Isotope ratio monitoring of small molecules and macromolecules by liquid chromatography coupled to isotope ratio mass spectrometry.
In the field of isotope ratio mass spectrometry, the introduction of an interface allowing the connection of liquid chromatography (LC) and isotope ratio mass spectrometry (IRMS) has opened a range of new perspectives. The LC interface is based on a chemical oxidation, producing CO2 from organic molecules. While first results were obtained from the analysis of low molecular weight compounds, the application of compound-specific isotope analysis by irm-LC/MS to other molecules, in particular biomolecules, is presented here. The influence of the LC flow rate on the CO2 signal and on the observed delta13C values is demonstrated. The limits of quantification for angiotensin III and for leucine were 100 and 38 pmol, respectively, with a standard deviation of the delta13C values better than 0.4 per thousand. Also, accuracy and precision of delta13C values for elemental analyser-IRMS and flow injection analysis-IRMS (FIA-LC/MS) were compared. For compounds with molecular weights ranging from 131 to 66,390 Da, precision was better than 0.3 per thousand, and accuracy varied from 0.1 to 0.7 per thousand. In a second part of the work, a two-dimensional (2D)-LC method for the separation of 15 underivatised amino acids is demonstrated; the precision of delta13C values for several amino acids by irm-LC/MS was better than 0.3 per thousand at natural abundance. For labelled mixtures, the coefficient of variation was between 1% at 0.07 atom % excess (APE) for threonine and alanine, and around 10% at 0.03 APE for valine and phenylalanine. The application of irm-LC/MS to the determination of the isotopic enrichment of 13C-threonine in an extract of rat colon mucosa demonstrated a precision of 0.5 per thousand, or 0.001 atom %.
Godin
Jean-Philippe
JP
Nestec Ltd., Nestlé Research Center, Department of Bioanalytical Science, PO Box 44, CH-1000 Lausanne 26, Switzerland. jean-philippe.godin@rdls.nestle.com
Hau
Jörg
J
Fay
Laurent-Bernard
LB
Hopfgartner
Gérard
G
D000596:Amino Acids / Q000737:chemistry*; D000805:Angiotensin III / Q000737:chemistry; D000818:Animals; D002138:Calibration; D002417:Cattle; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D007328:Insulin / Q000737:chemistry; D007554:Isotopes / Q000032:analysis*; D046911:Macromolecular Substances / Q000737:chemistry; D008297:Male; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D010316:Particle Size; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000737:chemistry
D000596:Amino Acids; D007328:Insulin; D007554:Isotopes; D046911:Macromolecular Substances; D012709:Serum Albumin; D000805:Angiotensin III