在线二维液相色谱-四极杆飞行时间质谱法检测二丁酰环磷腺苷钙的杂质谱

Impurity Profile of Calcium Dibutyryladenosine Cyclophosphate by 2D-LC-Q-TOF MS

建立了在线二维液相色谱-四极杆飞行时间质谱法(2D-LC-Q-TOF MS)分析二丁酰环磷腺苷钙的杂质谱。第一维色谱采用C18色谱柱,流动相为0.1 mol/L磷酸二氢钾溶液∶甲醇(60∶40),检测波长273 nm;第二维色谱采用Poroshell 120 EC-C18色谱柱,以0.1%甲酸水溶液和0.1%甲酸乙腈溶液为流动相,线性梯度洗脱。当第一维HPLC目标组分洗脱时,通过阀切换将目标组分准确切割并捕获在40μl定量环中,之后第二维HPLC泵将捕获的组分洗脱并进行质谱检测。采用正离子模式采集数据。对第一维谱图中的7个杂质进行了结构推测,并分析其来源和稳定性。本试验建立的2D-LC-Q-TOF MS法能用于二丁酰环磷腺苷钙杂质谱的分析,可为其生产工艺和质量控制提供参考。

A two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry （2D-LC- Q-TOF MS） method was established for profiling the impurities of calcium dibutyryladenosine cyclophosphate. ACts column was used in the first dimensional chromatography, with the mobile phase of 0.1 mol/L potassium dihydrogen phosphate buffer ： methanol （60 ： 40）, at the detection wavelength of 273 nm, A Poroshell 120 EC-C18 column was used in the second dimensional chromatography, with the mobile phase of water （containing 0.1% formic acid） and acetonitrile （containing 0.1% formic acid） for linear gradient elution. The target peak from the first dimensional LC was trapped in a 40 μl loop via switching valve, and then was separated using a MS compatible mobile phase from pump 2. The data was collected in positive ion mode. The ion source was Dual AJS ESI, and the temperature of dry gas was set at 200 ℃ and the electrospray ionization （ESI） needle voltage was set at 4 000 V. The nebulizer gas was set at 350℃. The molecular formulas were determined by their exact masses and isotope distributions. The structures were postulated by the protonated molecular ions and the manufacturing process of calcium dibutyryladenosine cyclophosphate. Seven impurities of calcium dibutyryladenosine cyclophosphate were characterized and the origination of the impurities was deduced. Four of them were unknown impurities to the best of our knowledge. 2D-LC-Q-TOF MS method established in this work can be used for profiling the impurities in calcium dibutyryladenosine cyclophosphate. The results obtained are referential experiences for the optimization of manufacturing process and quality control of calcium dibutyryladenosine cyclophosphate.