反相HPLC法同时测定人尿中茶碱及其两种代谢产物

Reversed-phase high performance liquid chromatographic method for simultaneous determination of theophylline and its two metabolites in human urine

目的:建立一种同时测定人尿中茶碱及其1,3-二甲基尿酸(1,3-DMU)和3-甲基噻嗪(3-MX)代谢产物的HPLC方法。方法:尿样用异丙醇/二氯甲烷(2/8)混合液提取,有机相在空气吹干,用流动相复溶后进行HPLC分析。色谱柱为Dia-monsil ODS C185μm,150mm×4.6mmI.D),流动相由0.1%甲酸液和乙腈(95:5)组成,流速1.0mL/min,测定波长280nm。测定12名受试者单剂量和多剂量口服茶碱后24h内尿中茶碱及其代谢物累计排泄量。结果:尿中茶碱及其代谢物1,3DMU和3-MX的线性范围分别为0.312~40.0、0.156~20.0、0.078~10.0μg/mL,最低可定量浓度分别为0.312、0.156、0.078μg/mL。批间和批内的变异小于15%,回收率大于70%。结论:该方法的特异性、灵敏度能够满足临床上对人尿中茶碱及其代谢产物同时测定的要求。

AIM:To develop an HPLC method with ultraviolet detection for simultaneous determination of theophylline and its two metabolites 1,3-dimethyluric acid(1,3-DMU)and 3-methylxanthine(3-MX)in human urine.METHODS:Urine samples were extracted with mixture of isopropanol and dichloromethane(2/8).The aliquot of the organic phase was removed and dried under air.The residue was dissolved in 0.2 mL of mobile phase,and 20 μL of aliquots were injected immediately into the column.The analytes were separated on a Diamonsil ODS C18,5 μm,150 mm×4.6 mm I.D.column with the mobile phase consisting of 0.1% formic acid/L and acetonitrile(95:5)at a flow rate of 1.0 mL/min and the eluent was detected at 280 nm.Concentrations of theophylline and its two metabolites 1,3-DMU and 3-MX in urine of 12 healthy subjects were determined,and accumulative amounts of excretion during 24 h urine were calculated following single dose and multidose of theophenlline.RESULTS:Linear quantitative response curve was generated over a concentration ranges of 0.312-40.0 μg/mL,0.156-20.0 μg/mL and 0.078-10.0 μg/mL in urine for theophylline,3-MX and 1,3-DMU respectively.Lowest limits of quantification for theophylline,3-MX and 1,3-DMU were measured to be 0.312,0.156 and 0.078 μg/mL,respectively.Intra-and inter-batch precision and accuracy were acceptable for all quality control samples,which RSD and relative standard errors were <15%.The mean recoveries from urine were>70%.CONCLUSION:The specific,sensitive and precise method is suitable for monitoring of theophylline and its two metabolites in urine of human.