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

Mar del Plata

Congreso; LXIV Reunion Anual de la Sociedad Argentina de Investigaciones Clinicas (SAIC); 2019

SAIC

During the transition period cows experience increases of metabolic demand and tissue oxygen requirements 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 plasma samples from dairy cows. An ultra-fast liquid chromatography system (LC-20AD, Shimadzu) consisting of a binary pump equipped with autosampler SIL-20ACXR (Shimadzu) was used. Chromatographic separation was performed with a Shimadzu Shim-pack GIST C18 column (100 mm x 4.0 mm inner diameter, 3.0 µm 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 International Conference on Harmonisation (ICH) guideline and linearity, accuracy and precision, recovery and matrix 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 (< 15%) and precision (% CV < 15) were according to the international criteria. The 3-NT showed long-term stability (-80 °C) for plasma and under freeze-thaw cycles. The recovery values achieved at different spiked 3-NT levels were within the range of 70-110 % and matrix effect calculated was < 20%. Plasma 3-NT concentrations increased from days 28 prepartum to day 28 postpartum (P = 0.05). A rapid and accurate LC-MS/MS method for 3-NT quantification in plasma samples from lactating dairy cows was successfully developed, suggesting to be a useful tool for early diagnosis of metabolic disorders.