We present the analysis of the stable carbon isotope compositions of 14 individual N-pivaloyl-isopropyl (NPP) amino acid esters by gas chromatography-combustion isotope ratio mass spectrometry (GC-C-IRMS).
The mean reproducibility of derivatization procedure and GC-C-IRMS analysis was 0.45‰ (range, 0.12-0.68), whereas the mean analytical error was 0.26‰ ␦ 13 C (range, 0.13-0.42). Furthermore, the ␦ 13 C values of Npivaloyl-isopropyl and N-acetyl-n-propyl (NAP) amino acid esters were compared. Due to a reproducible isotopic fractionation introduced by the derivatization process an empirical correction factor for each individual amino acid was derived separately for both derivatives (NPP, ؊1.13 to ؊2.52 (lysine, ؉2.09)‰ ␦ 13 C; NAP, ؊2.36 to ؊3.97 (lysine, ؉1.91)‰ ␦ 13 C), and the original ␦ 13 C value of the underivatized amino acid was calculated. Further, we performed an animal study where rats (n ؍ 5) ingested a mixed meal containing uniformly 13 C-labeled casein (indispensable amino acids 1.3 to 1.7 at.%). One hour after the meal ␦ 13 C values of protein-bound amino acids from small intestinal mucosa and liver and of free amino acids from mucosa and plasma were determined. Significant 13 C enrichments of indispensable amino acids of the free pools of mucosa and plasma (range, 0.0518 to 0.1700 at.% excess) and in mucosa and liver proteins (range, 0.0021 and 0.0161 at.% excess) were observed. The feasibility of various derivatives for the measurement of carbon isotopic composition is discussed.