二维超高效液相色谱-QTof质谱联用技术在盐酸博来霉素杂质谱研究中的应用

目的：建立二维超高效液相色谱-QTof质谱联用法研究盐酸博来霉素的杂质谱。方法：一维色谱采用Waters HSS T3（100 mm×2.1 mm，1.8 μm）色谱柱，以己烷磺酸钠溶液（取己烷磺酸钠4.7 g 与乙二胺四乙酸1.86 g，以0.08 mol·L^-1 乙酸溶液溶解并稀释至1 000 mL，用氨溶液调节pH 为4.3）为流动相A，甲醇-乙腈（70:30）为流动相B，梯度洗脱，流速0.4 mL·min^-1，柱温40 ℃，检测波长254 nm，进样量2 μL；二维色谱采用ACQUITY UPLC BEH C18（50 mm×2.1 mm，1.7 μm）色谱柱，以0.1% 甲酸水溶液为流动相A，含0.1% 甲酸的乙腈为流动相B，梯度洗脱，流速0.5 mL·min^-1，柱温40 ℃ ；质谱采用Xevo G2-S QTof MS系统，离子源为ESI源，正离子方式检测，离子源温度120 ℃，毛细管电压3.0 kV，雾化器温度500 ℃，扫描范围100~2 000 m/z，测定盐酸博来霉素中主要杂质的一级和二级质谱，进行结构解析。结果：盐酸博来霉素原料中检出了18个主要杂质，并推断了其中的16个杂质，进而分析了杂质的活性与毒性。结论：本法建立了二维超高效液相色谱-QTof质谱联用技术测定盐酸博来霉素杂质的方法，研究结果对其质量评价具有参考意义，为制定合理的杂质限度，更好地控制药品质量打下基础，也拓宽了液质联用在药物杂质研究中的应用范围。

Objective:To establifh a two-dimensional UPLC-QTof MS(2D UPLC-QTof MS)method for the study of impurity profile of bleomycin hydrochloride.Methods:One-dimensional chromatographic conditions were optimized as follows:Waters HSS T3 column(100 mm×2.1 mm,1.8 μm)was adopted;sodium hexane sulfonate solution was used(sodium hexane sulfonate 4.7 g and EDTA-2Na 1.86 g were dissolved in 0.08 mol·L^-1 acetic acid solution, then diluted to 1 000 mL,and pH was adjusted to 4.3 with concentrated ammonia solution)as mobile phase A and methanol-acetonitrile(70:30)as mobile phase B;the gradient elution was used,and the flow rate was 0.4 mL·min^-1; the column temperature was 40 ℃;the injection volume was 2 μL;the detection wave length was 254 nm. 2D UPLC-QTof MS conditions were as follows:ACQUITY UPLC BEH C18 column(50 mm×2.1 mm,1.7 μm)was adopted with solution containing 0.1% formic acid as mobile phase A and acetonitrile containing 0.1% formic acid as mobile phase B;gradient elution was also used;the flow rate was 0.5 mL·min^-1;the column temperature was 40 ℃;G2-S QTof MS Xevo system was used for mass spectrometry.Electrospray ionization(ESI)source was used as positive ion detection model,the temperature of ESI source was 120 ℃,capillary voltage was 3.0 kV,nebulizing temperature was 500 ℃,and the scanning range was 100-2 000 m/z.The primary and product MS spectra of the main impurities obtained were used for the structure elucidation.Results:18 main impurities were characterized in the samples,among which 16 impurities were deduced.The activity and toxicity of the impurities were speculated. Conclusion:This is the first study of impurity profile of bleomycin hydrochloride by two-dimensional UPLC-QT of MS method.This work provides useful information for the quality control of bleomycin hydrochloride,which can contribute to establishment of reasonable impurity limits.The application scope of LC-MS technology used for the impurity research of drug is expanded.