title: Simultaneous determination of natural-abundance δ15N values and quantities of individual amino acids in proteins from milk of lactating women and from infant hair using gas chromatography/isotope ratio mass spectrometry.
abstract: BACKGROUND
  In isotope tracer experiments used in nutritional studies, it is frequently desirable both to determine the (15)N/(14)N ratios of target compounds and to quantify these compounds. This report shows how this can be achieved in a single chromatographic run for protein amino acids using an isotope ratio mass spectrometer.
  
  
  METHODS
  Protein hydrolysis by acidic digestion was used to release amino acids, which were then derivatized as their N-pivaloyl-O-isopropyl esters. Suitable conditions for sample preparation were established for both hair and milk proteins. The N-pivaloyl-O-isopropyl esters of amino acids were separated by gas chromatography (GC) on a 60 m ZB-WAX column linked via a combustion interface to an isotope ratio mass spectrometer. The (15)N/(14)N ratios were obtained from the m/z 28, 29 and 30 peak intensities and the quantities from the Area All (Vs) integrated peak areas.
  
  
  RESULTS
  It is shown from a five-point calibration curve that both parameters can be measured reliably within the range of 1.0 to 2.0 mg/mL for the major amino acids derived from the hydrolysis of human maternal milk or hair samples. The method was validated in terms of inter-day and inter-user repeatability for both parameters for 14 amino acids. The amino acid percentage composition showed a good correlation with literature values. The method was applied to determine the variability in a population of lactating mothers and their infants.
  
  
  CONCLUSIONS
  It has been established that δ(15)N values can be simultaneously determined for a complex mixture of amino acids at variable concentrations. It is shown that the percentage composition obtained correlates well with that obtained by calculation from the protein composition or from literature values. This procedure should provide a significant saving in analysis time, especially in those cases where the GC run-time is necessarily long. It allows the satisfactory determination of the variation within a sample population.
authors.affiliation: Elucidation of Biosynthesis by Isotopic Spectrometry Group, CEISAM, LUNAM Université, University of Nantes-CNRS UMR6230, 2 rue de la Houssinière, BP 92208, F-44322 Nantes, France. illa.tea@univ-nantes.fr
authors.forename: Illa
authors.initials: I
authors.lastname: Tea
authors.forename: Adrien
authors.initials: A
authors.lastname: Le Guennec
authors.forename: Marine
authors.initials: M
authors.lastname: Frasquet-Darrieux
authors.forename: Maxime
authors.initials: M
authors.lastname: Julien
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Romek
authors.forename: Ingrid
authors.initials: I
authors.lastname: Antheaume
authors.forename: Régis
authors.initials: R
authors.lastname: Hankard
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Robins
mesh_terms: D000328:Adult; D000596:Amino Acids / Q000032:analysis*; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006197:Hair / Q000737:chemistry*; D006801:Humans; D007223:Infant; D007774:Lactation; D008297:Male; D008895:Milk, Human / Q000737:chemistry*; D009587:Nitrogen Isotopes / Q000032:analysis*; D011506:Proteins / Q000737:chemistry*; D055815:Young Adult
chemical_list: D000596:Amino Acids; D009587:Nitrogen Isotopes; D011506:Proteins