Simultaneous measurement of ¹³C‐ and ¹⁵N‐isotopic enrichments of threonine by mass spectrometry

Abstract

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. (13)C and (15)N 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 ([U(13)C] and (15)N) 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 [U(13)C]-Thr and (15)N-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 [U(13)C]-Thr and (15)N-Thr, respectively. Moreover, the accuracy of GC/MS (13)C-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 (13)C- and (15)N-isotopic enrichments with higher throughput, and using a lower amount of sample, than using GC/C/IRMS.

High‐precision mass spectrometric analysis using stable isotopes in studies of children

“…To study intestinal mucin synthesis, isotopically labeled threonine was administered because muc 2 is rich in threonine. Outcomes have been observed in rats (Faure et al, 2005(Faure et al, , 2007, pigs (Puiman et al, 2011), and neonates (Schaart et al, 2009;Godin et al, 2009). Nutritional interventions (e.g., the effects of pro-or prebiotics) could be studied using this method, but no clinical studies have yet been performed.…”

Analysis of carbon and nitrogen co-metabolism in yeast by ultrahigh-resolution mass spectrometry applying 13C- and 15N-labeled substrates simultaneously

“…In a first attempt, simultaneous measurements of 13 C-and 15 N-isotopic enrichments of threonine were carried out by GC/MS using an intense electron ionization (EI) fragment and applying mathematical corrections [21]. Popa et al used co-feeding of 13 C and 15 N (H 13 CO 3 -and 15 N 2 ) to study the relationship between CO 2 and N 2 fixation in filamentous nitrogen-fixing cyanobacteria [22].…”

Determination of the natural abundance δ15N of taurine by gas chromatography–isotope ratio measurement mass spectrometry

“…A number of protocols exploiting isotope ratio measurement by mass spectrometry (irm-MS) interfaced via a combustion furnace to gas chromatography (GC-irm-MS) for the determination of the d 13 C and d 15 N values of a-amino acids at or near natural abundance have been reported. [1][2][3][4] While this analytical approach has the advantage of not requiring the use of highly isotopically-enriched starting materials, it has the disadvantage of requiring the derivatization of amino acids to provide sufficient volatility to permit a GC separation prior to stable isotope determination. In order to measure the d 13 C and d 15 N values by GC-irm-MS, it is necessary that the derivatization reagents: (i) react quantitatively with all target analytes; (ii) lead to minimal isotopic dilution (i.e.…”

“…Methods have been reported that avoid the use of TMS derivatives. Andresen et al 17 used a large-scale multi-step reaction to convert taurine into N-carbobenzyloxytaurine amide for the MS analysis of 2% enriched [1,[2][3][4][5][6][7][8][9][10][11][12][13] C]taurine, while Irving and Klein 18 reported a method for the GC-MS analysis of the N,N-dimethylaminomethylene methyl ester of taurine. However, these derivatization reactions are not suitable for determining the d 15 N of taurine, since they involve the addition of one exogenous nitrogen atom, which would reduce the precision of measurement.…”

“…However, these derivatization reactions are not suitable for determining the d 15 N of taurine, since they involve the addition of one exogenous nitrogen atom, which would reduce the precision of measurement. The N-pentafluorobenzoyldi-n-butylamide derivative of taurine has also been employed in the GC-MS analysis of enriched [1,[2][3][4][5][6][7][8][9][10][11][12][13] C]-and [ 15 N]taurine. 19,20 Although this derivative has good chromatographic properties, not only is there the addition of one exogenous nitrogen atom but the fluorine released upon combustion of the N-trifluoroacetyl-i-propyl (TFA-IP) esters would poison the oxidation reactor Pt catalyst surface, resulting in the irreversible conversion of Cu and Ni into the stable metal halides Cu 2 F and Ni 2 F, respectively.…”