Validation of a reversed-phase high-performance liquid chromatography method for quantification of allantoin, creatinine, and uric acid in individual spot cow urine samples

The possibility of monitoring allantoin, creatinine, and uric acid in individual cow urine can provide insights into the physiological responses of dairy cows under heat stress conditions and offer information on nitrogen metabolism. The aim of this study was to validate a reversed-phase HPLC method for the simultaneous quantification of allantoin, creatinine, and uric acid in dairy cow urine. Urine samples were collected from 10 lactating Holstein cows and were analyzed in triplicate for 5 consecutive days, for an overall total of 150 analyses. The method demonstrated good repeatability and reproducibility performances. In particular, repeatability coefficients exceeded 99% for allantoin and creatinine, and surpassed 95% for uric acid. Among the compounds analyzed in the present study, creatinine and uric acid exhibited the highest and the lowest coefficient of reproducibility (99.39% and 78.03%, respectively). The method exhibited optimal linearity for dilutions between 1:20 (i.e., 1 part of urine and 20 parts of water) and 1:8 (i.e., 1 part of urine and 8 parts of water), with R² ranging from 0.98 to 0.99 for uric acid and allantoin, respectively. The best recovery rates were obtained at the highest spike level for allantoin (102.97%), at the medium spike level for creatinine (98.70%), and at the lowest spike level for uric acid (95.61%). This study may lay the groundwork for future research on the association between novel urine biomarkers and temperature and humidity indexes.