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

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).

Automated liquid handling extraction and rapid quantification of underivatized amino acids and tryptophan metabolites from human serum and plasma using dual-column U(H)PLC-MRM-MS and its application to prostate cancer study

“…For all metabolites except GLY (LLOQ: 2.46 µM), the dual-column U(H)PLC-MRM-MS method provided LLODs and LLOQs in the nanomolar range (Figure 1A, Table 3). The current gold standard for amino acid analysis in serum or plasma, namely ion exchange chromatography (IEX) followed by post-column ninhydrin derivatization and fluorescence detection, provided LLODs and LLOQs in the low µmolar range 46 . Our dual-column U(H)PLC-MRM-MS method provided lower LLOD and LLOQ concentrations for all amino acids analyzed and was 15 times faster than the gold standard (run time: 119 min) with total a run time of 7.9 min.…”

“…The dual-column U(H)PLC-MRM assay was found to be robust, reproducible, specific and sensitive enough for the quantification of free amino acids and TRP metabolites in plasma and serum. With LLOQs in the nanomolar range for all analytes except for GLY (LLOQ: 2.46 µM), our assay was more sensitive and 15 times faster than the gold standard for amino acid quantification, IEX, followed by post-column ninhydrin derivatization and fluorescence detection 46 . Compared to other LC-MS assays for the quantification of underivatized amino acids 32,33,47 and TRP metabolites 18 , our dual-column U(H)PLC-MRM-MS method yielded lower LLOQs for the amino acids in a 2-3 times shorter analysis time, and similar LLOQs for TRP metabolites.…”

Performance evaluation of the commercial aquafeeds available in the market of Pakistan on Channa marulius (Sole)

“…Amino acids profile of experimental fish muscle was analyzed by using amino acid analyzer (Biochrome 30 + Model) available at Dr. M. Yaqoob Malik amino acid analysis laboratory at UVAS, Ravi Campus Pattoki, using ion exchange chromatography described by Official Journal of European Communities using amino acids analyser (Biochrom 30+ Model) known as gold standard for AAA (Michel et al, 2020).…”

Metabolomics: A New Tool in Our Understanding of Congenital Heart Disease

“…Both methods proved to be reliable, although, in the Fontan patient group, concentrations significantly differed between the two techniques. Mass spectrometry is time-saving compared to liquid chromatography [ 25 ].…”