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.
We describe a new method of assessing, in a single run, (13)C 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 ((13)C-valine or (13)C-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 (13)C 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 per thousand. The (13)C 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.
Godin
Jean-Philippe
JP
Department of Bioanalytical Science, Nestlé Research Center, Nestec Ltd, Vers chez les Blanc, Lausanne 26, Switzerland. jean-philippe.godin@rdls.nestle.com
Faure
Magali
M
Breuille
Denis
D
Hopfgartner
Gérard
G
Fay
Laurent-Bernard
LB
D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D002247:Carbon Isotopes / Q000737:chemistry*; D004952:Esters / Q000737:chemistry*; D005019:Ethyl Ethers / Q000737:chemistry*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D007413:Intestinal Mucosa / Q000737:chemistry / Q000378:metabolism; D008297:Male; D014176:Protein Biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013912:Threonine / Q000032:analysis*; D013997:Time Factors; D014633:Valine / Q000032:analysis*
D000596:Amino Acids; D002247:Carbon Isotopes; D004952:Esters; D005019:Ethyl Ethers; D013912:Threonine; D014633:Valine