Comprehensive quantitative analysis of purines and pyrimidines in the human malaria parasite using ion-pairing ultra-performance liquid chromatography–mass spectrometry

• Quantification of 35 purines and pyrimidines accomplished in a 15 min runtime.
• Versatility to be customized for other metabolites with similar chemical properties.
• Broad spectrum of potential applications from drug discovery to cell biology.
• The method was validated and applied to malaria parasites and human erythrocytes.

Targeted metabolite profiling has aided in the understanding of a variety of biological processes in the malaria parasite as well as in drug discovery. A fast and sensitive analytical method, based on ion-pairing reversed phase ultra-high performance liquid chromatography tandem mass spectrometry (IP-RP–UPLC–MS/MS), was optimized for the simultaneous analysis of intracellular levels of 35 purine and pyrimidine nucleobases, nucleosides, and nucleotides. This analytical method allows for chromatographic separation of highly polar metabolites using reverse phase chemistry within 15 min. The analytical performance of the method was evaluated and successfully applied to the quantification of purines and pyrimidines in Plasmodium falciparum and its host cell, the human erythrocyte. In addition, this method can be customized to include other related metabolites such as NADPH and NADP, among others.