Comparison of reversed-phase, anion-exchange, and hydrophilic interaction HPLC for the analysis of nucleotides involved in biological enzymatic pathways

Allison Fabino Carr*a http://orcid.org/0000-0003-1437-8170, Darshan C. Patelbc, Diego Lopezbd, Daniel W. Armstrongb & Victor Ryzhov*aa Department of Chemistry and Biochemistry, Northern Illinois University, Dekalb, IL, USA; b Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, TX, USA; c Process Research & Development, AbbVie Inc, North Chicago, IL, USA; d AZYP LLC, Arlington, TX, USAIn accordance with Taylor & Francis policy and my ethical obligation as a researcher, I am reporting that I have received discounted columns from AZYP LLC, a company that may be affected by the research reported in the enclosed paper. I have disclosed those interests fully to Taylor & Francis, and I have in place an approved plan for managing any potential conflicts arising from that involvement.Supplemental data for this article can be accessed on the publisher’s website.CONTACT Allison Fabino Carr afabino1@niu.edu Department of Chemistry and Biochemistry, Northern Illinois University, 1425 W. Lincoln Hwy, Dekalb, 60115, IL, USAAbstractNucleotides and other phosphate-containing compounds are integral to enzymatic reactions such as those of the methylerythritol phosphate (MEP) pathway and glycolysis. Traditional chromatographic analysis of phosphates is often plagued by long run times and/or lack of MS compatibility. This study compares separation of five enzymatically-important nucleotides using ion-pair reversed phase (IP-RP), strong anion exchange (SAX), and hydrophilic interaction (HILIC) methods. These three methods were evaluated and compared based on separation parameters describing retention, resolution, efficiency, peak symmetry, selectivity, and inter- and intraday peak drift. Use of the FructoShell-N HILIC column led to separation of the five nucleotides isocratically with the shortest run time of all three methods tested. Additionally, the FructoShell HILIC method yielded a very low intraday variability and low peak asymmetry, issues that are often observed with HILIC separations on other stationary phases. To our knowledge, this column has not been applied to the separation of phosphates in biological samples and future work will focus on in vitro and in vivo analysis as well as broadening the applicability to other pathways. To this end, we have shown that the column will retain fructose bisphosphate, the substrate of the aldolase enzyme, under the same chromatographic conditions used for nucleotides.