title: Development, validation and application of a fast analytical methodology for the simultaneous determination of DNA- and RNA-derived urinary nucleosides by liquid chromatography coupled to tandem mass spectrometry.
abstract: In the present work we report the development and validation of a fast liquid chromatography-mass spectrometry method for the simultaneous determination of endogenous nucleosides derived from DNA and RNA in urine. The target compounds were 2'-deoxyguanosine and 8-hydroxy-2'-deoxyguanosine, derived from DNA, and the analogue 8-hydroxyguanosine, derived from RNA, together with adenosine, 1-methyladenosine, 7-methylguanosine and inosine. The method is based on the use of a chromatographic column packed with superficially porous particles for high-efficiency separation; further detection by MS/MS was accomplished with a triple quadrupole-mass spectrometer for analyte identification and accurate quantification. As a preliminary purification step, we developed a new procedure based on solid-phase extraction (SPE) with a mixed-sorbent prepared from three polymeric materials that facilitated the isolation of modified nucleosides, such as 2-deoxynucleosides, that are not retained by phenylboronic acid-based SPE. The proposed approach (SPE prior to LC-MS/MS) was validated in human urine in terms of linearity, the limit of detection, the limit of quantification, accuracy, recovery, repeatability, reproducibility and matrix-effects. For the SPE step, intra-day and inter-cartridge reproducibility were evaluated in natural and spiked urine samples, being ± 16.9% or below, with recoveries in the 74-125% range. No significant matrix effects were found in further MS/MS detection. The application of the present method to urine from healthy smoker and non-smoker volunteers is also reported in order to test its usefulness as a tool for clinical and toxicological trials.
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.affiliation: Departamento de Química Analítica, Nutrición y Bromatología, Facultad de Química, Universidad de Salamanca, 37008 Salamanca, Spain.
authors.forename: Leticia
authors.initials: L
authors.lastname: Herrero-Herrero
authors.affiliation: Departamento de Química Analítica, Nutrición y Bromatología, Facultad de Química, Universidad de Salamanca, 37008 Salamanca, Spain.
authors.forename: Diego
authors.initials: D
authors.lastname: García-Gómez
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D000328:Adult; D002853:Chromatography, Liquid / Q000379:methods*; D004247:DNA / Q000302:isolation & purification / Q000652:urine*; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D005260:Female; D006151:Guanosine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D008297:Male; D009705:Nucleosides / Q000302:isolation & purification / Q000652:urine*; D012313:RNA / Q000302:isolation & purification / Q000652:urine*; D052616:Solid Phase Extraction / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D055815:Young Adult
chemical_list: D009705:Nucleosides; D006151:Guanosine; C016578:7-methylguanosine; D012313:RNA; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D003849:Deoxyguanosine
keywords: Fast liquid chromatography; Mixed-sorbent; Non-smokers; Smokers; Superficially porous particles; Tandem mass spectrometry; Urinary nucleosides