DEVELOPMENT AND VALIDATION OF A LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY METHOD FOR DETERMINATION OF FREE 3-NT IN URINE AND LIVER SAMPLES OF LACTATING DAIRY CATTLE

ADDONA SM; BARCAROLO D; ORTEGA HH; ANGELI E; HEIN GJ

Mar del Plata

Congreso; LXVI ANNUAL MEETING OF SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA; 2021

SAIC

During the transition from pregnancy to lactation, cows experience higher energy demand and oxygen requirements leading to oxidative stress. The 3-nitrotyrosine (3-NT) has been described as a specific biomarker of oxidative damage mediated by peroxynitrite. This study aimed to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of 3-NT in urine and liver tissue samples from dairy cows and described the 3-NT concentration in plasma, urine, and liver during the transition period in the spring (SG) and winter (WG) seasons. The developed method was validated following the Food and Drug Administration guideline. The matrix effect was negligible for urine and there was a medium matrix effect for liver tissue. The calibration curve of 3-NT was fitted with a 1/x2 weighted linear regression over the concentration range of 0.24-19.31 mg/L, for urine, and 2.27-102.04 mg/L, for liver tissue. Regarding the dilution study, the factor was 1/5 for urine and 1/10 for liver tissue. The precision was < 15 % and the accuracy was in the range of 85?115 %. 3-NT was stable in the different matrices after short-term storage for 30 days at -80 °C, after long-term storage for six months at -80° C, and after three freeze and thaw cycles. In plasma, urine, and liver samples, 3-NT concentration was higher in cows of the SG than in those of the WG (p < 0.05). A high correlation was observed between 3-NT concentration in urine and liver (r = 0.74; p < 0.01) and a moderate correlation between plasma and urine (r = 0.61; P < 0.01) and between that in plasma and liver (r = 0.55; p < 0.01). This study describes the development and validation of an LC-MS/MS method for the quantification of an important oxidative stress biomarker in fluids and tissue from groups of animals with different heat stress, showing that it could be a very useful parameter to evaluate the comfort of dairy cows, especially during the transition period.