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.
References
1. Walker, V, Mills, G. Quantitative methods for amino acid analysis in biological fluids. Ann Clin Biochem 1995;32:28–57. https://doi.org/10.1177/000456329503200103.Search in Google Scholar PubMed
2. Song, Y, Xu, C, Kuroki, H, Liao, Y, Tsunoda, M. Recent trends in analytical methods for the determination of amino acids in biological samples. J Pharm Biomed Anal 2018;147:35–49. https://doi.org/10.1016/j.jpba.2017.08.050.Search in Google Scholar PubMed
3. Carling, RS, John, K, Churchus, R, Turner, C, Dalton, RN. Validation of a rapid comprehensive and clinically relevant amino acid profile by liquid chromatography tandem mass spectrometry. Clin Chem Lab Med 2020;58:758–68. https://doi.org/10.1515/cclm-2019-0604.Search in Google Scholar PubMed
4. Dietzen, DJ, Weindel, AL, Carayannopoulos, MO, Landt, M, Normansell, ET, Reimschisel, TE, et al.. Rapid comprehensive amino acid analysis by liquid chromatography/tandem mass spectrometry: comparison to cation exchange with post-column ninhydrin detection. Rapid Commun Mass Spectrom 2008;22:3481–8. https://doi.org/10.1002/rcm.3754.Search in Google Scholar PubMed
5. Phipps, WS, Crossley, E, Boriack, R, Jones, PM, Patel, K. Quantitative amino acid analysis by liquid chromatography-tandem mass spectrometry using low cost derivatisation and an automated liquid handler. JIMD Reports 2020;51:62–9. https://doi.org/10.1002/jmd2.12080.Search in Google Scholar PubMed PubMed Central
6. Smon, A, Cuk, V, Brecelj, J, Murko, S, Groselj, U, Zerjav Tansek, M, et al.. Comparison of liquid chromatography with tandem mass spectrometry and ion-exchange chromatography by post-column ninhydrin derivatization for amino acid monitoring. Clin Chim Acta 2019;495:446–5. https://doi.org/10.1016/j.cca.2019.05.007.Search in Google Scholar PubMed
7. Carling, RS, McDonald, BAC, Austin, D, Burden, D, Correia, J, Leung, J, et al.. Challenging the status quo: a comparison of ion exchange chromatography with liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry methods for the measurement of amino acids in human plasma. Ann Clin Biochem 2020;4:277–90. https://doi.org/10.1177/0004563220933303.Search in Google Scholar PubMed
8. Piraud, M, Vianey-Saban, C, Petritis, K, Elfakir, C, Steghens, J, Bouchu, D. Ion-pairing reversed-phase liquid chromatography/electrospray ionization mass spectrometric analysis of 76 underivatized amino acids of biological interest: a new tool for the diagnosis of inherited disorders of amino acid metabolism. Rapid Commun Mass Spectrom 2005;19:1587–602. https://doi.org/10.1002/rcm.1957.Search in Google Scholar PubMed
9. Prinsen, H, Schiebergen-Bronkhorst, B, Roeleveld, M, Jans, J, de Sain-van der Velden, M, Visser, G, et al.. Rapid quantification of underivatized amino acids in plasma by hydrophilic interaction liquid chromatography (HILIC) coupled with tandem mass-spectrometry. J Inherit Metab Dis 2016;39:651–60. https://doi.org/10.1007/s10545-016-9935-z.Search in Google Scholar PubMed PubMed Central
10. Salazar, C, Armenta, JM, Cortes, DF, Shulaev, V. Combination of an AccQ•Tag-ultra performance liquid chromatography method with tandem mass spectrometry for the analysis of amino acids. Methods Mol Biol 2012;828:13–28.10.1007/978-1-61779-445-2_2Search in Google Scholar PubMed
11. Pretorius, CJ, McWhinney, BC, Sipinkoski, B, Wilce, A, Cox, D, McWhinney, A, et al.. Rapid amino acid quantitation with pre-column derivatization; ultra-performance reverse phase liquid chromatography and single quadrupole mass spectrometry. Clin Chim Acta 2018;478:132–9. https://doi.org/10.1016/j.cca.2017.12.027.Search in Google Scholar PubMed
12. Bruno, C, Veyrat-Durebex, C, Lumbu Lukuntonda, CH, Andres, CR, Moreau, C, Bendavid, C, et al.. Validation of plasma amino acid profile using UHPLC-mass spectrometer (QDa) as a screening method in a metabolic disorders reference centre: performance and accreditation concerns. Clin Biochem 2021;92:34–45. https://doi.org/10.1016/j.clinbiochem.2021.03.004.Search in Google Scholar PubMed
13. Passing, HB, Bablok, W. A new biometrical procedure for testing the equality of measurements from two different analytical methods. Application of linear regression procedures for method comparison studies in clinical chemistry. Part I. J Clin Chem Clin Biochem 1983;21:709–20. https://doi.org/10.1515/cclm.1983.21.11.709.Search in Google Scholar PubMed
14. Lin, LA. A concordance correlation coefficient to evaluate reproducibility. Biometrics 1989;45:255–68. https://doi.org/10.2307/2532051.Search in Google Scholar
15. Liao, J, Lewis, JW. A note on concordance correlation coefficient. PDA J Pharm Sci Technol 2000;54:23–6.Search in Google Scholar
16. Parvy, P, Bardet, J, Gasquet, M, Rabier, D, Kamoun, SP. Stability of free amino acids in sulfosalicylic filtrates. Clin Chem 1995;41:465–6. https://doi.org/10.1093/clinchem/41.3.465.Search in Google Scholar
17. Friedman, M, Finley, J. Methods of tryptophan analysis. J Agric Food Chem 1972;19:626–31. https://doi.org/10.1021/jf60176a010.Search in Google Scholar PubMed
18. van der Crabben, SN, Verhoeven-Duif, NM, Brilstra, EH, Van Maldergem, L, Coskun, T, Rubio-Gozalbo, E, et al.. An update on serine deficiency disorders. J Inherit Metab Dis 2013;36:613–9. https://doi.org/10.1007/s10545-013-9592-4.Search in Google Scholar PubMed
19. Alfadhel, M, Alrifai, MT, Trujillano, D, Alshaalan, H, Al Othaim, A, Al Rasheed, S, et al.. Asparagine synthetase deficiency: new inborn errors of metabolism. JIMD Rep 2015;22:11–16. https://doi.org/10.1007/8904_2014_405.Search in Google Scholar PubMed PubMed Central
20. Tangeraas, T, Constante, JR, Backe, PH, Oyarzábal, A, Neugebauer, J, Weinhold, N, et al.. BCKDK deficiency: a treatable neurodevelopmental disease amenable to newborn screening. Brain 2023;146:3003–13.21. https://doi.org/10.1093/brain/awad010.Search in Google Scholar PubMed
21. Moat, SJ, Carling, R, Nix, A, Henderson, M, Briddon, A, Prunty, H, et al.. Multicentre age-related reference intervals for cerebrospinal fluid serine concentrations: implications for the diagnosis and follow-up of serine biosynthesis disorders. Mol Genet Metabol 2010;101:149–52. https://doi.org/10.1016/j.ymgme.2010.07.006.Search in Google Scholar PubMed
Supplementary Material
This article contains supplementary material (https://doi.org/10.1515/cclm-2025-0424).
© 2025 Walter de Gruyter GmbH, Berlin/Boston
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