中国受试者口服茶碱缓释片后尿中原形和代谢物定量分析

Quantitative analysis of theophylline and its metabolites in urine of Chinese healthy subjects after oral administration of theophylline sustained-release tablets

建立一种同时测定人尿中茶碱及其代谢物1,3-二甲基尿酸（1,3-DMU）、3-甲基黄嘌呤（3-MX）和1-甲基尿酸（1-MU）的液相色谱-加热电喷雾电离源串联质谱法（LC-HESI/MS/MS）,并用于人尿中茶碱及代谢物累积排泄量研究。本法采用d6-茶碱作为茶碱的内标,氟尿嘧啶作为1,3-DMU、3-MX和1-MU的内标。尿样用甲醇稀释并离心后,在Agilent XDB-Phenyl（150 mm×4.6 mm ID,5μm）色谱柱上进行色谱分离,以水-甲醇-甲酸（30∶70∶0.15）为流动相,流速为0.6 mL·min-1;采用加热电喷雾电离源（HESI）,选择反应监测（SRM）负离子扫描检测。4种分析物的标准曲线的线性范围均为1.0~250μg·mL-1。该方法快速、简便,可用于人尿中茶碱及其3种代谢物浓度的测定。结果表明,中国受试者口服茶碱缓释片后,尿中茶碱、1,3-DMU、3-MX和1-MU的排泄量比值约为1.0∶3.4∶0.92∶1.6,与已报道的白种人受试者结果相近。

To study the metabolite excretion of theophylline, a rapid and specific method by liquid chromatography with heated electrospray ionization tandem mass spectrometry（LC-HESI/MS/MS） method for simultaneous determination of theophylline, 1, 3-dimethyluric acid（1, 3-DMU）, 3-methylxanthine（3-MX） and 1-methyluric acid（1-MU） in human urine was developed using theophylline-d6 and 5-fluorouracil as internal standards. Selected reaction monitoring（SRM） with heated electrospray ionization（HESI） was used in the negative mode for mass spectrometric detection. After diluted with methanol and centrifuged, the analytes and ISs were separated on a XDB-Phenyl（150 mm × 4.6 mm, 5 μm） column with a mixture of water- methanol-formic acid（30∶70∶0.15） as mobile phase at a flow rate of 0.6 mL·min-1. The linear calibration curves for theophylline, 1, 3-DMU, 3-MX and 1-MU were obtained in the concentration range of 1.0-250 μg·mL-1, separately. The method herein described is effective and convenient, and can be used for determination of theophylline and its three metabolites. The results showed that urinary excretion ratio of theophylline, 1, 3-DMU, 3-MX and 1-MU is approximately 1∶3∶1∶2 in Chinese subjects, which is similar to the reported excretion pattern in Caucasian.