Development And Optimization Of Modified Nucleosides And Deoxynucleosides Simultaneous Extraction With The Use Of Design Of Experiments Approach

Modified nucleosides and deoxynucleosides are investigated as potential markers of pathological disorders. Due to different molecular properties, their concurrent extraction from biological matrices is challenging. The aim of the present work was the development of sample preparation procedure allowing for simultaneous extraction of 12 selected modified nucleosides and deoxynucleosides with the use of solid phase extraction (SPE) technique. Commercially available mixed-mode SPE cartridges based on phenylboronic acid and cation-exchange sorbents were utilized. In order to decrease the number of experiments and, consequently the volume of applied solvents, the developed method was optimized with the use of Design of Experiments approach. Optimization of SPE method was carried out by employing Fractional Factorial Design with selected input variables such as composition of conditioning, washing and elution solvents. Next, Full Factorial Design with conditioning, extraction and elution duration time as factors was employed. Evaluated responses in these two experimental sets were peak areas of selected analytes. Levels of assessed factors were determined. Developed and optimized SPE procedure allowed for simultaneous extraction of modified nucleosides and deoxynucleosides with mean recovery of 76.0%. Procedure was applied on the exemplary urine samples with the use of high performance liquid chromatography hyphenated with triple quadrupole mass spectrometry (HPLC-QqQ/MS).