高效液相色谱法同时测定人血清中茶碱及其代谢物1,3-二甲基尿酸浓度

Simultaneous detection of concentrations of theophylline and its metabolite 1,3-dimethyluric acid in human serum by HPLC

目的：建立一种同时测定人血清样品中茶碱及其代谢物1,3-二甲基尿酸血药浓度的HPLC方法。方法：血清样品经乙腈沉淀除去蛋白,选择对乙酰氨基酚为内标,真空浓缩、复溶后采用HPLC分析。色谱柱为：Agilent ZORBAX SB-C18柱（150 mm×4.6 mm,5μm）,流动相为乙腈∶0.3%甲酸溶液（5∶95）,检测波长为280 nm,流速为1 mL·min-1,进样量为20μL。结果：茶碱、1,3-二甲基尿酸的血清浓度线性范围分别为0.50~40.00μg·mL-1和0.10~12.80μg·mL-1,两者的线性关系均良好（R分别为0.998 8和0.999 7）,最低定量下限分别为0.50,0.10μg·mL-1。茶碱和1,3-二甲基尿酸低、中、高质量浓度的提取回收率分别为80.22%,87.25%,89.23%和87.03%,80.01%,87.95%,二者批内和批间RSD均小于15%。结论：该方法简便、准确,特异性和灵敏度能够满足临床测定人血清中茶碱及其代谢物1,3-二甲基尿酸浓度的要求,适用于评价和研究人体内茶碱的药动学影响,指导临床用药。

OBJECTIVE To establish a method for simultaneous detection of serum concentrations of theophylline and its metabolite 1,3-dimethyluric acid in human. METHODS Serum protein was removed by acetonitrile precipitation,4-Acetamido- phenol was added as internal standard substance, then its concentration and dissolution were detected by HPLC. HPLC was per- formed on Agilent ZORBAX SB-C18 reversed phase column （150 mm-1 4. 6 ram,5 μm） with a mobile phase consisting of acetoni- trile and 0. 3 % formic acid （5：95）. The flow rate was I mL. min-land the detection wavelength was 280 nm, and injection vol- ume was 20 μL. RESULTS The calibration graphs were linear in range of 0. 50-40. 00 μg· mL-1 for theophylline and 0. 10- 12. 80 μg·mL-1 for 1,3-dimethyluric acid （R = 0. 998 8 and 0. 999 7, respectively）. Limits of quantification of theophylline and 1,3-dimethyluric acid were 0. 50 and 0. 10 μg· mL-1, respectively. Extraction recoveries of low, medium and high concentrations were 80. 22~,87. 25% and 89. 23% for theophylline,and 87. 03%,80. 01% and 87. 95% for 1,3%dimethyluric acid,respective- ly. Either intra- or inter-batch RSD was less than 15%. CONCLUSION HPLC is simple,sensitive and reliable for detection of serum concentrations of theophylline and its metabolite 1,3-dimethyluric acid in human. Therefore, it is suitable research effects of drug on pharmacokinetics of theophylline in human, and direct reasonable medication in clinical practice.