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

KeyWord:theophylline  in vivo metabolites  LC-MSn  precolumn derivatization

Abstract:

      A solid phase extraction and precolumn chemical derivatization technique coupled with liquid chromatography-electrospray ionization/mass spectrometric£¨LC-ESI/MSn£© method was developed for the analysis of theophylline and its metabolites in human urine after oral administration of theophylline tablets to discover its metabolism pathway in vivo.The mass spectral fragmentation behaviors 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 C18 column.Methanol part was collected to be dried by N2 at 50 ¡æ,and then subjected to derivatization with N,N-dimethyl ethylamine chloride.The separation of urine samples was achieved on a Shimpack C18 column with methanol-water-formic acid£¨20¡Ã80¡Ã1£© as mobile phase,and the detection was fulfilled with electro spray ionization£¨ESI£© in positive mode.Through this method,theophylline and four metabolites£¨1-methyl uric acid,1,3-dimethyl uric acid,1-methyl-N-acetyl derivative and 3-methyl xanthine£© were identified or tentatively identified,in which one of these metabolites was firstly recovered in theophylline.Under positive mode,theophylline and its metabolites always lost 18 u£¬28 u or 57 u fragments in MS2 spectrum and their derivatives lost 45 u fragments regularly in MS2 spectrum.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.