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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,5μm),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,5μm),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 analyze the impurities in gentamicin raw materials and injection solutions by high performance liquid chromatography-evaporative light scattering. Methods: A SHISEIDO ACR C18 column (250 mm × 4.6 mm, 5 μm) was used with mobile phase of 1.0% trifluoroacetic acid and methanol (92: 8) as the mobile phase at a flow rate of 0.4 mL · min- The HPLC-ELSD analysis was carried out. The mobile phase was 0.4 mL · min-1 with 0.4% trifluoroacetic acid (TFA) -acetonitrile (95: 5) using XTerra TM MS C18 column (150 mm × 2.1 mm, LC-MS analysis of the gentamicin sub-fraction was carried out. The structural sub-components X2 and X3 were confirmed by 1D and 2D NMR spectra and D2O as the solvent. Based on the background noise and the theoretical plate number of the principal components, 2 Drift tube temperature and carrier gas flow rates are optimized for different types of ELSD instruments. Results: Eleven components were detected by ELSD, including four C components and seven minor impurities. The six minor impurities were identified by LC-MS and NMR, which were gentamycin A2, A , X, B1, sisomicin and gentamycin C2b. The chromatographic retention behavior of gentamicin components was affected by the polarities and steric hindrance of substituents. The order of the substituents was OH> NH2> H> NHCH3> CH3. For ELSD analysis, instrument parameters should be set for each specific detector. The two parameters, drift tube temperature and carrier gas flow rate, should be optimized. CONCLUSION: The HPLC-ELSD method of Chinese Pharmacopoeia can control the small components of gentamicin raw materials and preparations with apparent content greater than 0.5%. It is still relatively simple and practical to control the effect of gentamicin components / impurities effectively method.