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 |
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Authors: | Godin Jean-Philippe Faure Magali Breuille Denis Hopfgartner Gérard Fay Laurent-Bernard |
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Institution: | (1) Department of Bioanalytical Science, Nestlé Research Center, Nestec Ltd, Vers chez les Blanc, P.O. Box 44, 1000 Lausanne 26, Switzerland;(2) Life Sciences Mass Spectrometry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 20 bd d’Yvoy, 1211 Geneva 4, Switzerland;(3) Department of Nutrition, Nestlé Research Center, Nestec Ltd, Vers chez les Blanc, P.O. Box 44, 1000 Lausanne 26, Switzerland |
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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. |
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Keywords: | N(O S)-ethoxycarbonyl ethyl ester derivative 13C-amino acid derivatives 13C tracer study GC– C– IRMS |
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