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; ADONNA S; SANTIAGO G; REY F; ORTEGA HH; HEIN GJ

Mar del Plata

Congreso; LXIV Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2019

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 (100mm x 4.0mm innerdiameter, 3.0µmparticle size). Detection and quantitation were achieved by using a QTrap 3200triple quadrupole mass spectrometer (AB Sciex) equipped with an electrospray ionizationsource interface. The analytical technique was validated in plasma inaccordance with the International Conference on Harmonisation (ICH) guidelineand linearity, accuracy and precision, recovery and matrix effect and stabilitywere considered. After that, bloodfrom six grazing dairy cows of a commercial dairy farm was sampled at -28, -14,4, 14, 28 and 60 days relative to parturition and plasma 3-NT was evaluated inthose samples. Method showed linearity (r>0.992) over the working range (0.5to 15 ng/mL). Accuracy (<15%) and precision (%CV<15) wereaccording to the international criteria. 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 smooth (<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.