Method for the determination of 15NH3 enrichment in biological samples by gas chromatography/electron impact ionization mass spectrometry.

An alternative method for the determination of [15N]ammonia enrichment in biological fluids was developed. It is based on the use of glutamate dehydrogenase of bovine liver (EC 1.4.1.2.) with 2-oxopentanoic acid as substrate, to convert the ammonia present in the sample into norvaline, the enrichment of which can be measured by gas chromatography/mass spectrometry as its tertiary butyldimethylsilyl (TBDMS) derivative under electron impact selective ion recording (SIR) conditions. The principal advantage of the present approach is that it is simpler and quicker than the previously described methods, because the synthetic product, norvaline, is not present in biological fluids and pre-processing of the sample is unnecessary. The procedure includes a pre-incubation stage which allows removal of contaminant ammonia present in the reagents used for the enzyme reaction. The contributions of other sources of nitrogen to norvaline production have been checked and quantified: these may provide limitations of the technique when samples for analysis are low in ammonia (e.g. arterial or hepatic venous blood). To reduce these contributions, short times of incubation are proposed. The results from two experiments in vivo in which two sheep were infused with [15N]ammonium chloride in the mesenteric vein are presented and the biological implications which arise from the results are discussed. The validity of the procedures was demonstrated by the quantitative recovery from the mesenteric and portal veins of [15N]ammonia infused.