RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2006; 20: 137–150 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.2266 Analysis of urinary nucleosides. V. Identification of urinary pyrimidine nucleosides by liquid chromatography/electrospray mass spectrometry Alison Bond 1,5 , Edward Dudley 1 , Filip Lemie `re 2,3 , Robin Tuytten 1,2 , Salah El-Sharkawi 4 , A. Gareth Brenton 1,6 , Eddy L. Esmans 2,3 and Russell P. Newton 1,5 * 1 Biomolecular Analysis Mass Spectrometry (BAMS) Facility, Grove Building, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK 2 Department of Chemistry, Nucleoside and Mass Spectrometry Research Unit, University of Antwerp, Groenenborgerlaan 171, B-2020, Antwerp, Belgium 3 Centre for Proteome Analysis and Mass Spectrometry (CeProMa), University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium 4 Oncology Unit, Singleton Hospital Trust, Sketty, Swansea SA2 8QA, UK 5 Biochemistry Group, School of Biological Sciences, Wallace Building, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK 6 Mass Spectrometry Research Unit, Department of Chemistry, Grove Building, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK Received 4 October 2005; Revised 24 October 2005; Accepted 24 October 2005 Modified urinary nucleosides are potentially invaluable in cancer diagnosis, as they reflect altered RNA turnovers. High-performance liquid chromatography (HPLC) was combined with full-scan mass spectrometry, tandem mass spectrometry, MS n analysis and accurate mass measurements in order to identify pyrimidine nucleosides purified from urine. Potential nucleosides were assessed by their evident UV absorbance in the HPLC chromatogram and then further examined by the various mass spectrometric techniques. In this manner numerous pyrimidine nucleosides were identified in the urine samples from cancer patients including pseudouridine, cytidine, two methylcytidines and an acetylcytidine. Furthermore, a number of novel modified pyrimidine nucleosides were tentatively identified via critical interpretation of the combined mass spectro- metric data. Copyright # 2005 John Wiley & Sons, Ltd. A number of past studies have indicated that modified nucleosides excreted in the urine have the potential to act as cancer biomarkers with increased levels arising from disease onset and progression. 1–4 Modified nucleosides differ from ‘normal’ nucleosides usually by a minor modification, with the most common alteration being methylation. Modification of nucleosides occurs after incorporation into nucleic acid molecules of which transfer RNA (tRNA) is the most com- monly modified. During tRNA turnover these compounds are released from the nucleic acid molecules and, due to the absence of salvage or degradation pathways and the potential toxicity of their accumulation, they are excreted via the urine. Although the levels of these compounds have been studied over a number of decades it is only recently that mass spectro- metric means have been employed for the purpose. 5–10 Mass spectrometry (MS) has been successfully applied to analyse nucleosides from various sources including ancient seeds 11 and RNA hydrolysates 12 and has been assessed by our group for the detection of the urinary modified nucleo- sides. Electrospray ionisation tandem mass spectrometry (ESI-MS/MS), when coupled with high-performance liquid chromatography (HPLC), has the advantage over previously utilised methods of detection, which include HPLC with UV absorbance detection, 1,2 capillary electrophoresis 13 and fluorescence spectroscopy, 14 that more structural data can be obtained, thereby allowing improved certainty on the identity of the nucleosides being studied. The need to employ MS analysis as opposed to HPLC alone can readily be gauged once the number and diversity of naturally occurring modified nucleosides are appreciated, as detailed in two previous reviews. 15,16 Nucleoside identification by UV absorbance and HPLC retention time, typically over an hour run time, can clearly lead to co-elution and erroneous identification if such a large number of similar compounds is present. MS analysis will allow the discriminative detection of co-eluting nucleosides as long as they differ (sufficiently) in mass. Therefore, using MS it is less likely that the results from two co-eluting nucleosides will accidentally be assigned as being that of one nucleoside. Furthermore, many mass spectrometers can acquire product ion spectra of the nucleo- sides and these can provide structural information on these compounds. The fragmentation of these compounds and their protonated heterocyclic base units [BH þ ] by MS means Copyright # 2005 John Wiley & Sons, Ltd. *Correspondence to: R. P. Newton, Biochemistry Group, School of Biological Sciences, Wallace Building, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK. E-mail: r.p.newton@swansea.ac.uk Contract/grant sponsor: Evan Davies Trust Fund. Contract/grant sponsor: Flemish Government GOA Action; contract/grant numbers: FWO-Vlaanderen 1.5.139.00 and G.2133.94.