摘要
目的建立重组人血白蛋白产品中异丙醇残留量检测的气相色谱法。方法色谱条件:DB-624毛细管色谱柱(30 m×0.53 mm,3μm);进样口温度150℃;检测器为FID,温度200℃;空气流量400 ml/min;氢气流量40 ml/min;载气为高纯氮气,流量6.0 ml/min;程序升温:起始温度60℃保留5 min,以40℃/min升至120℃,保持3 min;分流进样,分流比为5∶1;进样量为1μl。以正丁醇为内标,对建立的方法进行专属性、精密度、准确度、耐用性验证,确定该方法的线性范围和检测限。用建立的方法检测3批重组人血白蛋白样品中异丙醇残留量。结果异丙醇浓度在10~300μg/ml范围内,与异丙醇与正丁醇峰面积比的线性关系良好(r=0.999 9),最低检出限为0.09μg/ml;该检测条件下与异丙醇性质相似的甲醇和乙醇对异丙醇的测定基本无影响;异丙醇与正丁醇峰面积比3次检测结果的RSD为0.4%,精密度良好;不同浓度异丙醇分别与重组人血白蛋白等体积混合,3次检测结果的回收率在94.2%~105.0%之间,准确度良好;不同检测器温度和不同进样口温度条件下测定的色谱峰面积的平均RSD分别为1.6%和2.2%,对测定结果基本无影响。3批重组人血白蛋白样品均未检测出异丙醇残留,符合《中国药典》三部(2015版)推荐标准。结论建立的气相色谱法快速、准确,可用于检测重组人血白蛋白中异丙醇的残留量。
Objective To develop a gas chromatographic method for determination of residual 2-propanol content in recombinant human serum albumin（HSA）. Methods DB-624 capillary column（30 m × 0. 53 mm, 3 μg） was used at an injector temperature of 150 ℃. Hydrogen flame ionization detector（FID） was used at a temperature of 200 ℃. The flexes of air and hydrogen were 400 and 40 ml/min respectively. The carrier gas was nitrogen at a flow rate of 6. 0 ml/min.The original temperature was set as 60 ℃ and maintained for 5 min, which was raised to 120 ℃ at a rate of 40 ℃/min and maintained for 3 min. The split ratio for sample loading was 5 ∶ 1. The sample load was 1 μl. The developed method was verified for specificity, precision, accuracy and robustness using n-butyl alcohol as an internal standard, and determined for linear range and detection limit. The residual 2-propanol content in three batches of recombinant human HSA was determined by the developed method. Results The linear range of the method was 10 ~ 300 μg/ml（r = 0. 999 9）,while the minimum detection limit was 0. 09 μg/ml. Under the condition, methanol and ethanol, with similar properties to that of 2-propanol, showed no significant effect on the determination of 2-propanol. The RSD of three test results of peak area ratio of 2-propanol and n-butyl alcohol was 0. 4%, indicating a high precision. The recovery rates of mixtures of 2-propanol and HSA at equal volumes in three tests were 94. 2% ~ 105. 2%, indicating a high accuracy. The mean RSDs of chromatographic peak areas determined at various detector and injector temperatures were 1. 6% and 2. 2% respectively,indicating no significant influence on the determination result. No residual 2-propanol was detected in three batches of recombinant human HSA, which met the requirements in Chinese Pharmacopoeia（Volume Ⅲ, 2015 edition）. Conclusion The developed gas chromatographic method was rapid and accurate, and might be used for the determination of residual 2-propanol content in recombinant human HSA.
出处
《中国生物制品学杂志》
CAS
CSCD
2017年第8期847-849,854,共4页
Chinese Journal of Biologicals
基金
重大新药创制课题(2012ZX09304010)
关键词
气相色谱
白蛋白
异丙醇
允许残留量
Gas chromatography
Albumin
2-Propanol
Permitted residue
