Analysis of urinary nucleosides. I. Optimisation of high performance liquid chromatography/electrospray mass spectrometry

Dudley, Edward G.; El-Sharkawi, Salah; Games, David E.; Newton, Russell P.

doi:10.1002/1097-0231(20000730)14:14<1200::aid-rcm10>3.0.co;2-i

Cited by 43 publications

(36 citation statements)

References 13 publications

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“…Exceptionally, pseudouridine did not produce base ions [BH 2 ] þ due to the presence of the stable C-C glycosidic bond; instead the major fragmentation involved loss of two water molecules to give m/z 209, and an additional fragment at m/z 155 arising from loss of a part of the 2-deoxyribose moiety. The ion at m/z 179 was probably formed by loss of formaldehyde (CH 2 O) and two water molecules 19 (see Table 2, Fig. 3).…”

Section: Analysis Of Positive Ionization Of Nucleosides By Lc/msmentioning

confidence: 99%

“…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 extraction and/or derivatization steps. An earlier LC/MS method 19 involved extraction and workup steps and used LC/MS (rather than LC/MS/MS) for quantification of a more limited number of nucleosides. Using our new method we studied the urinary profiles of nucleosides in normal healthy subjects.…”

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confidence: 99%

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

Lee

Jung

Kim

et al. 2004

Rapid Comm Mass Spectrometry

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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...

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Section: Analysis Of Positive Ionization Of Nucleosides By Lc/msmentioning

confidence: 99%

mentioning

confidence: 99%

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

Lee

Jung

Kim

et al. 2004

Rapid Comm Mass Spectrometry

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“…The nucleosides of lower basicity, especially the ones with pyrimidine base, show a higher LOD than purines, as shown in Table°2.°The°resolution°usually°varies°in°a°range°from 5000 to 9000. Dudley et al determined LOD between 19 and 110 pmol for nucleoside standard solutions measured°by°LC-ESI°MS° [20].°Compared°with°these°results, MALDI-TOF MS is the most suitable mass spectromet- [20].°Analo-gous fragmentation was seen in our PSD spectra. The nucleosides decay under loss of 132 Da (ribose), except pseudouridine, because of the stronger carbonOcarbon bond between sugar and nucleic base.…”

Section: Resultsmentioning

confidence: 70%

“…The nucleosides decay under loss of 132 Da (ribose), except pseudouridine, because of the stronger carbonOcarbon bond between sugar and nucleic base. Dudley et al observed the fragments m/z 209, 179, and 155°for°pseudouridine° [20].°In°our°PSD-spectrum°of pseudouridine,°which°is°shown°in°Figure°3,°these masses also occurred. The PSD spectra of the other standard nucleosides show a characteristic decay between nucleic base and sugar.…”

Section: Resultsmentioning

confidence: 98%

MALDI-TOF MS analysis of urinary nucleosides

Kammerer¹,

Frickenschmidt²,

Gleiter³

et al. 2005

J. Am. Soc. Mass Spectrom.

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As RNA turnover seems to be impaired in cancer patients, modified nucleosides have been evaluated as potential tumor markers. Modified nucleosides are mainly formed posttranscriptionally in tRNA, set free during RNA metabolism, and excreted in urine. Especially methylated nucleosides play an important role, as their levels are higher in urine from cancer patients. For structural elucidation of known and unknown nucleosides from urine samples of cancer patients, MALDI-TOF MS and MALDI-PSD were used for the first time. This technique generally ensures high sensitivity, mass resolution, and accuracy. In our analytical approach we prepurified nucleosides from urine by affinity chromatography and subsequently separated them by semipreparative high performance liquid chromatography. The different fractions were collected separately and analyzed by MALDI-TOF MS and PSD-MALDI using a mixture of six low molecular weight calibrants for internal or external calibration. The molecular totals formulas based on a mass accuracy of 10 ppm and below were calculated and a systematic data base search was performed. The inherent problem of the MALDI-technique, the reduced sensitivity for low molecular weight substances caused by matrix suppression effects, was reduced by our technique. We identified several nucleosides in urine, which were previously identified via retention times and UV spectra of standards after HPLC analysis. Eight further nucleosides were observed. This work demonstrates for the first time the potential of MALDI-TOF and PSD-MALDI in combination with semipreparative HPLC for assignment of nucleosides in urine. The particularly high mass accuracy of this mass spectrometric method provides opportunities for identifying unknown compounds. (J Am Soc Mass Spectrom 2005, 16, 940 -947)

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“…Different methods have been applied for identifying and quantifying nucleosides using high-performance liquid chromatography (HPLC) with UV detection and capillary electrophoresis (CE) [13]. Recently, the coupling of HPLC, gas chromatography or capillary liquid chromatography with mass spectrometric detection via electrospray ionization ion trap mass spectrometry (ESI-IT MS) [14], ESI tandem MS [15] and fast atom bombardment (FAB MS) [16] has been applied. ESI-TOF MS [17] and offline mass spectrometric techniques like MALDI-TOF MS [18] have proven valuable tools for the identification of nucleoside structures.…”

Section: Introductionmentioning

confidence: 97%

Metabolic signature of breast cancer cell line MCF–7: Profiling of modified nucleosides via LC-IT MS coupling

Neubauer¹,

Bullinger²,

Fehm³

et al. 2008

Geburtsh Frauenheilk

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Background: Cancer, like other diseases accompanied by strong metabolic disorders, shows characteristic effects on cell turnover rate, activity of modifying enzymes and DNA/RNA modifications, resulting also in elevated amounts of excreted modified nucleosides. For a better understanding of the impaired RNA metabolism in breast cancer cells, we screened these metabolites in the cell culture supernatants of the breast cancer cell line MCF-7 and compared it to the human mammary epithelial cells MCF-10A. The nucleosides were isolated and analyzed via 2D-chromatographic techniques: In the first dimension by cis-diol specific boronate affinity extraction and subsequently by reversed phase chromatography coupled to an ion trap mass spectrometer.

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Analysis of urinary nucleosides. I. Optimisation of high performance liquid chromatography/electrospray mass spectrometry

Cited by 43 publications

References 13 publications

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

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

MALDI-TOF MS analysis of urinary nucleosides

Metabolic signature of breast cancer cell line MCF–7: Profiling of modified nucleosides via LC-IT MS coupling

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