柱前衍生化结合LC-MS^n分析人尿中茶碱及其代谢物

Analysis of Theophylline and Its Metabolites in Human Urine by Precolumn Derivatization Coupled with Liquid Chromatography Tandem Mass Spectrometry

采用固相萃取结合化学衍生化的样品柱前预处理方法,利用液相色谱-电喷雾/多级质谱联用技术(LC-ESI/MSn),对人经口服给药后尿液中的茶碱及其代谢产物进行分析,探讨了茶碱、代谢物及相关衍生物的质谱裂解行为,推测茶碱在人体内的代谢途径。尿样经Bond Elute C18小柱固相萃取,收集甲醇洗脱部分在50℃下氮气吹干,以N,N-二甲氨基氯乙烷为衍生试剂进行衍生化。以Shimpack C18为色谱柱、甲醇-水-甲酸(体积比20∶80∶1)为流动相,在正离子模式下对尿样中茶碱、代谢物及其衍生化产物进行LC-ESI/MSn分析。采用上述方法,共分析鉴定了人尿中的茶碱和4种代谢物(1-甲基尿酸、1,3-二甲基尿酸、1-甲基-N-乙酰化物、3-甲基黄嘌呤),其中1种代谢物未见文献报道。在正离子模式下,茶碱及代谢物的二级质谱大多丢失18 u,28 u或57 u片段生成一系列碎片离子,而衍生化产物的二级质谱均有规律地丢失45 u片段生成一系列碎片离子。通过与未经衍生化的尿样比较发现,衍生化可明显增强茶碱、1-甲基尿酸和3-甲基黄嘌呤的质谱检测灵敏度。本研究补充了茶碱在人体内的代谢轮廓,可为灵敏检测生物样本中茶碱、代谢物及其结构类似物提供借鉴。

A solid phase extraction and precolumn uid chromatography - eleetrospray ionization/mass chemical derivatization technique coupled with liq- spectrometric （ LC - ESI/MS ） method was devel- oped for the analysis of theophylline and its metabolites in human urine after oral administration of the- ophylline tablets to discover its metabolism pathway in vivo. The mass spectral fragmentation behav- iors of theophylline, metabolites and their derivatives were also discussed. Human urine between 0 hour and 6 hour was collected immediately after administration of theophylline tablets. Then the urine samples were pretreated by solid phase extraction through a Bond Elute C^s column. Methanol part was collected to be dried by N2 at 50 ~C, and then subjected to derivatization with N, N-dimethyl ethylamine chloride. The separation of urine samples was achieved on a Shimpack Czs column with methanol -water- formic acid（20 ： 80 ： 1 ） as mobile phase, and the detection was fulfilled with e- lectro spray ionization（ESI） in positive mode. Through this method, theophylline and four metabo- lites （1-methyl uric acid, 3-dimethyl uric acid, 1-methyl-N-acetyl derivative and 3-methyl xan-thine） were identified or tentatively identified, in which one of these metabolites was firstly recovered Through the comparison with the underivatized urine samples, the samples derivatized were found to be able to enhance the sensitivity of theophylline, 1-methyl uric acid and 3-methyl xanthine in mass spectrometry. This method replenished the metabolite file of theophylline in human, and provided a new approach for the identification of theophylline, metabolites and their structural analogues with low concentration in biosamples.