Determination of ¹³C isotopic enrichment of valine and threonine by GC–C–IRMS after formation of the N(O,S)-ethoxycarbonyl ethyl ester derivatives of the amino acids

Abstract

We describe a new method of assessing, in a single run, ¹³C isotopic enrichment of both Val and Thr by gas chromatography–combustion–isotope-ratio mass spectrometry (GC–C–IRMS). This method characterised by a rapid one-step derivatisation procedure performed at room temperature to form the N(O,S)-ethoxycarbonyl ethyl ester derivatives, and a polar column for GC. The suitability of this method for Val and Thr in in-vivo samples (mucosal hydrolysate) was demonstrated by studying protein metabolism with two tracers (¹³C-valine or ¹³C-threonine). The intra-day and inter-day repeatability were both assessed either with standards or with in-vivo samples at natural abundance and at low ¹³C isotopic enrichment. For inter-day repeatability CVs were between 0.8 and 1.5% at natural abundance and lower than 5.5% at 0.112 and 0.190 atom% enrichment for Val and Thr, respectively. Overall isotopic precision was studied for eleven standard amino acid derivatives (those of Val, Ala, Leu, Iso, Gly, Pro, Asp, Thr, Ser, Met, and Phe) and was assessed at 0.32‰. The ¹³C isotopic measurement was then extended to the other amino acids (Ala, Val, Leu, Iso, Gly, Pro, Thr, and Phe) at natural abundance for in-vivo samples. The isotopic precision was better than 0.002 atom% per amino acid (for n = 4 rats). This analytical method was finally applied to an animal study to measure Thr utilization in protein synthesis.