Determination of13C- and15N-enrichment of glutamine by gas chromatography/mass spectrometry and gas chromatography/combustion/isotope ratio mass spectrometry after N(O,S)-ethoxycarbonyl ethyl ester derivatisation

Abstract

To measure 13C- and 15N-Gln enrichments in plasma samples by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) we investigated the use of the N(O,S)-ethoxycarbonyl ethyl ester derivative. We found that this derivative is very stable at room temperature, even after 5 days storage. It allows reproducible and accurate 13C- and 15N-isotopic enrichment determinations with values of RSD below 0.8 and 3.2% for GC/MS and GC/C/IRMS, respectively. This derivative enables high sample throughput analyses when automated GC/MS and GC/C/IRMS are used. The methodology was applied to measure isotopic enrichments in rat plasma after oral force-feeding with [2,5-15N2]-Gln and in human plasma samples obtained after intravenous infusion of [1-13C]-Gln. In rats after oral force-feeding, we found that the maximal appearance of [2,5-15N2]-Gln occurred between 40 and 80 min in the two compartments studied (portal and peripheral blood). In humans infused intravenously with [1-13C]-Gln for 4 h, the plateau in 13C-Gln enrichments in plasma was reached after 3 h of infusion and coefficients of variation between samples were 1.7 and 12.4% for the two subjects. Using the N(O,S)-ethoxycarbonyl ethyl ester derivative, 13C- and 15N-isotopic enrichment can be determined in the range 2–100 ± 0.3 MPE by GC/MS and 0.3–2 ± 0.02 MPE by GC/C/IRMS. Copyright © 2001 John Wiley & Sons, Ltd.