人胰岛素及其类似物中残留溶剂测定方法研究

Determination of residual solvents in human insulin and its analogues

目的建立人胰岛素及其类似物中有机溶剂残留测定方法,用同一方法测定目前在人胰岛素及类似物生产工艺中普遍存在的甲醇、乙醇、异丙醇、乙腈和正丙醇5种残留溶剂。方法采用顶空气相色谱法,使用DB-624毛细管柱(30 m×0.53 mm,3μm),载气为氮气,流速为2.5 ml/min,检测器为氢火焰离子化检测器,检测器温度为250℃,进样口温度为200℃,分流比为5:1,顶空平衡温度为85℃,平衡时间为20 min,柱温程序为:起始温度40℃,保持10 min,随后以5℃/min升至80℃,随后以20℃/min升至200℃。结果甲醇、乙醇、异丙醇、乙腈及正丙醇峰之间分离良好;专属性、耐用性、精密度良好;甲醇、乙醇、异丙醇、乙腈和正丙醇分别在1.5~60.8、1.1~98.9、0.8~100.4、0.7~10.4、0.8~100.1μg/ml浓度范围内呈良好的线性关系(r2均大于0.99);甲醇、乙醇、异丙醇、乙腈和正丙醇检出限分别为0.6、0.3、0.2、0.2、0.2μg/ml,定量限分别为1.5、1.1、0.8、0.7、0.8μg/ml;各溶剂在人胰岛素及其类似物中平均加标回收率(n=9)均在90%~110%范围内,准确度良好。结论该方法可操作性强、准确、灵敏度高,适用于人胰岛素及其类似物中甲醇、乙醇、异丙醇、乙腈和正丙醇5种残留溶剂测定。

Objective To establish a method for the determination of organic solvent residues in human insulin and its analogues,and to determine the residual solvents of methanol,ethanol,isopropanol,acetonitrile and N-propanol simultaneously.Methods Headspace gas chromatography using DB-624 capillary column(30 m×0.53 mm,3μm)with conditions as follows:①the carrier gas is nitrogen;②the flow rate is 2.5 ml/min;③the detector is a flame ionization detector;④the detector temperature is 250℃;⑤the injection port temperature is 200℃;⑥the split ratio is 5:1;⑦the headspace equilibrium temperature is 85℃;⑧the equilibrium time is 20 min.The column temperature program is as follows:①the initial temperature is 40℃,and maintained for 10 min;②it is raised to 80℃at 5℃/min,and then it is raised to 200℃at 20℃/min.Results The peaks of methanol,ethanol,isopropanol,acetonitrile and N-propanol were well separated with good specificity,durability and precision.Methanol,ethanol,isopropanol,acetonitrile and N-propanol show a good linear relationship in the concentration range of 1.5-60.8,1.1-98.9,0.8-100.4,0.7-10.4,and 0.8-100.1μg/ml,respectively(r2>0.99).The detection limits of methanol,ethanol,isopropanol,acetonitrile and N-propanol were 0.6,0.3,0.2,0.2,0.2μg/ml,respectively.The limits of quantification were 1.5,1.1,0.8,0.7,0.8μg/ml,respectively.The average recoveries(n=9)of all solvents in human insulin and its analogues were in the range of 90%-110%,and the accuracy was good.Conclusion The method is operable,accurate and sensitive,and is suitable for the determination of methanol,ethanol,isopropanol,acetonitrile and N-propanol in human insulin and its analogues.