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. 2020 Dec 15;16(12):128.
doi: 10.1007/s11306-020-01741-8.

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

Miriam Michel  1   2 , Christina Salvador  3 , Verena Wiedemair  4 , Mark Gordian Adam  5 , Kai Thorsten Laser  2 , Karl-Otto Dubowy  2 , Andreas Entenmann  3 , Daniela Karall  3 , Ralf Geiger  1 , Manuela Zlamy  6 , Sabine Scholl-Bürgi  3
Affiliations

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

Miriam Michel et al. Metabolomics. .

Abstract

Introduction: Metabolomics studies are not routine when quantifying amino acids (AA) in congenital heart disease (CHD).

Objectives: Comparative 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.

Methods: PM: 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.

Results: Both 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.

Conclusion: Serum 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 number: ClinicalTrials.gov Identifier NCT03886935, date of registration March 27th, 2019 (retrospectively registered).

Keywords: Amino acid metabolism; Congenital heart disease; Fontan; Metabolomics; Pattern recognition; Tandem mass spectrometry.

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Conflict of interest statement

All authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Amino acid serum concentrations (means and standard deviations) by client group (Fontan vs. control) and method (PM vs. TMS). White boxes: controls, PM; white boxes with black stripes: controls, TMS; grey boxes: Fontan patients, PM; black boxes: Fontan patients, TMS. Ala, alanine; alpha.AAA, alpha-aminoadipic acid; Arg, arginine; Asn, asparagine; Asp, aspartic acid; Cit, citrulline; Gln, glutamine; Glu, glutamic acid; Gly, glycine; His, histidine; OH-Pro, hydroxyproline; Ile, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; Orn, ornithine; Phe, phenylalanine; Pro, proline; Ser, serine; Tau, taurine; Thr, threonine; Trp, tryptophan; Tyr, tyrosine; Val, valine
Fig. 2
Fig. 2
Bland–Altman-plot comparing serum concentrations of analytes in Fontan patients determined by PM with those determined by TMS. Diff, difference; LOA, limit of agreement. LOA indicates 95% LOA
Fig. 3
Fig. 3
Bland–Altman-plot comparing serum concentrations of analytes in controls determined by PM with those determined by TMS. Diff, difference; LOA, limit of agreement. LOA indicates 95% LOA
See this image and copyright information in PMC

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