Fast quantification of methylated nucleosides in urine by triple quadrupole mass spectrometry

Modified nucleosides and nucleobases have raised great scientific interest due to their possible use as diagnostic and prognostic markers for various diseases. One of the most common types of modification is methylation. Sensitive and accurate methods are essential for the quantification of these compounds in biological samples. In this work we propose, for the first time, a rapid non-separative method for the determination of methylated nucleosides in urine by direct injection on electrospray ionization triple quadrupole mass spectrometer (ESI-MS/MS). The proposed method has been applied to the simultaneous determination of five methylated nucleosides in human urine samples. A separative method based on HILIC-MS/MS has been developed in parallel with the same instrumental setup to confirm the results obtained by the non-separative method. Both methods were validated in terms of linearity, matrix effect, limits of quantification, precision and recovery. The proposed method was successfully applied to the determination of the selected analytes in the urine of healthy volunteers and was compared with the results obtained by the separative method. The proposed non-separative method considerably reduced the required analysis time and simplified the sample treatment, which is a remarkable advantage over the chromatographic methods described. Therefore, this method is suitable for rapid, simple, and reliable determination of endogenous biomarkers in urine samples for routine or high-throughput analysis. The separative method is recommended as a complementary tool for confirmation in complex situations or in cases of abnormally high biomarker levels, maintaining specificity and robustness without compromising the practical advantages of the rapid method.