基于2D-LC-HR-MS/MS法的头孢丙烯原料药杂质谱分析

Impurity profile analysis of cefprozil using 2D-LC-HR-MS/MS

目的基于二维液相色谱-高分辨串联质谱(2D-LC-HR-MS/MS)法分析头孢丙烯原料药杂质谱。方法一维色谱条件进行样品色谱图采集,确认各杂质的出峰位置,采用YMC Hydrosphere色谱柱(250 mm×4.6 mm,5μm),以11.5 g·L^(-1)磷酸二氢铵(用磷酸调节pH为4.4)为流动相A,乙腈-流动相A(50∶50)为流动项B,梯度洗脱;柱温40℃;体积流量1.0 mL·min^(-1);检测波长230 nm;进样量4μL。二维液相脱盐后切进高分辨串联质谱进行分析,根据结果推断杂质结构及生成机制,采用Waters BEH C_(18)色谱柱(50 mm×2.1 mm,1.7μm),以0.01%甲酸水溶液为流动相A,乙腈为流动相B,切峰后开始A相由98%到1%,柱温40℃,体积流量0.3 mL·min^(-1),质谱采用Xevo G2-XS QTof MS系统,离子源为ESI源,毛细管电压3.0 kV,雾化器温度450℃,扫描范围m/z 100~2000。结果头孢丙烯样品中存在9个杂质色谱峰,其中5个杂质为已知杂质,峰3为杂质B(头孢羟氨苄)、峰5为杂质D、峰6为杂质F、峰7为杂质G、峰9为杂质I;对其中3个未知杂质可能的结构式进行了初步推测以及探讨了可能的生成途径,峰2分子式为C_(18)H_(19)N_(3)O_(6)S,该化合物比头孢丙烯多一个氧,分析其为头孢丙烯的氧化杂质;峰4分子式为C_(16)H_(15)N_(3)O_(6)S,与杂质B相比增加了1个氧原子,减少了2个氢原子,判断其为7-ACA内酯与对羟基苯甲甘氨酸甲酯(HPGM)反应产物中的硫原子继续发生了氧化生成的;峰8分子式为C_(8)H_(9)NO_(2)S,该组分为头孢丙烯分子结构的一部分。峰1有待进一步研究。结论该方法有效解决了头孢丙烯流动相中含不挥发性磷酸盐的色谱体系与色谱-质谱快速鉴定杂质不兼容的难题,可以简单、快速地对头孢丙烯有关物质进行定性分析及杂质谱研究。

Objective To analyze the impurity profile of cefprozil API using 2D-LC-HR-MS/MS.Methods The sample chromatogram was acquired by one-dimensional chromatography conditions to confirm the peak positions of each impurity.The YMC Hydrosphere column(250 mm×4.6 mm,5μm)was used,with11.5g·L^(-1)ammonium dihydrogen phosphate(pH adjusted to 4.4 with phosphoric acid)as solvent A,a mixture of acetonitrile and solvent A(50:50)as solvent B,and gradient elution;column temperature was 40℃;flow rate was 1.0mL·min^(-1);detection wavelength was 230 nm;injection volume was 4μL.The impurity profile was analyzed by 2D-LC desalting and high-resolution tandem mass spectrometry(HR-MS/MS)after cutting into the mass spectrometer.Based on the results,the structures and formation mechanisms of the impurities were infered.The Waters BEH C_(18) column(50 mm×2.1 mm,1.7μm)was used,with 0.01%formic acid aqueous solution as solvent A and acetonitrile as solvent B.The peaks were cut and the A phase was gradually increased from 98%to 1%starting from the cut.The column temperature was 40℃,the flow rate was 0.3 mL·min^(-1),and the mass spectrometer used was the Waters Xevo G2-XS QTof MS system with an ESI ion source,a capillary voltage of 3 kV,a vaporizer temperature of 450℃,and a scanning range of m/z 100-2000.Results Nine impurity peaks were identified in the cefprozil sample,of which five were known impurities.Peak 3 was impurity B(cefhydroxime),peak 5 was impurity D,peak 6 was impurity F,peak 7 was impurity G,and peak 9 was impurity I.Preliminary structural hypotheses were made for the three unknown impurities and the possible generation routes were discussed.Peak 2 has a molecular formula of C_(18)H_(19)N_(3)O_(6)Swhich was one oxygen atom more than cefprozil.It was analyzed as an oxidation impurity of cefprozil.Peak 4 has a molecular formula of C_(16)H_(15)N_(3)O_(6)S which was one oxygen atom and two hydrogen atoms less than impurity B.It was judged that it was the sulfur atom of the reaction product between 7-ACA lactam and HPGM(hydroxyphenylglycine methyl ester)continuing to undergo oxidation.Peak 8 has a molecular formula of C_(8)H_(9)NO_(2)S which is part of the molecular structure of cefprozil.Peak 1 needs further study.Conclusion This method effectively solved the problem of the chromatographic system with non-volatile phosphate in the mobile phase of cefprozil flowing phase and the incompatibility of chromatography-mass spectrometry rapid identification of impurities.It can simply and quickly determine the qualitative analysis and impurity spectrum of cefprozil-related substances.