Synthesis and physicochemical characterization of the one-carbon carrier 10-formyltetrahydrofolate; a reference standard for metabolomics

Abstract

Introduction Metabolomics analysis depends on the identification and validation of specific metabolites. This task is significantly hampered by the absence of well-characterized reference standards. The one-carbon carrier 10-formyltetrahydrofolate acts as a donor of formyl groups in anabolism where it is a substrate in formyltransferase reactions in purine biosynthesis. It has been reported as an unstable substance and is currently unavailable as a reference standard for metabolomics analysis. Objectives The current study was undertaken to provide the metabolomics community thoroughly characterized 10-formyltetrahydrofolate along with analytical methodology and guidelines for its storage and handling. Methods Anaerobic base treatment of 5,10-methenyltetrahydrofolate chloride in the presence of anti-oxidant was utilized to prepare 10-formyltetrahydrofolate. Results Pure 10-formyltetrahydrofolate has been prepared and physicochemically characterized. Conditions toward maintaining the stability of a solution of the dipotassium salt of 10-formyltetrahydrofolate in solution have been determined. Conclusion This study describes the facile preparation of pure (>90%) 10-formyltetrahydrofolate, its qualitative physicochemical characterization, as well as conditions to enable its use as a reference standard in physiologic samples.