高效液相色谱与电喷雾飞行时间质谱联用鉴定胰岛素注射液

Analysis of Commercial Insulin Injection Using High Performance Liquid Chromatography Combined with Electrosprary Ionization-Time of Flight-Mass Spectrometry

用反相高效液相色谱(RP-HPLC-UV)和电喷雾飞行时间质谱(ESI-TOF/MS)鉴定商品化胰岛素注射液中的胰岛素。使用反相C18微柱(2.1mm×30mm,3.5μm),1%醋酸的水/乙腈(66∶34,V/V)为流动相,胰岛素能在1min之内实现快速分离检出。通过改变流动相流速、酸度以及质谱各参数获得胰岛素检测的最优化条件,在该条件下,胰岛素可获得最佳电离效率,样品量仅为0.174pmol即可获得有效质谱信号。同时在ESI正离子全范围扫描中主要形成带有+3、+4、+5、+6、+7等不同电荷数的质谱峰。经去卷积计算可得胰岛素的精确分子量,与理论值的相对误差可至1.72×10-7以下。

A new method based on reversed phase high performance liquid chromatography （HPLC）, positive ion electrospray time-of-flight mass spectrometry （ESI-TOF/MS） has been developed for the characterization of insulin in commercial insulin injection. With reversed phase C18 microcolumn（2.1 mm × 30 mm, 3.5 μm） and water and acetonitrile （66：34, V/V） containing 1% HAe as mobile phase, insulin and other compounds could be separated rapidly within 1 min. The factors including flow rate, pH, fragmentor affecting the MS signal of insulin have been optimized. Under the optimum conditions, insulin could be ionized efficiently and only 0. 174 pmol sample was required to satisfiy the quantitative analysis of insulin. Meanwhile, MS peaks with ＋ 3, ＋ 4, ＋ 5, ＋ 6 and ＋ 7 charges were formed in mass spectrum under the ESI positive scan mode. After deconvolution analysis, the exact mass of insulin in different samples could be obtained. Compared to the theoretical mass, the relative error of the mass could be down to 1.72 × 10^-7