Simultaneous analysis of the main markers of nitrogen status in dairy cowʹs urine using hydrophilic interaction chromatography and tandem mass spectrometry detection

Microbial protein synthesis and nitrogen balance status in ruminants can be evaluated by the presence of metabolites in urine. This work aims to develop and validate a simple and sensitive method for simultaneous determination of nine markers of nitrogen status in ruminantʹs urine using hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS). The LC–ESI-MS/MS system, operated in multiple reaction monitoring (MRM) in positive mode, was used for determining selected purine (allantoin, uric acid, xanthine, and hypoxanthine) and pyrimidine derivatives (β-aminobutyric acid and β-alanine), which are used to estimate rumen microbial protein synthesis – the main source of protein for ruminants. Creatinine, creatine and urea, three other metabolites involved in nitrogen metabolism were also measured by this method. The procedure was based on a simple dilution of urine samples in acetonitrile, followed by LC–ESI-MS/MS analysis. Chromatographic separation was tested with three different columns. A zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) column provided an optimal separation for all metabolites. Precision of the method was typically below 10%, and accuracy was above 90% with the exceptions of allantoin and urea at pH 6 and 3, and β-alanine at pH 3. Metabolites were stable after 3 months of storage at −20 °C, except for xanthine, hypoxanthine and β-aminobutyric acid that lost up to 48, 50 and 39% of initial concentration after only 1 month of storage in acidified urine. This LC–ESI-MS/MS method is more specific, unequivocally detecting target metabolites at lower detection limits than methods using UV detection. The method was suitable for the determination of all metabolites tested. The developed method was subsequently used to compare total and spot urine sampling obtained from dairy cows fed diets with contrasting levels of protein.