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; SANTIAGO G; HEIN GJ; ADDONA S; ORTEGA HH; ANGELI E; REY F

Mar del Plata

Conferencia; Sociedad Argentina de Investigación Clínica (SAIC); 2019

During the transition period cows experienceincreases ofmetabolic demand andtissue oxygenrequirements leading to an oxidative stress.The 3-nitrotyrosine (3-NT) is thought to be a relatively specific biomarker of oxidative damage mediated by peroxynitrite.The aim of this study was to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of 3-NT in plasmasamples from dairy cows.An ultra-fast liquid chromatography system (LC-20AD, Shimadzu) consisting of a binary pump equipped with autosamplerSIL-20ACXR(Shimadzu) was used. Chromatographic separation was performed with a Shimadzu Shim-pack GIST C18 column (100mm x 4.0mm inner diameter, 3.0µm particle size). Detection and quantitation were achieved by using a QTrap 3200 triple quadrupole mass spectrometer (AB Sciex) equipped with an electrospray ionization source interface.The analytical technique was validated in plasma in accordance with the InternationalConference on Harmonisation (ICH) guideline andlinearity, accuracy and precision, recovery andmatrix effect and stability were considered.After that, blood from 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 in those samples. Method showed linearity (r>0.992) over the working range (0.5 to 15 ng/mL). Accuracy (