{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,2]],"date-time":"2026-03-02T18:33:24Z","timestamp":1772476404201,"version":"3.50.1"},"reference-count":29,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2021,7,9]],"date-time":"2021-07-09T00:00:00Z","timestamp":1625788800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["PD\/BDE\/114426\/2016"],"award-info":[{"award-number":["PD\/BDE\/114426\/2016"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Robust and selective quantification methods are required to better analyze feed supplementation effectiveness with specific amino acids. In this work, a reversed-phase high-performance liquid chromatography method with fluorescence detection is proposed and validated for lysine quantification, one of the most limiting amino acids in ruminant nutrition and essential towards milk production. To assess and widen method applicability, different matrices were considered: namely Li2CO3 buffer (the chosen standard reaction buffer), phosphate buffer solution (to mimic media in cellular studies), and rumen inoculum. The method was validated for all three matrices and found to be selective, accurate (92% \u00b1 2%), and precise at both the inter- and intra-day levels in concentrations up to 225 \u00b5M, with detection and quantification limits lower than 1.24 and 4.14 \u00b5M, respectively. Sample stability was evaluated when stored at room temperature, 4 \u00b0C, and \u221220 \u00b0C, showing consistency for up to 48 h regardless of the matrix. Finally, the developed method was applied in the quantification of lysine on real samples. The results presented indicate that the proposed method can be applied towards free lysine quantification in ruminant feeding studies and potentially be of great benefit to dairy cow nutrition supplementation and optimization.<\/jats:p>","DOI":"10.3390\/molecules26144173","type":"journal-article","created":{"date-parts":[[2021,7,9]],"date-time":"2021-07-09T04:14:57Z","timestamp":1625804097000},"page":"4173","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Validation of a Simple HPLC-Based Method for Lysine Quantification for Ruminant Nutrition"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5647-3005","authenticated-orcid":false,"given":"Jo\u00e3o","family":"Albuquerque","sequence":"first","affiliation":[{"name":"LAQV, REQUIMTE, Departamento de Ci\u00eancias Qu\u00edmicas, Faculdade de Farm\u00e1cia, Universidade do Porto, Rua Jorge Viterbo Ferreira n.\u00b0 228, 4050-313 Porto, Portugal"},{"name":"LAQV, REQUIMTE, ICBAS, Instituto de Ci\u00eancias Biom\u00e9dicas Abel Salazar, Universidade do Porto, Rua Jorge Viterbo Ferreira n.\u00b0 228, 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1686-3850","authenticated-orcid":false,"given":"Susana","family":"Casal","sequence":"additional","affiliation":[{"name":"LAQV, REQUIMTE, Departamento de Ci\u00eancias Qu\u00edmicas, Faculdade de Farm\u00e1cia, Universidade do Porto, Rua Jorge Viterbo Ferreira n.\u00b0 228, 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9685-231X","authenticated-orcid":false,"given":"Rebeca","family":"Cruz","sequence":"additional","affiliation":[{"name":"LAQV, REQUIMTE, Departamento de Ci\u00eancias Qu\u00edmicas, Faculdade de Farm\u00e1cia, Universidade do Porto, Rua Jorge Viterbo Ferreira n.\u00b0 228, 4050-313 Porto, Portugal"}]},{"given":"Ingrid","family":"Van Dorpe","sequence":"additional","affiliation":[{"name":"PREMIX-Especialidades Agr\u00edcolas e Pecu\u00e1rias, Lda, Parque Ind\u00fastrial II\u2013Neiva, 4935-232 Viana do Castelo, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1716-6205","authenticated-orcid":false,"given":"Margarida Rosa Garcez","family":"Maia","sequence":"additional","affiliation":[{"name":"LAQV, REQUIMTE, ICBAS, Instituto de Ci\u00eancias Biom\u00e9dicas Abel Salazar, Universidade do Porto, Rua Jorge Viterbo Ferreira n.\u00b0 228, 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6237-417X","authenticated-orcid":false,"given":"Ant\u00f3nio Jos\u00e9 Mira","family":"Fonseca","sequence":"additional","affiliation":[{"name":"LAQV, REQUIMTE, ICBAS, Instituto de Ci\u00eancias Biom\u00e9dicas Abel Salazar, Universidade do Porto, Rua Jorge Viterbo Ferreira n.\u00b0 228, 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1904-0358","authenticated-orcid":false,"given":"Ana Rita Jord\u00e3o","family":"Cabrita","sequence":"additional","affiliation":[{"name":"LAQV, REQUIMTE, ICBAS, Instituto de Ci\u00eancias Biom\u00e9dicas Abel Salazar, Universidade do Porto, Rua Jorge Viterbo Ferreira n.\u00b0 228, 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4262-5837","authenticated-orcid":false,"given":"Ana Rute","family":"Neves","sequence":"additional","affiliation":[{"name":"LAQV, REQUIMTE, Departamento de Ci\u00eancias Qu\u00edmicas, Faculdade de Farm\u00e1cia, Universidade do Porto, Rua Jorge Viterbo Ferreira n.\u00b0 228, 4050-313 Porto, Portugal"},{"name":"CQM\u2014Centro de Qu\u00edmica da Madeira, Campus da Penteada, Universidade da Madeira, 9020-105 Funchal, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0736-2835","authenticated-orcid":false,"given":"Salette","family":"Reis","sequence":"additional","affiliation":[{"name":"LAQV, REQUIMTE, Departamento de Ci\u00eancias Qu\u00edmicas, Faculdade de Farm\u00e1cia, Universidade do Porto, Rua Jorge Viterbo Ferreira n.\u00b0 228, 4050-313 Porto, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2021,7,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/0301-6226(91)90145-G","article-title":"Limiting amino acids in dairy cows given casein as the sole source of protein","volume":"28","author":"Fraser","year":"1991","journal-title":"Livest. 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