Determination of free amino acid concentrations in bovine plasma using high-pressure liquid chromatography with electrospray ionization mass spectroscopy detection

Authors: Reinhardt, Laurie; SVAREN, LEVI - Oak Ridge National Laboratory; MARATHE, RITVIK - Oak Ridge National Laboratory; Zanton, Geoffrey; Sullivan, Michael

Submitted to: Rapid Communications in Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/7/2025
Publication Date: 3/19/2025
Citation: Reinhardt, L.A., Svaren, L., Marathe, R., Zanton, G.I., Sullivan, M.L. 2025. Determination of free amino acid concentrations in bovine plasma using high-pressure liquid chromatography with electrospray ionization mass spectroscopy detection. Rapid Communications in Mass Spectrometry. https://doi.org/10.1002/rcm.10027.
DOI: https://doi.org/10.1002/rcm.10027

Interpretive Summary: Measurement of individual amino acid levels in blood plasma is an important tool in assessing health and production responses of dairy cattle under various management regimes, especially diet. Here we developed a robust, accurate method to measure amino acid levels in bovine plasma using chemical derivatization followed by high pressure liquid chromatography with mass spectroscopy detection. This method will aid in evaluating efficacy of dietary interventions to maximize production and minimize the environmental footprint of diary production systems while maintaining animal health. Additionally, the method should be broadly applicable to other ruminant and food animal production systems.

Technical Abstract: Rationale: Quantification of free amino acid concentrations in plasma has become an important tool in monitoring the health of dairy cows and health of their offspring under various management regimes, especially diet. Consequently, it was desirable to develop a robust, accurate, medium-throughput method to quantitate free amino acid concentrations in bovine plasma. Methods: Bovine plasma was deproteinated with methanol and amino acids partially purified using cation exchange resin. Samples were then subjected to pre-column derivatization with phenyl isothiocyanate, followed by high-pressure liquid chromatography with positive electrospray ionization single quadrupole mass spectrometry detection for analysis. The corresponding 13C and 15N labeled amino acids (mass unit difference >3) were used as internal standards, while deuterium labeled standards were used for other metabolites. Results: All 20 amino acids showed linear fits to their individual calibration curves (correlation coefficients >0.99) with concentration range of amino acids measured from 5 to 600 uM. Coefficient of variation (CV) values for the concentrations measured for all amino acids ranged from 2.0 to 6.7 for intra-day aliquots and from 1.0 to 4.6 for inter-day aliquots with the exception of aspartic acid (11.1 and 12.6 for intra- and inter-day, respectively). Conclusions: The use of a stable isotope labeled version of each amino acid analyte as internal standard added to plasma samples at the beginning of the procedure corrected for any losses, instrument variability, and chemistry of derivatization. Use of this method to quantify bovine plasma amino acids will allow better understanding of physiological processes underlying nutritional interventions in dairy production systems and may be more broadly applicable to ruminant and other animal production systems.