Rapid amino acid quantitation with pre-column derivatization; ultra-performance reverse phase liquid chromatography and single quadrupole mass spectrometry

Abstract

BackgroundWe optimized a quantitative amino acid method with pre-column derivatization, norvaline (nva) internal standard and reverse phase ultra-performance liquid chromatography by replacing the ultraviolet detector with a single quadrupole mass spectrometer (MSnva). MethodWe used 13C15N isotopically labeled amino acid internal standards and a C18 column with 1.6μm particles to optimize the chromatography and to confirm separation of isobaric compounds (MSlis). We compared the analytical performance of MSnva with MSlis and the original method (UVnva) with clinical samples. ResultsThe chromatography time per sample of MSnva was 8min, detection capabilities were <1μmol/L for most components, intermediate imprecisions at low concentrations were <10% and there was negligible carryover. MSnva was linear up to a total amino acid concentration in a sample of approximately 9500μmol/L. The agreements between most individual amino acids were satisfactory compared to UVnva with the latter prone to outliers and suboptimal quantitation of urinary arginine, aspartate, glutamate and methionine. MSnva reliably detected argnininosuccinate, β-alanine, citrulline and cysteine-s-sulfate. ConclusionMSnva resulted in a more than fivefold increase in operational efficiency with accurate detection of amino acids and metabolic intermediates in clinical samples.