高效液相色谱法同时测定人血浆中多种喹诺酮类药物浓度

Simultaneous Determination of Various Quinolone Antimicrobial Agents in Human Plasma by HPLC

目的建立一种可同时测定人血浆中多种喹诺酮类药物浓度的高效液相色谱法。方法以Kromasil C8柱(250 mm×4.6 mm,5μm)为色谱柱,含1%三乙胺的0.02 mol/L磷酸二氢钾缓冲液(用磷酸调pH至3.0)-甲醇-乙腈(65∶20∶15)为流动相,流速1.0 mL/min,柱温30℃,检测波长依受检药物而定,进样量20μL,不同喹诺酮类药物互为内标。采用二氯甲烷以液液萃取法提取样品,氮气流吹干后以流动相复溶进样。结果在上述色谱条件和样品处理条件下,加替沙星、莫西沙星等几种喹诺酮类药物可得到有效分离和检测。以莫西沙星为模型药物,对建立的测定方法进行验证,莫西沙星在血药质量浓度0.05～5.0μg/mL范围内线性关系良好(r=0.999 8),最低定量限为0.05μg/mL;高、中、低质量浓度方法回收率为99.37%～104.50%,提取回收率为80.48%～91.25%;日内精密度RSD为4.03%～5.73%,日间精密度RSD为4.53%～6.83%。样品在低温冷冻(-20℃)条件下至少1个月内稳定。结论所建立的高效液相色谱法准确可靠、简便快速、灵敏度较高,可用于莫西沙星等多种喹诺酮类药物的血药浓度测定。

Objective To develop a HPLC method for the simultaneous determination of various quinolone antimicrobial agents in human plasma.Methods The separation was conducted on the Kromasil C8 column.The mobile phase consisted of 0.02 mol/L KH2PO4 buffer solution（containing 1%triethylamine,adjusting to pH 3.0 with phosphoric acid）-methanol-acetonitrile with a volume ratio of 65 ∶20 ∶15.The flow rate was 1.0 mL/min and the detection wavelength depended on the determined quinolone antibiotics.The column temperature was 30 ℃and the injection volume was 20 μL.The determined quinolone antimicrobial agents were able to be chosen as reciprocal internal standard.Liquid-liquid extraction with methylene chloride was screened for the sample processing.After dried under nitrogen,the residue was redissolved with mobile phase and injected into the sampler.Results Under the optimized chromatographic conditions,various quinolone antimicrobial agents,such as moxifloxacin and gatifloxacin,could be separated effectively without mutual interference.The established HPLC method was validated with model drug of moxifloxacin and internal standard of gatifloxacin.The calibration curve for moxifloxacin was linear in the concentration range of 0.05-5.0 μg/mL.The limit of quantitation（LOQ） for moxifloxacin was 0.05 μg/mL.The methodological recovery of low,medium and high concentrations was 99.37%-104.50%.The recovery of extraction was 80.48%-91.25%.The relative standard deviations for intra-day and inter-day assay were 4.03%-5.73%and 4.53%-6.83%,respectively.The sample was stable at least within one month under-20 ℃.Conclusion The established HPLC method is accurate,simple and rapid with high sensitivity,which could be used for the simultaneous determination of various quinolone antimicrobial agents.