Amino acid analysis in biological fluids is essential for the study of inborn errors of metabolism (IEM) and other diseases.
Our aim was to develop a UPLC-MS/MS procedure for the analysis of 25 amino acids and identification of 17 related compounds.
Sample treatment conditions were optimized for plasma, urine, cerebrospinal fluid (CSF) and dried blood spots. Amino acids and related compounds were analyzed on a Waters ACQUITY UPLC H-class instrument with a reversed-phase C-18 column using water and acetonitrile with 0.1% formic acid as the mobile phases (run time = 9 min). The detection was performed with a Waters Xevo TQD triple-quadrupole mass spectrometer using positive electrospray ionization in the multiple reaction monitoring mode.
The method linearity, intra-assay and inter-assay precision, detection limit, quantification limit and trueness analysis displayed adequate results in both physiological and pathological conditions. Method comparison was performed between UPLC-MS/MS and ion exchange chromatography (IEC) with ninhydrin derivatization, and the methods showed good agreement, except for 4-hydroxyproline, aspartate and citrulline. Paediatrics age-related reference values in plasma, urine and CSF were established and patients with different IEM were easily identified.
We report a modified UPLC-MS/MS procedure for the analysis of 42 amino acids and related compounds in different specimens. The method is fast, sensitive and robust, and it has been validated to be an alternative to the traditional IEC procedure as the routine method used in metabolic laboratories. The method greatly decreases the run time of the analysis while displaying good metrological results.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Allan, J. D., Cusworth, D. C., Dent, C. E., & Wilson, V. K. (1958). A disease, probably hereditary, characterized by severe mental deficiency and a constant gross abnormality of amino acid metabolism. Lancet, 271, 182–187.
Arnold, G. L., Greene, C. L., Stout, J. P., & Goodman, S. I. (1993). Molybdenum cofactor deficiency. Journal of Pediatrics, 123, 595–598.
Checa-Moreno, R., Manzano, E., Mirón, G., & Capitán-Vallvey, L. F. (2008). Revisitation of the phenylisothiocyanate-derivatives procedure for amino acid determination by HPLC-UV. Journal of Separation Science, 31, 3817–3828.
Chen, J., Hou, W., Han, B., Liu, G., Gong, J., Li, Y., et al. (2016). Target-based metabolomics for the quantitative measurement of 37 pathway metabolites in rat brain and serum using hydrophilic interaction ultra-high-performance liquid chromatography-tandem mass spectrometry. Analytical and Bioanalytical Chemistry, 408, 2527–2542.
Cohen, S. A. (2003). Amino acid analysis using pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Analysis of hydrolyzed proteins and electroblotted samples. Methods in Molecular Biology, 211, 143–154.
Corso, G., Cristofano, S., Sapere, N., la Marca, G., Angiolillo, A., Vitale, M., et al. (2017). Serum amino acid profiles in normal subjects and in patients with or at risk of Alzheimer dementia. Dementia and Geriatric Cognitive Disorders, 7, 143–159.
DeArmond, P. D., Dietzen, D. J., & Pyle-Eilola, A. L. (2017). Amino acids disorders. In U. Garg & L. D. Smith (Eds.), Biomarkers in inborn errors of metabolism: Clinical aspects and laboratory determination (pp. 25–64). Amsterdam: Elsevier.
Duran, M. (2009). Amino acids. In N. Blau, M. Duran & K. M. Gibson (Eds.), Laboratory guide to the methods in biochemical genetics (pp. 53–90). Germany: Springer.
Fekkes, D. (1996). State-of-the-art of high-performance liquid chromatographic analysis of amino acids in physiological samples. Journal of Chromatography B: Biomedical Sciences and Applications, 682, 3–22.
Ferguson, J. F., & Wang, T. J. (2016). Branched-chain amino acids and cardiovascular disease: Does diet matter? Clinical Chemistry, 62, 545–547.
Filee, R., Schoos, R., & Boemer, F. (2014). Evaluation of physiological amino acids profiling by tandem mass spectrometry. Journal of Inherited Metabolic Disease Reports, 13, 119–128.
Freeto, S., Mason, D., Chen, J., Scott, R. H., Narayan, S. B., & Bennett, M. J. (2007). A rapid ultra performance liquid chromatography tandem mass spectrometric method for measuring amino acids associated with maple syrup urine disease, tyrosinaemia and phenylketonuria. Annals of Clinical Biochemistry, 44, 474–481.
Gray, N., Plumb, R. (2016) A validated method for the quantification of amino acids in mammalian urine. In: Waters, the science of what’s possible. Accessed January 31, 2018, from http://www.waters.com/waters/library.htm?cid=511436&lid=134820198&locale=es_ES.
Gerrits, G. P., Trijbels, F. J., Monnens, L. A., Gabreels, F. J., De Abreu, R. A., Theeuwes, A. G., et al. (1989). Reference values for amino acids in cerebrospinal fluid of children determined using ion-exchange chromatography with fluorimetric detection. Clinical Chimica Acta, 182, 271–280.
Gregory, D. M., Sovetts, D., Clow, C. L., & Scriver, C. R. (1986). Plasma free amino acid values in normalchildren and adolescents. Metabolism, 35, 967–969.
Krumpochova, P., Bruyneel, B., Molenaar, D., Koukou, A., Wuhrer, M., Niessen, W. M., et al. (2015). Amino acid analysis using chromatography-mass spectrometry: An inter platform comparison study. Journal of Pharmaceutical and Biomedical Analysis, 114, 398–407.
Leah, J. M., Palmer, T., Griffin, M., Wingad, C. J., Briddon, A., & Oberholzer, V. G. (1986). Urine amino acid analysis by HPLC in the investigation of inborn errors of metabolism. Journal of Inherited Metabolic Disease, 9, 250–253.
Ma, H., Hasim, A., Mamtimin, B., Kong, B., Zhang, H. P., & Sheyhidin, I. (2014). Plasma free amino acid profiling of esophageal cancer using high-performance liquid chromatography spectroscopy. World Journal of Gastroenterology, 20, 8653–8659.
Mengerink, Y., Kutlán, D., Tóth, F., Csámpai, A., & Molnár-Perl, I. (2002). Advances in the evaluation of the stability and characteristics of the amino acid and amine derivatives obtained with the o-phthaldialdehyde/3-mercaptopropionic acid and o-phthaldialdehyde/N-acetyl-l-cysteine reagents: High-performance liquid chromatography-mass spectrometry study. Journal of Chromatography A, 949, 99–124.
Miyagi, Y., Higashiyama, M., Gochi, A., Akaike, M., Ishikawa, T., Miura, T., et al. (2011) Plasma free amino acid profiling of five types of cancer patients and its application for early detection. PLoS ONE, 6, e24143.
Morton, D. H., Strauss, K. A., Robinson, D. L., Puffenberger, E. G., & Kelley, R. I. (2002). Diagnosis and treatment of maple syrup disease: A study of 36 patients. Pediatrics, 109, 999–1008.
Parvy, P. R., Bardet, J. I., Rabier, D. M., & Kamoun, P. P. (1988). Age-related reference values for free amino acids in first morning urine specimens. Clinical Chemistry, 34, 2092–2095.
Piraud, M., Vianey-Saban, C., Bourdin, C., Acquaviva-Bourdain, C., Boyer, S., Elfakir, C., et al. (2005). A new reversed-phase liquid chromatographic/tandem mass spectrometric method for analysis of underivatised amino acids: Evaluation for the diagnosis and the management of inherited disorders of amino acid metabolism. Rapid Communications in Mass Spectrometry, 19, 3287–3297.
Plecko, B., Paul, K., Paschke, E., Stoeckler-Ipsiroglu, S., Struys, E., Jakobs, C., et al. (2007). Biochemical and molecular characterization of 18 patients with pyridoxine-dependent epilepsy and mutations of the antiquitin (ALDH7A1) gene. Human Mutation, 28, 19–26.
Pollitt, R. J., Jenner, F. A., & Merskey, H. (1968). Aspartylglycosaminuria: An inborn error of metabolism associated with mental defect. Lancet, 292, 253–255.
Powell, G. F., Rasco, M. A., & Maniscalco, R. M. (1974). A prolidase deficiency in man with iminopeptiduria. Metabolism, 23, 505–513.
Prinsen, H. C., Schiebergen-Bronkhorst, B. G., Roeleveld, M. W., Jans, J. J., de Sain-van der Velden, M. G., Visser, G., et al. (2016). Rapid quantification of underivatized amino acids in plasma by hydrophilic interaction liquid chromatography (HILIC) coupled with tandem mass-spectrometry. Journal of Inherited Metabolic Disease, 39, 651–660.
Ruiz-Canela, M., Toledo, E., Clish, C. B., Hruby, A., Liang, L., Salas-Salvadó, J., et al. (2016). Plasma branched-chain amino acids and incident cardiovascular disease in the PREDIMED trial. Clinical Chemistry, 62, 582–592.
Salazar, C., Armenta, J. M., & Shulaev, V. (2012). An UPLC-ESI-MS/MS assay using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization for targeted amino acid analysis: Application to screening of Arabidopsis thaliana mutants. Metabolites, 2, 398–428.
Sharma, G., Attri, S. V., Behra, B., Bhisikar, S., Kumar, P., Tageja, M., et al. (2014). Analysis of 26 amino acids in human plasma by HPLC using AQC as derivatizing agent and its application in metabolic laboratory. Amino Acids, 46, 1253–1263.
Shingyoji, M., Iizasa, T., Higashiyama, M., Imamura, F., Saruki, N., Imaizumi, A., et al. (2013). The significance and robustness of a plasma free amino acid (PFAA) profile-based multiplex function for detecting lung cancer. BMC Cancer, 13, 77.
Singh, A. K., & Ashraf, M. (1988). Analysis of amino acids in brain and plasma samples by sensitive gas chromatography-mass spectrometry. Journal of Chromatography A, 425, 245–255.
Uutela, P., Ketola, R. A., Piepponen, P., & Kostiainen, R. (2009). Comparison of different amino acid derivatives and analysis of rat brain microdialysates by liquid chromatography tandem mass spectrometry. Analytica Chimica Acta, 633, 223–231.
Wang, T. J., Larson, M. G., Vasan, R. S., Cheng, S., Rhee, E. P., McCabe, E., et al. (2011). Metabolite profiles and the risk of developing diabetes. Nature Medicine, 17, 448–453.
Waterval, W. A., Scheijen, J. L., Ortmans-Ploemen, M. M., Habets-van der Poel, C. D., & Bierau, J. (2009). Quantitative UPLC-MS/MS analysis of underivatised amino acids in body fluids is a reliable tool for the diagnosis and follow-up of patients with inborn errors of metabolism. Clinica Chimica Acta, 407, 36–42.
The Department of Clinical Biochemistry is part of the CIBERER-ISCIII and ‘Centre Daniel Bravo de Diagnòstic i Recerca en Malalties Minoritàries’. R. Artuch and M. Casado are funded by Programa de intensificación de la actividad investigadora from the ISCIII, Spain.
Conflict of interest
Authors declare no conflict of interests.
The patient samples were collected for diagnostic and treatment monitoring purposes. The study was conducted following the Helsinki Declaration of 1975 as revised in 2013. The ethical committee of the Hospital Sant Joan de Déu approved the study. For genetic diagnosis, informed consent was collected from patients or their guardians.
Electronic supplementary material
Below is the link to the electronic supplementary material.
About this article
Cite this article
Casado, M., Sierra, C., Batllori, M. et al. A targeted metabolomic procedure for amino acid analysis in different biological specimens by ultra-high-performance liquid chromatography–tandem mass spectrometry. Metabolomics 14, 76 (2018). https://doi.org/10.1007/s11306-018-1374-4
- Amino acids
- Inborn errors of metabolism
- Tandem mass spectrometry