Development and validation of a LC-MS/MS method to quantify plasma 3-nitrotyrosine in lactating dairy cows as a biomarker of oxidative stress.

BARCAROLO, D; ANGELI, E.; ADDONA, S; SANTIAGO, G; REY, F.; ORTEGA, H.H.; HEIN, G.J.

Mar del Plata

Congreso; LXIV Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica.; 2019

Sociedad Argentina de Investigación Clínica

During thetransition period cows experience increases of metabolic demand and tissue oxygenrequirements leading to an oxidative stress. The 3-nitrotyrosine (3-NT) isthought to be a relatively specific biomarker of oxidative damage mediated byperoxynitrite. The aim of this study was to develop a liquidchromatography-tandem mass spectrometry (LC-MS/MS) method for thequantification of 3-NT in plasma samples from dairy cows. An ultra-fast liquidchromatography system (LC-20AD, Shimadzu) consisting of a binary pump equippedwith autosampler SIL-20ACXR (Shimadzu) was used. Chromatographic separation wasperformed with a Shimadzu Shim-pack GIST C18 column (100 mm x 4.0 mm innerdiameter, 3.0 µm particle size). Detection and quantitation wereachieved by using a QTrap 3200 triple quadrupole mass spectrometer (AB Sciex)equipped with an electrospray ionization source interface. The analyticaltechnique was validated in plasma in accordance with the International Conferenceon Harmonisation (ICH) guideline and linearity, accuracy and precision,recovery and matrix effect and stability were considered. After that, blood from six grazingdairy cows of a commercial dairy farm was sampled at -28, -14, 4, 14, 28 and 60days relative to parturition and plasma 3-NT was evaluated in those samples. Methodshowed linearity (r > 0.992) over the working range (0.5 to 15 ng/mL). Accuracy(< 15%) and precision (% CV < 15) were according to the internationalcriteria. The 3-NT showed long-term stability (-80 °C) for plasma and under freeze-thaw cycles. Therecovery values achieved at different spiked 3-NT levels were within the rangeof 70-110 % and matrix effect calculated was < 20%. Plasma 3-NT concentrations increased from days28 prepartum to day 28 postpartum (P = 0.05). A rapid and accurate LC-MS/MS method for 3-NT quantificationin plasma samples from lactating dairy cows was successfully developed, suggestingto be a useful tool for early diagnosis of metabolic disorders.