A rapid and sensitive method for quantitation of nucleosides in human urine using liquid chromatography/mass spectrometry with direct urine injection

Abstract

Oxidized nucleosides are biochemical markers for tumors, aging, and neurodegenerative diseases. However, during the last decade, the analytical methods for nucleosides by gas chromatography/ mass spectrometry (GC/MS) and high-performance liquid chromatography (HPLC) with singleparameter detectors like electron-capture detection (ECD) have not been sufficiently rapid or reliable to detect nucleosides in urine and to analyze clinical samples. It has been reported (Dudley et al., Rapid Commun Mass Spectrom. 2000; 14: 1200) that liquid chromatography with electrospray mass spectrometry (LC/ESI-MS) is more specific and sensitive for analysis of nucleosides than HPLC with conventional detectors; however, this method required complex extraction steps. In the present work a direct LC/ESI-MS method for nucleosides without extraction of urine samples has been developed. Analysis of nucleosides using positive-ion mode with selected reaction monitoring effectively eliminated potential interferences from endogenous constituents of the urine. This highly selective and sensitive method made it possible to analyze urinary nucleosides with a lower limit of quantitation of 0.2 nmol/mL. The method has been validated, with both excellent linearity and reproducibility, in the calibration range from 0.2-400 nmol/mL. The correlation coefficients of the calibration curves were higher than 0.987. The coefficients of variation were in the range 0.03-14.92% (inter-day) and 0.54-14.39% (intra-day), respectively. Copyright # 2004 John Wiley & Sons, Ltd.Oxidized nucleosides represent excellent biomarkers for determining the extent of damage in genetic material, which has long been of interest in understanding the mechanism of aging, neurodegenerative diseases, and carcinogenesis. [1][2][3][4][5] Various methods have been developed for quantifying modified nucleosides, based on an enzyme-linked immunosorbent assay (ELISA), 6,7 32 P-post-labelling, 8 capillary electrophoresis, 9,10 HPLC with photodiode array detection 11,12 or ECD, 13 and GC/MS. 14-16 However, GC/MS analysis faced additional problems in the hydrolysis and derivatization steps. 17 HPLC/ECD analysis has limitations in that the requirement for electroactive components meant that the method was mainly applicable to the detection of only 8-oxodeoxyguanosine. 18 Due to their chemical characteristics in HPLC analysis, various kinds of nucleosides could not be analyzed using just one kind of detector.To overcome these problems, in the present work, we developed an analytical method for determining many kinds of nucleosides at one time using LC/MS with ESI (electrospray ionization) detection. We confirmed that mass spectrometric detection with ESI is more specific and sensitive than any other HPLC detection method. We also developed a direct analytical method for nucleosides without extracting urine samples. This approach is both more convenient and more efficient in conducting studies with large sample numbers, e.g., a clinical study, than analytical methods involving complex extrac...

Rapid and selective simultaneous quantitative analysis of modified nucleosides using multi-column liquid chromatography-tandem mass spectrometry

“…Descriptions of selected reaction monitoring (SRM) transitions can be found in previous studies and are summarized in Table 3. (Lee et al 2004;Su et al 2014;Fan et al 2006) The LC-MS/MS system was controlled by Xcalibur software (Thermo Fisher Scientific), which was also used to collect data.…”

“…2 and demonstrate sufficient separation using an ADME column. Simultaneous quantitative analysis of modified nucleosides was previously performed using the ODS column (Djukovic et al 2010;Russell and Limbach 2013;Lee et al 2004;Basanta-Sanchez et al 2016;Rodríguez-Gonzalo et al 2016). Chromatographic separation on the ODS column depends on the hydrophobic interaction, which enables separation by the number of methyl groups (Núñez et al 2007).…”

“…In addition, biomarker evaluation and discovery necessitates the analysis of large sample sizes to ensure statistical reliability (Koulman et al 2009); thus, the analytical methods applied must have a high throughput. Analytical methods for modified nucleosides include liquid chromatography (LC), ultra-performance LC (UPLC), gas chromatography, or capillary electrophoresis combined with ultraviolet or mass spectrometry (MS) (Russell and Limbach 2013;Struck et al 2011;Lee et al 2004;Basanta-Sanchez et al 2016). Of these methods, LC-tandem MS (LC-MS/MS) analysis is the most frequently used for modified nucleoside determination due to its high selectivity and quantitative performance.…”

“…Of these methods, LC-tandem MS (LC-MS/MS) analysis is the most frequently used for modified nucleoside determination due to its high selectivity and quantitative performance. Several studies have reported good chromatographic separation; however, these methods require long times to separate nucleosides (Djukovic et al 2010;Cho et al 2009;Struck et al 2011;Lee et al 2004). Rodríguez-Gonzalo et al (2016) reported an analytical method that quickly and simultaneously determined nucleosides; however, this method has not been used to study significant cancer biomarkers or cellular stress markers, such as m 5 C, Cm, and m 2 2 G. (Nakano et al 1993;Djukovic et al 2010;Cho et al 2009;Chan et al 2010) m 3 C Alkylating stress (Chan et al 2015) m 5 C Oxidative stress, replication stress (Chan et al 2010(Chan et al , 2012 Cm Oxidative stress, replication stress (Chan et al 2010) s 2 C Interference with codon-anti-codon interaction (Agris 2015) m 5 U Replication stress (Endres et al 2015) Ψ Gastric cancer, intestinal cancer, lung cancer, AIDS, leukemia, lymphoma (Nakano et al 1993;Itoh et al 1992) (Korfmacher et al 1999;Cass et al 2001;Oertel et al 2002;Orton et al 2013;Lee et al 2015;Patel et al 2016).…”

Detection of 1,N2-propano-2′-deoxyguanosine in human urine by stable isotope dilution UHPLC–MS/MS analysis

“…Various methods have been developed for the detection of urinary nucleosides, including electrochemical method [18], immunoassays [19,20], capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) [17,[21][22][23][24], matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) [25,26], liquid chromatography tandem mass spectrometry (LC-MS) [27][28][29][30][31][32]. In comparison to other methods, the LC-MS based methodologies take the advantages of high sensitivity and direct analysis with structural information from MS measurements [33].…”