An improved analytical method for the determination of urea nitrogen isotopomers in biological samples utilizing continuous flow isotope ratio mass spectrometry

Abstract

Over the past few years numerous dual inlet isotope ratio mass spectrometry (IRMS) applications have been adapted to continuous flow systems which allow the automation of sample admission and a higher throughput. The isotopomer analysis of urea nitrogen by IRMS requires the offline conversion of urea into nitrogen gas before analysis. The oxidation of urea with LiOBr results in the monomolecular degradation of urea, which preserves the identity of the parent urea molecule, and has to be conducted under vacuum to prevent contamination with atmospheric nitrogen. We have developed an offline system of urea degradation utilizing disposable Exetainers, in which atmospheric nitrogen is displaced by helium. Recovery of urea nitrogen was linear within the range of the standards tested (0 to 420 microg nitrogen) and standard curves for 15N15N-urea standards showed high coefficients of determination (R2 > 0.9998). A small portion of urea degrades in a non-monomolecular fashion and has been shown to depend on the concentration of urea in the sample. Long-term storage of prepared samples showed a decline in 15N15N enrichment, suggesting air contamination. However, samples were stable for 24 h, which allows for the analysis of large sample batches. Interest in urea metabolism, particularly in ruminant species, has increased recently due to the environmental implications of urea and nitrogen excretion by farm animals. This novel analytical method will allow for accurate measurements and the rapid throughput needed in order to support these field studies.