Nowadays, mass spectrometry is very important and widely applied tool in nucleotide analysis. As a result of technological advances in sample preparation, separation and mass spectrometry detection, the developed methods allow sensitive and selective measurement of polar compounds occurring in low levels in various biological matrices. This enables more potential uses in clinical field. Direct methods require no special sample pre‐treatment before analysis in contrast to indirect methods, where fractionation, dephosphorylation and purification are needed. The use of ion‐pairing agent, ion exchange chromatography with pH gradient, porous graphitic carbon columns and HILIC in liquid chromatography represents the most common methods of nucleotide analysis. High separation efficiency is also achieved with the use of CE with MS detection. Analysis of nucleotides was also described by the means of MALDI‐TOF, but poor reproducibility and lack of applications make a limitation for this approach. The chapter summarizes different techniques and approaches for determination of endogenous nucleotides and its analogues in various clinical applications
