Derivatization for LC-electrospray ionization-MS: a tool for improving reversed-phase separation and ESI responses of bases, ribosides, and intact nucleotides

We have developed a method for analyzing polar compounds by reversed-phase LC-ESI-MS following esterification of the analytes' free hydroxyl groups with propionyl or benzoyl acid anhydride. The method was applied to members of the plant hormone group cytokinins, which includes adenine bases, ribosides/glycosides, and nucleotides substituted at N-6 with an isoprenoid side chain, spanning a wide range of polarity. It was also used to analyze other compounds of biological importance, e.g., the nucleotides AMP, ADP, and ATP. The formation of more hydrophobic derivatives had a significant impact on two aspects of the analysis. The retention on a reversed-phase material was greatly increased without the use of any acetate/formate buffer or ion pairing reagent, and the ESI response was enhanced, due to the higher surface activities of the derivatives. Detection limits of propionylated cytokinins were in the high-attomole to low-femtomole range, an improvement by factors of 10-100 compared to previously reported figures. Using an automated SPE-based purification method, 12 endogenous cytokinins were quantified in extracts from 20- to 100-mg samples of leaves (from the plant Arabidopsis thaliana) with high accuracy and precision. Furthermore, the chromatographic properties of the benzoylated AMP, ADP, and ATP in the reversed-phase LC-MS system were much better in terms of retention, separation, and sensitivity than those of their underivatized counterparts, even without the use of any ion pairing reagent. Our data show that derivatization followed by LC-ESI-MS is an effective strategy for analyzing low molecular weight compounds, enabling compounds with a wide range of polarity to be determined in a single-injection LC-MS analysis.