Isotope ratio monitoring of small molecules and macromolecules by liquid chromatography coupled to isotope ratio mass spectrometry

Abstract

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 %.

Online δ13C analysis of volatile fatty acids in sediment/porewater systems by liquid chromatography-isotope ratio mass spectrometry

“…Sample size requirements-The sensitivity of the irm-LC/MS method averages around 0.25 Vs ng -1 C injected on the HPLC column (Table 2) and is comparable to previously reported values (Krummen et al 2004;Godin et al 2005). During evaluation of our method, peak areas showed a constant signal response to the injected amount of carbon for a given flow rate.…”

“…For the concentration ranges reported in Table 2, we observed a linear correlation between signal area and injected amount of carbon as reported previously for benzoic acid (Krummen et al 2004), angiotensin III, and leucine (Godin et al 2005). Therefore, irm-LC/MS can be used to combine stable carbon isotopic and quantitative analysis of VFAs.…”

Comparison of rRNA and Polar-Lipid-Derived Fatty Acid Biomarkers for Assessment of 13 C-Substrate Incorporation by Microorganisms in Marine Sediments

“…Further improvements could result from analyzing rRNA nucleotides by HPLC-IRMS (39) or GC-c-IRMS, which have much better transfer efficiencies and allow direct identification of the compounds being analyzed (45). Furthermore, the HPLC-IRMS interface can be used as a micro-EA-IRMS if very small samples are injected (31,60). Use of 14 C-labeled substrates with detection by scintillation counting or autoradiography could also increase the sensitivity.…”

Isotope ratio mass spectrometry coupled to liquid and gas chromatography for wine ethanol characterization

“…This process has been successfully applied to sugars 22 and amino acids, 21,23,24 and has been shown to be quantitative and without isotopic fractionation.…”

“…In LC/IRMS, the target molecule containing the heavy 13 C isotope is eluted after the molecule containing the light one ( 12 C). 23,24 The chromatographic isotope effect in LC seems to be related either to the nature of the compounds, to the stationary phases, or to both, and needs to be carefully investigated.…”