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目的:参考《欧洲药典》的HPLC-脉冲安培电化学法建立高效液相色谱-柱后衍生化法测定庆大霉素C组分含量及其有关物质,同时与电化学法进行比较研究。方法:采用亲水性Hydrosphere C18(250 mm×4.6 mm,5μm)色谱柱,以含0.7%三氟乙酸及0.025%五氟丙酸的50 mmol·L-1氢氧化钠溶液(pH 2.6)-乙腈(98.5∶1.5)为流动相。衍生化法反应温度30℃,荧光激发波长340 nm,发射波长430 nm。电化学法采用脉冲安培检测器,金电极为工作电极,四电位波形工作模式。结果:两种方法测定的庆大霉素C组分(C1a,C2,C2a和C1)分别在5.82~233.00,6.92~277.00,4.00~160.00和6.23~249.00μg·ml-1的浓度范围内线性关系良好(r均≥0.999 3),检测限和定量限范围分别为0.92~3.28 ng和1.37~5.19 ng。结论:两种方法的检测结果无明显差异。
OBJECTIVE: To establish a method for the determination of gentamycin C component and its related substances by high performance liquid chromatography - post-column derivatization with reference to the “European Pharmacopoeia” HPLC-pulsed amperometric electrochemical method and compare with electrochemical method. Methods: A 50 μmol·L -1 sodium hydroxide solution (pH 2.6) containing 0.7% trifluoroacetic acid and 0.025% pentafluoropropionic acid was applied to a hydrophilic Hydrosphere C18 column (250 mm × 4.6 mm, 5 μm) Acetonitrile (98.5: 1.5) is the mobile phase. Derivatization reaction temperature 30 ℃, fluorescence excitation wavelength 340 nm, emission wavelength 430 nm. Electrochemical method using pulsed ampere detector, gold electrode for the working electrode, four-potential waveform mode of operation. Results: The gentamicin components (C1a, C2, C2a and C1) determined by the two methods were linear within the range of 5.82 ~ 233.00, 6.92 ~ 277.00, 4.00 ~ 160.00 and 6.23 ~ 249.00 μg · ml- (R ≥ 0.999 3). The limits of detection and quantification ranged from 0.92 to 3.28 ng and from 1.37 to 5.19 ng, respectively. Conclusion: There is no significant difference between the two methods.