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

## TL;DR

The study developed and validated a reliable HPLC method to measure allantoin, creatinine, and uric acid in cow urine, which could help understand how cows respond to heat stress.

## Contribution

A validated reversed-phase HPLC method for simultaneous quantification of allantoin, creatinine, and uric acid in cow urine is introduced.

## Key findings

- The HPLC method showed high repeatability and reproducibility for allantoin and creatinine.
- Optimal linearity was achieved for dilutions between 1:20 and 1:8.
- Recovery rates were excellent, especially for allantoin and creatinine.

## Abstract

Summary: The aim of this study was to validate a chromatographic method to detect and quantify allantoin, creatinine, and uric acid in cow urine samples. The method featured a simple chromatographic separation, yielding well-defined peaks. High repeatability and reproducibility were achieved, with optimal linearity performances. Excellent recovery rates were obtained, especially for allantoin and creatinine. The findings of this study confirm the effectiveness of the method in analyzing target compounds, laying the foundation for a better understanding of physiological responses to heat stress, one of the major challenges facing the future of the livestock sector.

Summary: The aim of this study was to validate a chromatographic method to detect and quantify allantoin, creatinine, and uric acid in cow urine samples. The method featured a simple chromatographic separation, yielding well-defined peaks. High repeatability and reproducibility were achieved, with optimal linearity performances. Excellent recovery rates were obtained, especially for allantoin and creatinine. The findings of this study confirm the effectiveness of the method in analyzing target compounds, laying the foundation for a better understanding of physiological responses to heat stress, one of the major challenges facing the future of the livestock sector.

•Allantoin, creatinine, and uric acid were quantified in individual spot cow urine.•High repeatability and reproducibility were achieved.•Robust linearity was found across the tested dilution ranges.•High recovery rates were reached for allantoin, creatinine, and uric acid.

Allantoin, creatinine, and uric acid were quantified in individual spot cow urine.

High repeatability and reproducibility were achieved.

Robust linearity was found across the tested dilution ranges.

High recovery rates were reached for allantoin, creatinine, and uric acid.

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.