Podium Presentations for Lindsay Bazydlo

A Selective Amino Acid Panel to Support Testing for Inborn Errors of Metabolism Lindsay Bazydlo (Presenter) University of Virginia

To be presented in Track 3 (Montreal 4) on Wednesday at 13:30

INTRODUCTION:
Clinical analysis of plasma amino acids is an important diagnostic and prognostic tool in patients with inborn errors of metabolism (IEM). More recently, liver transplants have been explored as a treatment option for patients with specific IEMs, such as urea cycle disorders and maple syrup urine disease. In an effort to support clinical teams performing transplants as a therapeutic option in these patients, we developed a fast, selective amino acid panel to provide the concentrations of 8 amino acids to be used in the management of patients undergoing liver transplants for IEMs.

METHODS:
Plasma samples were prepared using a protein precipitation followed by a filter step. The chromatographic separation of amino acids was performed by hydrophilic interaction liquid chromatography (HILIC) followed by tandem mass spectrometry. A Waters Acquity UPLC BEH Amide Column (2.1 × 100 mm, 1.7 µm particle size) was used with a gradient of 85% acetonitrile, 15% water (solvent A) and Water (solvent B) supplemented with 10 mM ammonium formate and 0.15% formic acid in both cases. The gradient consisted of a 12-minute run of decreasing solvent A concentration preceded by a 6 min equilibration period.1 The mass spectrometry data was collected in positive mode and two transitions were chosen for each amino acid.

RESULTS:
A LC-MSMS method was developed that was able to adequately separate 8 different amino acids: glutamine, citrulline, arginine, alanine, glycine, leucine, isoleucine, and valine. This was accomplished using HILIC, which showed reproducible retention times and adequate separation of leucine and isoleucine. The lower limit of quantitation (LLOQ) varied per amino acid, with all amino acids being able to achieve at least 5 µM except for alanine, which showed a LLOQ of 50 µM. Sample preparation was performed using a simple protein precipitation and the amino acids were analyzed without derivatization. The solvents used in the protein precipitation were optimized to provide desirable recovery and compatibility with the starting solvents used in the chromatography. Matrix-matched calibrators were designed by removing the relevant amino acids from plasma to provide a blank matrix similar to patient samples.

CONCLUSION: We developed a rapid LC-MSMS assay for the measurement of select amino acids using a HILIC separation. This assay can be used in patients who need emergent care where a full amino acid panel is not necessary, particularly in the setting of liver transplants for IEMs.

REFERENCES:
1. Prinsen, H.C.M.T., B.G.M. Schiebergen-Bronkhorst, M.W. Roeleveld, J.J.M. Jans, M.G.M. de Sain-van der Velden, G. Visser, P.M. van Hasselt, and N.M. Verhoeven-Duif. Rapid quantification of underivatized amino acids in plasma by hydrophilic interaction liquid chromatography (HILIC) coupled with tandem mass-spectrometry. J Inherit Metab Dis, 2016 39;651-660.