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

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

Mar del Plata

Congreso; LXVI Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2021

SAIC

During the transition from pregnancy to lactation, cows experience higher energy demand andoxygen requirements leading to oxidative stress. The 3-nitrotyrosine (3-NT) hasbeen described as a specific biomarker of oxidative damage mediated byperoxynitrite. This study aimed to develop a liquid chromatography-tandem massspectrometry (LC-MS/MS) method for the quantification of 3-NT in urine andliver 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 DrugAdministration guideline. The matrix effect was negligible forurine and there was a medium matrix effect for liver tissue. The calibrationcurve of 3-NT was fitted with a 1/x2 weighted linear regression overthe 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 and1/10 for liver tissue. The precision was < 15 % and the accuracywas in the range of 85?115 %. 3-NT was stable in the different matricesafter short-term storage for 30 days at -80 °C, after long-term storagefor six months at -80° C, and after three freeze and thaw cycles. Inplasma, urine, and liver samples, 3-NT concentration was higher in cows of theSG than in those of the WG (p < 0.05). A high correlation wasobserved 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 forthe quantification of an important oxidative stress biomarker in fluids and tissue from groups ofanimals with different heat stress, showing that it could be a very usefulparameter to evaluate the comfort of dairy cows, especially during thetransition period.