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.
