Targeted metabolomics: Liquid chromatography coupled to mass spectrometry method development and validation for the identification and quantitation of modified nucleosides as putative cancer biomarkers

A notable change in the body fluids nucleosides of cancer patients has been actively highlighted in searches for new biomarkers to early cancer detection. For this reason, improvements of bioanalytical methods for these compounds focused on a noninvasive sampling trend are of great importance. Therefore, this work aimed firstly to develop efficient methods for nucleoside analysis in urine and serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS), applying different strategies to quantify nine nucleosides, and further identify other untargeted nucleosides. Sample preparation was based on protein precipitation and affinity-solid phase extraction (SPE), whereas quantification was performed using a triple quadrupole (QqQ) mass analyzer operating in the selected reaction monitoring (SRM) mode. Surrogates matrices were proposed as an alternative to standard addition calibration. Specifically, to quantitate creatinine, a simple LC-MS/MS method was validated and used for normalization of urinary metabolites quantitation. To identify the other nucleosides, LC methods using different MS scans modes were evaluated on a quadrupole-time of flight (Q-TOF) or a hybrid triple quadrupole linear ion trap (Q-trap). Validation was performed for nucleosides quantification using the synthetic matrices of urine and serum, and selectivity, linearity, accuracy, reproducibility, matrix effect, LOD's and LOQ's were accessed, providing trustworthy results for bioanalysis purposes. Both LC-Q-Trap/MS and LC-Q-TOF/MS methods showed proper sensitivity for structural characterization on assays with urine and serum samples from healthy volunteers and could also be used in the identification of untargeted nucleosides. The investigated approaches delivered in-depth results and seem promising for future applications on urine and serum samples analyses aiming to validate nucleosides as cancer biomarkers.