目的 建立一种用于直接测定硫酸巴龙霉素含量和有关物质的高效液相色谱-脉冲安培电化学检测器(HPLC-PAD)法。方法 采用Agilent Pursuit®PFP(4.6 mm×250 mm,5 μm)色谱柱,以体积分数2.0%三氟乙酸溶液[含0.15%(体积分数)五氟丙酸,用50%氢氧化钠溶液(质量分数)调pH值至3.5]的水溶液为流动相,积分脉冲安培电化学检测器的四电位波形测定硫酸巴龙霉素有关物质。结果 五氟苯基柱比传统的十八烷基硅烷键合硅胶填充色谱柱分离效果更优,代表性样品中分离出21个峰,有巴龙霉素Ⅱ与其同分异构体巴龙霉素Ⅰ和另外的19个有关物质;在0.49~118.30 μg·mL-1内巴龙霉素质量浓度与峰面积呈良好的线性关系(r>0.999 3),检测限(LOD)为0.49 μg·mL-1,定量限(LOQ)为0.99 μg·mL-1,重复性和精密度RSD均小于2.0%。结论 建立的HPLC-PAD方法专属性好、灵敏度高、稳定性强、耐用性好,为巴龙霉素质量控制和工艺优化研究提供了可靠的分析手段。
Abstract
OBJECTIVE To establish a high performance liquid chromatography-pulsed amperometric electrochemical detector(HPLC-PAD) method for the direct determination of paromomycin sulfate raw material. METHODS The separation was performed on Agilent® Pursuit PFP(4.6 mm×250 mm, 5 μm). The mobile phase consisted of 2.0%(V/V) trifluoroacetic acid solution [containing 0.15%(V/V) pentafluoropropionic acid, adjusted to pH 3.5 with 50% sodium hydroxide(W/W)] to determine the substances related to paromomycin sulfate with the four-potential waveform of an integrated pulse amperometric electrochemical detector. RESULTS The established HPLC-PAD method using pentafluorophenyl column, was more suitable for the analysis of polar substances than octadecylsilane bond and silica gel filled column. Twteney-one peaks were separated, including paromomycin Ⅱ and its isomer paromomycin I and 19 other impurities. In the range of 0.49-118.30 μg·mL-1, the concentration of paromomycin showed a good linear relationship with the peak area(r>0.999 3). The limit of detection(LOD) was 0.49 μg·mL-1, the limit of quantitation(LOQ) was 0.99μg·mL-1, and the RSDs of repeatability and precision were less than 2.0%. CONCLUSION The developed HPLC-PAD method has good specificity, sensitivity, stability, and durability, providing a reliable analytical means for the quality control and process optimization of paromomycin.
关键词
硫酸巴龙霉素 /
脉冲安培电化学检测器 /
五氟苯基柱 /
含量测定 /
有关物质
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Key words
paromomycin sulfate /
high performance liquid chromatography combined with pulsed amperometric detection(HPLC-PAD) /
pentafluorophenyl column /
content determination /
related substance
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中图分类号：
R917
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参考文献
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