HPLC法测定地特胰岛素有关物质及主要杂质鉴别

目的：探索和建立地特胰岛素的纯度分析测定方法，并对主要杂质进行鉴别。方法：采用RP-HPLC方法分离分析地特胰岛素的有关物质，使用Protronavi C₄色谱柱（250 mm×4.6 mm，5 μm，300 Â；），流动相A为0.2 mol·L^-1硫酸钠缓冲液（pH 2.3）-乙腈（82：18），流动相B为水-乙腈（40：60），洗脱梯度（0 min，35%B；45 min，67%B；65 min，67%B；66 min，35%B；80 min，35%B），流速1.0 mL·min^-1，检测波长214 nm，柱温45 ℃。同时收集该色谱条件下的最大杂质组分，利用LC-MS/MS方法进行鉴别。液质条件为：液相色谱分离采用Acquity UPLC BEH300 C₁₈柱（2.1 mm×100 mm，1.7 μm），流动相A为含0.1%甲酸的水溶液，流动相B为含0.1%甲酸的乙腈溶液，30 min内B相由5%升至30%，流速为300 nL·min^-1，洗脱时间为30 min。质谱离子源为纳升级电喷雾离子源，采用正离子模式检测。结果：建立的RP-HPLC纯度分析方法具有良好的方法学验证结果，能准确测定样品中的有关物质；LC-MS/MS鉴定了地特胰岛素中的最大杂质为A链第21位天冬酰胺的脱氨产物。结论：建立的RP-HPLC和LC-MS/MS方法可准确有效地对地特胰岛素有关物质进行定量测定和鉴别分析，可作为地特胰岛素的质量研究和质控分析方法。

Objective: To explore and establish a method for determination of purity of insulin detemir and to identify the main impurity. Methods: The related substances in insulin detemir were separated and analyzed by RP-HPLC.The column was Protronavi C₄ (250 mm×4.6 mm,5 μm,300 Â);the mobile phase A was 0.2 mol·L^-1 sodium sulphate buffer (pH 2.3)-acetonitrile (82:18);the mobile phase B was water-acetonitrile (40:60) with gradient elution (0 min,35%B;45 min,67%B;65 min,67%B;66 min,35%B;80 min,35%B).The flow rate was 1.0 mL·min^-1;the detection wavelength was set at 214 nm;the column temperature was 45 ℃.Chromatographic separation was achieved on an Acquity UPLC BEH300 C₁₈column (2.1 mm×100 mm,1.7 μm) with a gradient solvent system composed of 0.1% aqueous formic acid (A) and acetonitrile with 0.1% formic acid (B),and the gradient profile was 0-30 min,5% B→30% B.The flow rate was 300 nL·min^-1 with 30 min elution.The samples were analyzed with a nanoliter electrospray ionization interface set in positive ionization mode.The main impurity was collected and identified using LC-MS/MS. Results: The analytical procedures were validated and the results showed that the method was qualified and could be applied to accurate determination of the related substances in insulin detemir.The main impurity was identified by LC-MS/MS as the deaminated product of the 21st asparagine residue in A chain of insulin detemir. Conclusion: The RP-HPLC and LC-MS/MS methods established are accurate and effective for qualitative determination and identification of related substances in insulin detemir,and can be applied to specification research and quality control of insulin detemir.