Method comparison of HPLC-ninhydrin-photometry and UHPLC-PITC-tandem mass spectrometry for serum amino acid analyses in patients with complex congenital heart disease and controls

Abstract

IntroductionMetabolomics studies are not routine when quantifying amino acids (AA) in congenital heart disease (CHD).ObjectivesComparative analysis of 24 AA in serum by traditional high-performance liquid chromatography (HPLC) based on ion exchange and ninhydrin derivatisation followed by photometry (PM) with ultra-high-performance liquid chromatography and phenylisothiocyanate derivatisation followed by tandem mass spectrometry (TMS); interpretation of findings in CHD patients and controls.MethodsPM: Sample analysis as above (total run time, ~ 119 min). TMS: Sample analysis by AbsoluteIDQ® p180 kit assay (BIOCRATES Life Sciences AG, Innsbruck, Austria), which employs PITC derivatisation; separation of analytes on a Waters Acquity UHPLC BEH18 C18 reversed-phase column, using water and acetonitrile with 0.1% formic acid as the mobile phases; and quantification on a Triple-Stage Quadrupole tandem mass spectrometer (Thermo Fisher Scientific, Waltham, MA) with electrospray ionisation in the presence of internal standards (total run time, ~ 8 min). Calculation of coefficients of variation (CV) (for precision), intra- and interday accuracies, limits of detection (LOD), limits of quantification (LOQ), and mean concentrations.ResultsBoth methods yielded acceptable results with regard to precision (CV < 10% PM, < 20% TMS), accuracies (< 10% PM, < 34% TMS), LOD, and LOQ. For both Fontan patients and controls AA concentrations differed significantly between methods, but patterns yielded overall were parallel.ConclusionSerum AA concentrations differ with analytical methods but both methods are suitable for AA pattern recognition. TMS is a time-saving alternative to traditional PM under physiological conditions as well as in patients with CHD.Trial registration numberClinicalTrials.gov Identifier NCT03886935, date of registration March 27th, 2019 (retrospectively registered).