HPLC-ELSD法分析庆大霉素中小组分杂质

目的：采用高效液相色谱-蒸发光散射法分析庆大霉素原料及注射液中的小组分杂质。方法：采用SHISEIDO ACR C₁₈色谱柱（250 mm×4.6 mm，5 μm），以1.0%三氟乙酸溶液-甲醇（92：8）为流动相，流速0.4 mL·min^-1，漂移管温度108℃，进行HPLC-ELSD分析;采用XTerra TMMS C₁₈色谱柱（150 mm×2.1 mm，5 μm），以0.4%三氟乙酸（TFA）-乙腈（95：5）为流动相，流速0.4 mL·min^-1，对庆大霉素小组分进行LC-MS分析;采用1D和2D核磁光谱，以D₂O为溶剂，对制备的小组分X2和X3进行结构确证;依据背景噪音和主成分的理论板数，进行2种不同型号ELSD仪器的漂移管温度和载气流速进行优化。结果：ELSD法共检出11个组分，其中包括4个C组分和7个小组分杂质，LC-MS及NMR法推断出其中6个小组分杂质，分别为庆大霉素A2、A、X、B1，西索米星，庆大霉素C2b;庆大霉素组分的色谱保留行为受取代基极性和空间位阻的综合影响，取代基影响的大小顺序为OH>NH₂>H>NHCH₃>CH₃;ELSD分析时应针对每种具体的检测器设定仪器参数，重点对漂移管温度和载气流速这2个参数进行优化。结论：中国药典的HPLC-ELSD方法能够对庆大霉素原料及制剂中表观含量大于0.5%的小组分进行控制，目前仍是相对简便、实用的控制庆大霉素组分/杂质的有效方法。

Objective: To establish an HPLC-ELSD method for the determination of the minor component impurities in gentamicin. Methods: HPLC-ELSD analysis was applied on a column of SHISEIDO ACR C₁₈ column(250 mm×4.6 mm,5 μm), 1.0% trifluoroacetic acid solution-acetonitrile(92: 8) as the mobile phase with the flow rate of 0.4 mL·min^-1 and the drift tube temperature of 108℃.LC-MS analysis was applied on a column of XTerra TMMS C₁₈ column(150 mm×2.1 mm,5 μm),0.4% trifluoroacetic acid solution-acetonitrile (95: 5) as the mobile phase with the flow rate of 0.4 mL·min^-1.The structures of prepared minor component impurities X2 and X3 were illustrated by 1D and 2D NMR spectra with D₂O as solvent.The drift tube temperatures and gas flow rates of two different types of ELSD were optimized separately on the basis of background noise and a number of theoretical plates. Results: Totally 11 components were detected in gentamicin by ELSD,including 4 C components and 7 minor components.The structures of 6 minor components were determined by LC-MS and NMR,which were gentamicin A2,A,X,B1,sisomicin and gentamicin C2b respectively.The chromatographic retention behaviors of gentamicin components were influenced by polar substituent groups combined with steric hindrance effects.The effect order of substituent groups was OH>NH₂>H>NHCH₃>CH₃.Instrument parameters should be set for each detector respectively,with emphasis on optimizing two parameters such as drift tube temperature and carrier gas flow during ELSD analysis. Conclusion: The proposed HPLC-ELSD method is sensitive and suitable for the determination of minor component impurities greater than 0.5% in API and preparations of gentamicin,which is still a relatively simple and practical control method of gentamicin components/impurities.