Clinical validation of a liquid chromatography single quadrupole mass spectrometry (LC-MS) method using Waters Kairos™ Amino Acid Kit reagents
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Rowan Hellier
Abstract
Objectives
Quantitation of plasma amino acids (AA) is critical for the diagnosis and monitoring of inherited disorders of AA metabolism. AA analysis using ion-exchange chromatography (IEC) with post-column ninhydrin derivatization is time consuming, with run times of ∼2 h, limiting sample throughput. Liquid chromatography mass-spectrometry can potentially address some of the current challenges.
Methods
Performance of components of the Waters Kairos Amino Acid Kit using liquid chromatography single quadrupole mass-spectrometry (LC-MS) following derivatization of samples with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ•Tag™ Ultra Derivatization Reagent) was evaluated. Results were compared with the Biochrom-IEC method using patient specimens (n=115), ClinChek® control and external quality assessment (EQA) material.
Results
The kit reagents and our developed method had a 19-min analysis time, demonstrated acceptable inter-assay imprecision (CV<10 %) and bias vs. IEC-method (overall mean bias <2 %). Excellent correlation (concordance correlation coefficient (CCC) >0.99) with IEC was demonstrated for 10/23 analytes, good correlation (CCC >0.95) for 10/23, with the remaining three amino acids (aspartate, histidine and tryptophan) demonstrating moderate concordance (CCC ≥0.90 but <0.95). 1/23 AAs had a mean bias >10 % using EQA material. The method demonstrated a lower limit of quantitation of ≤2.5 μmol/L for all AA, making this assay suitable for CSF analysis. Calibration stability bias was <5 % over 12-weeks. Derivatized AAs were stable for ≤17 days. The analytical column supplied demonstrated good retention time stability (<0.4 %) and was capable of >2000 injections.
Conclusions
The tested methodology demonstrated good analytical performance and correlation with IEC. This approach confers practical advantages over IEC, including analytical selectivity and workflow time efficiency.
Acknowledgments
The reagent kits were received as a gift from Waters for the evaluation. The authors thank Peter Christensen, Padhraic Rossiter, Adam Cryar, Alex Hunt, Lisa Calton and Heather Brown from Waters for technical support and scientific advice. The authors would like to thank Grace Heffernan, of Waters Wexford, who were instrumental in the development and manufacture of the kit and materials provided for the evaluation. SJM is supported by the UK Medical Research Council-Rare Disease Research Platform grant number MR/Y008057/1.
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Research ethics: Not applicable.
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Informed consent: Not applicable.
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Author contributions: SJM, conceived and planned the study. SJM and RH wrote the first draft of the manuscript. RH, LG, CS and AR undertook sample processing, performed all the laboratory analyses. All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
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Use of Large Language Models, AI and Machine Learning Tools: None declared.
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Conflict of interest: The authors state no conflict of interest.
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Research funding: None declared.
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Data availability: Not applicable.
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Supplementary Material
This article contains supplementary material (https://doi.org/10.1515/cclm-2025-0424).
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