title: Capillary electrophoresis-mass spectrometry for direct determination of urinary modified nucleosides. Evaluation of synthetic urine as a surrogate matrix for quantitative analysis.
abstract: This work describes the development of a fast and reliable method based on capillary zone electrophoresis coupled with electrospray ionization-mass spectrometry (CZE-ESI-MS) for the determination of modified nucleosides in untreated human urine. The target compounds were guanine, 1-methyl-guanine, 7-methyl-guanine, 9-methyl-guanine, adenosine, 1-methyl-adenosine, cytidine, guanosine, 7-methyl-guanosine. As internal standards, ribose-2-(13)C-adenosine and 8-(13)C-guanine were used. The CZE separation was carried out in acidic medium (pH 2.5). MS detection with a single quadrupole, with ESI operating in positive-ion mode, was optimized. For the analysis of urine samples, owing to the endogenous character of these analytes different quantification strategies were explored. The standard additions method, matrix-matched calibration in synthetic urine and calibration in pure aqueous medium were compared in order to evaluate the endogenous levels of these compounds in human urine. The results obtained showed that calibration in synthetic urine as a surrogate matrix was an appropriate alternative to the method of standard additions for the accurate quantitation of compounds such as guanine, 1-methyl-guanine, 7-methyl-guanine, adenosine, 1-methyl-adenosine and cytidine by CE-ESI-MS directly in the urine matrix; values in the range 0.1μg/mL for cytidine and 6.4μg/mL for 7mGua, as the lowest and the highest level, were found in untreated urine from healthy volunteers. These results were confirmed by LC-MS/MS detection. It can be concluded that the electrophoretic CZE-ESI-MS methodology offers a valid and reliable alternative for the determination of urinary nucleosides at naturally occurring levels in healthy individuals.
authors.affiliation: Departamento de Química Analítica, Nutrición y Bromatología, Facultad de Química, Universidad de Salamanca, 37008 Salamanca, Spain. Electronic address: erg@usal.es.
authors.forename: Encarnación
authors.initials: E
authors.lastname: Rodríguez-Gonzalo
authors.forename: Raquel
authors.initials: R
authors.lastname: Hernández-Prieto
authors.forename: Diego
authors.initials: D
authors.lastname: García-Gómez
authors.forename: Rita
authors.initials: R
authors.lastname: Carabias-Martínez
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D016014:Linear Models; D008954:Models, Biological; D009705:Nucleosides / Q000737:chemistry* / Q000652:urine*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D009705:Nucleosides
keywords: Capillary electrophoresis; Direct analysis; Mass spectrometry; Nucleosides; Surrogate matrix; Synthetic urine