A novel, high throughput, and low-cost method for the detection of 40 amines relevant to inborn errors of metabolism, in under 60 minutes, using reverse phase high performance liquid chromatography

Abstract

AbstractObjectivesAn assessment of amino acid and amine concentrations is important for the diagnosis and management of inborn errors of metabolism (IEMs). Methods exist that measure these biologically important metabolites but are cost-prohibitive and/or time consuming. We therefore sought to develop a novel methodology, applicable to IEMs, that is both high-throughput and low cost.MethodsPreviously, we developed a methodology for rapid, repeatable, and cost-efficient separation of approximately 20 amines as a proof of concept and now expand it to amines relevant to IEMs. We describe our separation methodology using reverse phase high performance liquid chromatography with ultraviolet-visible spectrum absorbance paired with precolumn derivatization witho-phthaladehyde.ResultsWe show reproducibility via concentration assessments, in triplicate, for each amine. We assess amines in prepared standard solutions and in biologic samples from patients with IEMs. We also detected and assessed the amino group containing compounds oxidized and reduced glutathione and ammonia. Validation was established using absolute area under the curve (AUC) and via comparison using a single internal standard.ConclusionsWe report good separation of 40 primary amino group containing metabolites, in a single, less than 60-minute run. This rapid, low cost, and accurate methodology only requires a small volume of sample and can greatly increase availability and access. Finally, the numerous disease associated amines (ie homocitrulline, trimethyllysine, alloisoleucine) and unique compounds detected in our single run has broad research and clinical utility and can increase efficiency, important as the need for analysis of amines grows globally.