多中心切割2D-HPLC-QTOF法对表柔比星脂质体制剂中未知杂质的分离与结构推断

目的 建立多中心切割(MHC)二维高效液相色谱-四级杆飞行时间质谱(2D-HPLC-QTOF)法分离和推断表柔比星脂质 体制剂中未知杂质的方法。方法 一维液相色谱采用Symmetry-C18 (250 mm×4.6 mm，5 μm)为色谱柱，以乙腈和甲醇(6:1)为流 动相A，以加入十二烷基硫酸钠的磷酸缓冲液为流动相B，流动相A与流动相B体积比为57:43进行等度洗脱；二维高效液相色谱 采用Agilent Eclipse-C18 (50 mm×2.1 mm，1.8 μm)为色谱柱，以0.1%的甲酸水和乙腈为流动相，采用梯度洗脱。将一维液相色谱 条件下分离的杂质峰进行多中心切割储存在定量环中，并转移至二维进行分离，以四极杆飞行时间质谱(QTOF)进行结构推断。 结果 该方法可以使表柔比星与杂质峰较好地分离，并且可以得到高质量杂质峰进行结构推断。在表柔比星脂质体制剂中对6 个未知杂质进行有效的推断。结论 建立的多中心切割二维高效液相色谱-四极杆飞行时间质谱法可高效准确的分离并推断表柔 比星脂质体制剂中的未知杂质，特别是微量和难以分离的杂质，对表柔比星制剂的质量控制提供了新方法。

Separation and inference of unknown impurities in epirubicin liposome by multiple heart-cutting 2D-HPLC-QTOF

Abstract Objective To establish a method called multiple heart-cutting 2D-HPLC-QTOF to separate and identify the unknown impurities in epirubicin liposome. Methods One-dimension (1D) chromatographic condition was Symmetry-C18 column using acetonitrile and methanol (6:1) as mobile phase A and phosphate buffer solution with sodium lauryl sulfate as mobile phase B. The volume ratio of mobile phase A to mobile phase B was 57:43. Two-dimension (2D) chromatographic condition was Agilent Eclipse-C18 column using 0.1% formic acid solution as mobile phase A and acetonitrile as mobile phase B (gradient elution). Multiple impurity peaks separated by 1D liquid chromatography were cut by multi hearting-cutting and stored in quantitative loops, and then transferred to 2D for separation. The structures were identified by quadrupole time-of-flight mass spectrometry (QTOF). Results Using this method, good separation of epirubicin from impurity peaks was achieved, and high-quality impurity peaks wereobtained for structural identification. Six unknown impurities were effectively identified in the epirubicin liposome. Conclusion The established method can efficiently and accurately separate and identify unknown impurities in the epirubicin liposome, especially the trace and hard-to-separate impurities, and provides a new method for quality control of the Epirubicin preparation.