Simultaneous measurement of¹³C- and¹⁵N-isotopic enrichments of threonine by mass spectrometry

Under conditions of high isotopic dilution, e.g. in a tracer study, the ability to determine accurately and quantitatively small variations in isotopic enrichments of differently labelled chemical compounds (e.g. C-13 and N-15 in threonine) in a single run by gas chromatography/mass spectrometry (GC/MS) is desirable but remains a technological challenge. Here, we report a new, rapid and simple GC/MS method for simultaneously measuring the isotopic enrichments of doubly labelled threonine ([(UC)-C-13] and N-15) with isotopic enrichment lower than 1.5 Molar Percent Excess (MPE). The long-term reproducibility measured was around 0.09 MPE for both tracers (throughout a 6 week period). The intra-day repeatability was lower than 0.05 and 0.06 MPE for [(UC)-C-13]-Thr and N-15-Thr, respectively. To calculate both isotopic enrichments, two modes of calculations were used: one based on work by Rosenblatt et al. in 1992 and the other one using a matrix approach. Both methods gave similar results (ANOVA, P >0.05) with close precision for each mode of calculation. The GC/MS method was then used to investigate the differential utilization of threonine in different organs according to its route of administration in minipigs after administration of both tracers. In plasma samples, the lowest isotopic enrichment measured between two successive time points was at 0.01 and 0.02 MPE for [(UC)-C-13]-Thr and N-15-Thr, respectively. Moreover, the accuracy of GC/MS C-13-isotopic enrichment measured was validated by analyzing the same plasma samples by gas chromatography/combustion/isotope ratio mass spectrometry-(GC/C/IRMS). Statistical analysis showed that both techniques gave the same results (ANOVA, P >0.05). This new GC/MS method offers the possibility to measure C-13- and N-15-isotopic enrichments with higher throughput, and using a lower amount of sample, than using GC/C/IRMS. Copyright (C) 2009 John Wiley & Sons, Ltd.