL^-1；定量限分别为0.25、0.23、0.24、0.24 μg·mL^-1；平均回收率分别为99.2%、100.0%、100.1%、98.8%，RSD分别为1.3%、1.2%、1.0%、0.90%(n=9)。结论: 该方法可用于检查胸腺法新及其制剂中的杂质。

Objective: To determine the impurities in thymalfasin and thymalfasin for injection,and to identify the structures of impurities with the content exceeding 0.1%. Methods: An HPLC method was developed to detect the impurities in thymalfasin and thymalfasin for injection.For those impurities whose contents were more than 0.1% which was the quality control limit according to ICH standard,the eluate was collected respectively,and then concentrated and desalted immediately with a C₁₈ solid phase extraction column.After that,the MS¹ and MS² spectrograms of the impurities were acquired using MALDI-TOF/TOF,and the structures of the impurities were inferred.After synthetizing the impurity standards,the same HPLC method was used to validate the identified structures of the impurities in thymalfasin and thymalfasin for injection.Then a methodology study of the determination of impurities was made and the correction factors of impurities relative to thymalfasin were calculated. Results: The analysis of several batches of thymalfasin and thymalfasin for injection in the market showed that there were mainly 4 impurities whose contents were more than 0.1%.Impurity 1 was the peptide with desorption of the third alanine of thymalfasin; impurity 2 was the peptide with the right racemization of the first serine; impurity 3 was the peptide with the right racemization of the last asparagine; impurity 4 was the peptide with hydrolysis of the last asparagine to aspartic acid.The method had excellent precision,repeatability,stability and durability.The linearities of impurity 1,2,3,4 were fine in their own linear ranges (r≥0.9997),and the correction factors relative to thymalfasin were 0.98,1.06,1.24 and 1.30,respectively.The LOD of impurity 1,2,3,4 were 0.083,0.077,0.080 and 0.080 μg·mL^-1,respectively,and the LOQ were 0.25,0.23,0.24 and 0.24 μg·mL^-1,respectively.The average recoveries of impurity 1,2,3,4 were 99.24%,99.97%,100.13% and 98.82%,respectively,with RSD of 1.33%,1.20%,1.04% and 0.90%,respectively (n=9).Conclusion: This method can be used to determine impurities of thymalfasin and thymalfasin for injection.