Evaluation of urinary ribonucleoside profiling for clinical biomarker discovery using constant neutral loss scanning liquid chromatography/tandem mass spectrometry

Abstract

The patterns and levels of urinary excreted ribonucleosides which reflect RNA turnover and metabolism in humans offer the potential for early detection of disease and monitoring of therapeutic intervention. A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method employing constant neutral loss (CNL) scanning for the loss of the ribose moiety (132 u) was used to detect ribonucleosides in human urine and to evaluate this analytical platform for biomarker research in clinical trials. Ribonucleosides were stable and not influenced by the time spent at room temperature prior to freezing or long-term storage at -80 °C. Matrix effects caused variation in the mass spectrometer response which was dependent on the concentration of the analysed urine sample. For the use of urinary ribonucleoside profiling in clinical biomarker studies, adjustment of the urine samples to a common concentration prior to sample preparation is therefore advocated. Changes in the mass spectrometer response should be accounted for by the use of an internal standard added after sample preparation. Diurnal variation exceeded inter-day variation of an individual's ribonucleoside profile, but inter-person differences were predominant and allowed the separation of individuals against each other in a multivariate space. Due to considerable diurnal variation the use of spot urine samples would introduce unnecessary variation and should be replaced by the collection of multiple spot urine samples across the day, where possible. Should such a protocol not be feasible, biological intra-day and inter-day variation must be considered and accounted for in the data interpretation.

Evaluating the reproducibility of quantifying modified nucleosides from ribonucleic acids by LC–UV–MS

“…The reproducibility of LC/UV and various LC/MS techniques for the quantitative analysis of urinary nucleosides has been reported[29–34]. Those methods were characterized using a set of urinary nucleoside standards and yielded reproducibilities in the range of 1–15% RSD using either UV or MS as the detector.…”

“…Among the various MS/MS techniques used for quantification, Wang et al characterized nine urinary nucleosides with a reproducibility ranging from 5 to 16% RSD using selected ion monitoring MS [33]. More recently, Teichert et al used constant neutral loss scans to quantify 35 urinary nucleosides with reproducibilities ranging from 6 to 50% RSD [34]. …”

Polyethyleneimine-grafted boronate affinity materials for selective enrichment of cis -diol-containing compounds

“…According to the previous reports [22,26,27], urine samples contain various ribonucleosides that act as the biomarkers of malignant diseases (e.g., cancer). Fortunately, most urinary ribonucleosides are cis-diol-containing biomolecules, and a neutral mass loss of the ribose moiety (132.04 Da) has been widely used for their identification.…”

Analysis of urinary nucleosides as potential cancer markers determined using LC–MS technique

“…Different approach to extraction was proposed by Teichert et al [50]. The authors performed extraction procedure using PBA sorbent bed and compared it with another one performed with Oasis HLB cartridges that are polymeric reverse-phase columns (a copolymer of divinylbenzene-co-N-vinylpyrrolidone).…”

“…Besides, application of mass spectrometry detection gives the possibility of applying various types of ionization as well as analyzers. Hence, for analysis of nucleosides three types of ionization source has been employed, namely, fast atom bombardment (FAB) [42], matrix assisted laser desorption ionization (MALDI) [43,44,63] and electrospray ionization (ESI) [7,18,21,25,26,[45][46][47][48][49][50]52,54,55,62]. All of them belong to so-called soft ionization technique.…”