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

Objectives

An assessment of amino acid and amine concentrations is important for the diagnosis and management of inherited metabolic disorders (IMDs). 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 IMDs, that is both high-throughput and low cost.

Methods

Previously, 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 IMDs. We describe our separation methodology using reverse phase high performance liquid chromatography with ultraviolet-visible spectrum absorbance paired with pre-column derivatization with o-pthalaldehyde.

Results

We show reproducibility via concentration assessments, in triplicate, for each amine. We assess amines in prepared standard solutions and in biologic samples from patients with IMDs. We also detected and assessed the amino group containing compounds glutathione (oxidized and reduced forms) and ammonia. Validation was established using absolute area under the curve (AUC) and via comparison using a single internal standard.

Conclusions

We report good separation of 40 primary amino group containing metabolites, in a single, 53 min 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 amines and unique compounds detected in our single run has large utility and can potentially increase clinical efficiency and broaden access to research, both important as the need for analysis of amines grows globally.