Rapid amino acid quantitation with pre-column derivatization; ultra-performance reverse phase liquid chromatography...

We 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 (MS_nva).

We 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 (MS_lis). We compared the analytical performance of MS_nva with MS_lis and the original method (UV_nva) with clinical samples.

The chromatography time per sample of MS_nva was 8min, detection capabilities were <1μmol/L for most components, intermediate imprecisions at low concentrations were <10% and there was negligible carryover. MS_nva 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 UV_nva with the latter prone to outliers and suboptimal quantitation of urinary arginine, aspartate, glutamate and methionine. MS_nva reliably detected argnininosuccinate, β-alanine, citrulline and cysteine-s-sulfate.

MS_nva resulted in a more than fivefold increase in operational efficiency with accurate detection of amino acids and metabolic intermediates in clinical samples.