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 CO 2 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 CO 2 signal and on the observed δ 13 C 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 δ 13 C values better than 0.4‰. Also, accuracy and precision of δ 13 C 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‰, and accuracy varied from 0.1 to 0.7‰. 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 δ 13 C values for several amino acids by irm‐LC/MS was better than 0.3‰ 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 13 C‐threonine in an extract of rat colon mucosa demonstrated a precision of 0.5‰, or 0.001 atom %. Copyright © 2005 John Wiley & Sons, Ltd.