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目的建立测定硫酸庆大霉素中4个C组分含量的高效液相色谱-串联质谱方法,并考察各组分对质谱检测器的响应因子。方法利用Synergi Polar-RP C18色谱柱(50mm×2.0mm,4μm,80?),以妥布霉素为内标,以流动相0.1%甲酸溶液-甲醇等度洗脱(10:90,V/V),流速0.2mL/min,柱温35℃,分析时间5min。在电喷雾离子化电离源上以选择反应监测方式进行正离子检测,用于定量分析的离子反应分别为m/z 478.2→322.2(C1),464.2→322.2(C2+C2a),450.2→322.2(C1a),和m/z 468.2→163.1(内标)。结果庆大霉素浓度的线性范围为0.5~500μg/L,庆大霉素的定量下限为0.5μg/L。C1、C2+C2a、C1a 4个组分的批内和批间RSD分别为0.2%~8.1%和3.6%~6.4%,批内和批间准确度分别为93.1%~107.1%和97.6%~104.9%。庆大霉素4个C组分各自标准曲线的斜率值无显著性差异,4个C组分在质谱检测器上具有相同的响应因子。结论该方法特异性高、准确度好、精密度高,且4个C组分对质谱的响应因子一致,可以利用高效液相色谱-串联质谱对庆大霉素制剂中各组分进行准确定量。
Objective To establish a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) method for the determination of 4 C components in gentamicin sulfate and investigate the response factors of each component to mass spectrometry detector. Methods Synergi Polar-RP C18 column (50 mm × 2.0 mm, 4 μm, 80 μm) was used as the internal standard. The mobile phase consisted of 0.1% formic acid and methanol (10:90, V / V), flow rate 0.2mL / min, column temperature 35 ℃, analysis time 5min. The positive ion detection was carried out on the electrospray ionization source by selective reaction monitoring. The ion reactions for quantitative analysis were m / z 478.2 → 322.2 (C1), 464.2 → 322.2 (C2 + C2a), 450.2 → 322.2 ( C1a), and m / z 468.2 → 163.1 (internal standard). Results The linear range of gentamicin concentration was 0.5-500 μg / L, and the lower limit of quantitation of gentamicin was 0.5 μg / L. The intra-assay and inter-assay RSD for the four components of C1, C2 + C2a and C1a were 0.2% -8.1% and 3.6% -6.4%, respectively, and the intra-assay and inter-assay RSDs were 93.1% -107.1% and 97.6% 104.9%. There was no significant difference in the slope of the standard curve for each of the four C components of gentamicin. The four C components had the same response factor on the mass spectrometer. Conclusion The method has high specificity, good accuracy and high precision, and the response factors of four C components are consistent with the mass spectrometry, and the components in gentamicin preparation can be accurately quantified by high performance liquid chromatography-tandem mass spectrometry .