RP-HPLC荧光法测定人尿中巴洛沙星的含量

目的建立反相高效液相色谱荧光法测定人尿中巴洛沙星质量浓度。方法采用DiamonsilODS C18柱(200 mm×4.6 mm,5μm),柱温:室温,流动相:乙腈-0.05 mol.L-1KH2PO4缓冲液(体积比为25∶75,三乙胺和H3PO4调pH为3.4),流速:1.0 mL.min-1。尿液样品用0.1 mol.L-1KH2PO4(KOH调pH为7.0)缓冲液稀释100倍,然后与二氯甲烷旋涡混合后,提取有机层,在40℃条件下氮气吹干,残渣用流动相溶解进样,荧光检测器(λex=295 nm,λem=500 nm)检测,以加替沙星作内标,按内标法定量。结果标准曲线在50~6 000μg.L-1内线性良好,最低检测限为10μg.L-1,巴洛沙星及内标的保留时间分别为5.42、4.05 min,日内和日间RSD均小于10.0%,提取回收率和方法回收率分别在93%~96%和98%~108%。结论本法适用于人尿液中巴洛沙星质量浓度测定及药物动力学研究。

Determination of balofloxacin in human urine by RP-HPLC with fluorescence detection

Objective To establish an RP-HPLC method with fluorescence detection for the determination of balofloxacin in human urine.Methods A fluorescence detector and Diamonsil ODS C18 analysis column(200 mm×4.6 mm,5 μm) were used.The urine sample was diluted by 0.1 mol·L-1 potassium dihydrogen phosphate(pH=7) and extracted with 5.0 mL dichloromethane.After evaporation of the organic layer,the residue was dissolved in 200 μL mobile phase and 20 μL was injected into the column.The mobile phase of acetonitrile-0.05 mol·L-1 potassium dihydrogen phosphate(V∶V=25∶75) was eluted at rate of 1.0 mL·min-1 through the column.The detector at high sensitivity was set at λex 295 nm and λem 500 nm.Results The retention time for balofloxacin and internal standard(gatifloxacin) were 5.42 min and 4.05 min,respectively.The standard curve was linear over the concentration range of 50 to 6 000 μg·L-1.The lowest concentration of detection in plasma was 10 μg·L-1.The extraction recoveries and method recoveries were 93%-96% and 98%-108%,respectively.The intra-day RSD and inter-day RSD were less than 10%.Conclusions This method is simple,rapid,sensitive and accurate and suitable for clinical practice application.