Development of an HPLC method for the quantification of allantoin, creatinine and uric acid in Holstein cows’ urine

Many phenotypes related to heat stress can be identified in cows’ urine, including compounds from protein me tabolism, such as urea, creatinine, uric acid and allantoin. This study aimed to qualitatively and quantitatively determine allantoin, creatinine, and uric acid in Holstein cows’ urine through reverse-phase HPLC. A total of 21 commercial herds located in Northern Italy were visited, and 1,275 individual urine and milk samples of Holstein Friesian cows were collected during morning milking. Cows produced on average 35.20 kg/d of milk, with 3.75% of fat and 3.40% of protein. Aliquots of 1 mL of each urine sample were centrifuged at 13,000xg for 15 min at room temperature. The supernatant was diluted in the proportion 1:10 with ultrapure water and filtered using a 0.22 μm syringe filter. Analysis of allantoin, creatinine, and uric acid was carried out using HPLC station (Agilent Technologies, Santa Clara, SA) equipped with reversed-phase analytical column C18 (Poroshell 120 EC-C18, Agilent Technologies). Linear gradient elution was carried out with a solution of 10 mM potassium dihydrogen phosphate. Among the studied analytes, allantoin showed the greatest concentration, averaging 3071.10 μg/mL, followed by creatinine and uric acid, averaging 505.91 and 225.05 μg/mL, respectively. The strongest Pearson’s correlations were observed between allantoin and creatinine (r = 0.76; p < 0.001). Uric acid was moderate-to-low positively correlated with allantoin (r = 0.31; p < 0.001), and creatinine (r = 0.28; p < 0.001). Creatinine negatively correlated with milk yield (r = -0.18; p < 0.001) and weakly positively correlated with milk protein content (r = 0.10; p < 0.05).