Measurement of 15N enrichment in multiple amino acids and urea in a single analysis by gas chromatography/mass spectrometry

Abstract

A precise and accurate procedure to measure the 15N isotopic enrichment of 18 common plasma amino acids and singly (15N1) and doubly (15N2) labeled urea in a single analysis by selected ion monitoring electron impact ionization gas chromatography/mass spectrometry analysis is presented. The choice of tert-butyldimethylsilyl derivatives allowed the enrichments in the amide and amino nitrogens of glutamine to be resolved. The ions monitored contained all the nitrogen atoms from the parent compounds except for arginine, which lost one guanidino nitrogen. Isotope ratios were determined with a coefficient of variation (within-assay precision) of 0.35% (range, 0.1-1.0%) on replicate measures averaged over all components; thus, the standard deviation associated with a nominal [m + 1]/[m + 0] isotope ratio of 0.2000 was 0.0007. The average error between measured and theoretical [m + 1]/[m + 0] isotope ratios was +0.0001 +/- 0.0086 for samples at natural abundance isotopic composition. The utility of the procedure is demonstrated by monitoring the incorporation of 15N into 18 plasma amino acids and urea during a 6 h oral administration of 15NH4Cl to a human volunteer. Highest levels of enrichment were achieved in arginine and urea, followed by glutamine. Approximately 80% of the label in glutamine was in the amino nitrogen. Excess 15N enrichment was observed in all plasma amino acids monitored with the exception of the essential amino acids phenylalanine, lysine and histidine. This method will facilitate the measurement of isotopic enrichment of multiple amino acids by a single analysis when it is necessary to monitor multiple stable-isotopically labeled amino acids in studies of amino acid and protein metabolic kinetics.