Low‐abundance plasma and urinary [ 15 N]urea enrichments analyzed by gas chromatography/combustion/isotope ratio mass spectrometry

We report a method for determining plasma und urinary [ 15 N]urea enrichments in an abundance range between 0.37 and 0.52 15 N atom% (0–0.15 atom% excess (APE) 15 N) using a dimethylaminomethylene derivative. Compared with conventional off‐line preparation and 15 N analysis of urea, this method requires only small sample volumes (0.5 ml of plasma and 25 µl of urine). The 15 N/ 14 N ratio of urea derivatives was measured by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Two peaks were separated; one was identified by gas chromatography/mass spectrometry (GC/MS) as the complete derivatized urea. Calibration of the complete urea derivative was performed by linear regression of enrichment values of known standard mixtures. Replicate standard (6–465‰ δ 15 N) derivatizations showed a relative standard deviation ranging from 0.1 to 7%. In order to test the feasibility of the method, human subjects and rats ingested a single meal containing either 200 mg of [ 15 N]glycine (95 AP 15 N) or 0.4 mg of [ 15 N]‐α‐lysine (95 AP 15 N), respectively. Urine and plasma were collected at hourly intervals over 7 h after the meal intake. After 15 N glycine intake, maximum urinary urea 15 N enrichments were 330 and 430‰ δ 15 N (0.12 and 0.16 APE 15 N) measured by GC/C/IRMS, whereas plasma [ 15 N]glycine enrichments were 2.5 and 3.3 APE 15 N in the two human subjects 2 h after the meal. 15 N enrichments of total urine and urine samples devoid of ammonia were higher enriched than urinary [ 15 N]urea measured by GC/C/IRMS, reflecting the presence of other urinary N‐containing substances (e.g. creatinine). In rats plasma urea 15 N enrichments were 15–20 times higher than those in urinary urea (10–20‰ δ 15 N). The different [ 15 N]urea enrichments observed after ingestion of [ 15 N]‐labeled glycine and lysine confirm known differences in the metabolism of these amino acids. Copyright © 2002 John Wiley & Sons, Ltd.