目的 建立基于HPLC-MS/MS对硫酸多黏菌素B中未知杂质的结构分析方法。方法 采用Agilent 1260-6550 Q/TOF-MS液质联用仪,选择Diamonsil Plus C18色谱柱(4.6 mm×250 mm,5 μm),以0.01 mol·L-1 CF3COOH-CH3CN(95∶5)溶液为流动相A,0.1%甲酸的CH3CN溶液为流动相B,流动相A-流动相B=79∶21,等度洗脱,流速为1 mL·min-1,检测波长为215 nm。质谱检测器采用电喷雾离子源(ESI),正离子扫描,一级质谱及二级质谱扫描范围(m/z ):50~1 700。通过对比已知组分如多黏菌素B1、B2等与未知组分的二级质谱的异同,推断杂质结构。采用254 nm低压汞灯作为光化学反应(帕特诺-比希反应)的光源,以丙酮-水(50/50,V/V)作为反应试剂。结果 推断出硫酸多黏菌素B原料药及制剂中7个未知杂质的结构,其中含量最大的未知杂质经光化学反应结合二级质谱推测结构为双键化多黏菌素B1(命名为7′-烯基多黏菌素B1),双键位于酰基脂肪链末端。结论 7′-烯基多黏菌素B1为首次报道,本方法为药物中含有双键未知杂质的结构鉴定提供了一种新颖的解决思路。
Abstract
OBJECTIVE To establish an HPLC-MS/MS method to identify the unknown impurities in polymyxin B sulfate. METHODS The analysis was performed on Agilent 1260-6550 Q/TOF-MS with a Diamonsil Plus C18 column(4.6 mm×250 mm, 5 μm). Mobile phase A was 0.01 mol·L-1 trifluoroacetic acid-acetonitrile(95∶5), and mobile phase B was acetonitrile containing 0.1% formic acid. Mobile phase A and B were set at the volume ratio of 79∶21 at a flow rate of 1 mL·min-1 under isocratic elution. The detection wavelength was set at 254 nm. ESI source was used. Positive ion scanning was conducted in the range of m/z 50-1 700 for MS and MS/MS. The unknown components were identified by comparing the MS and MS/MS with the known reference standards like polymyxin B1 and B2. The photochemical Paterno-Büchi reaction was performed using a low-pressure mercury lamp as the light source at emission wavelength of 254 nm with acetone/water(50/50, V/V) as the reaction solvent. RESULTS The structures of seven unknown related substances in polymyxin B sulfate were identified. The most abundant impurity was identified to be vinyl polymyxin B1, for which the double bond was at the end of the fatty acyl residue. CONCLUSION Vinyl polymyxin B1 is reported for the first time. The method provides a good idea for the identification of related substances in drugs.
关键词
硫酸多黏菌素B /
未知杂质 /
高效液相色谱质谱联用法 /
光化学反应
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Key words
polymyxin B sulfate /
unknown impurity /
HPLC-MS/MS /
photochemical reaction
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中图分类号：
R917
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参考文献
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脚注
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基金
上海市科学技术委员会科研计划项目资助(17DE1910404)
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