Measurement of oxygen isotope ratios (18O/16O) of aqueous O2 in small samples by gas chromatography/isotope ratio mass spectrometry

RationaleOxygen isotope fractionation of molecular O-2 is an important process for the study of aerobic metabolism, photosynthesis, and formation of reactive oxygen species. The latter is of particular interest for investigating the mechanism of enzyme-catalyzed reactions, such as the oxygenation of organic pollutants, which is an important detoxification mechanism. MethodsWe developed a simple method to measure the O-18 values of dissolved O-2 in small samples using automated split injection for gas chromatography coupled to isotope ratio mass spectrometry (GC/IRMS). After creating a N-2 headspace, the dissolved O-2 partitions from aqueous solution to the headspace, from which it can be injected into the gas chromatograph. ResultsIn aqueous samples of 10mL and in diluted air samples, we quantified the O-18 values at O-2 concentrations of 16M and 86M, respectively. The chromatographic separation of O-2 and N-2 with a molecular sieve column made it possible to use N-2 as the headspace gas for the extraction of dissolved O-2 from water. We were therefore able to apply a rigorous O-18 blank correction for the quantification of O-18/O-16 ratios in 20nmol of injected O-2. ConclusionsThe successful quantification of O-18-kinetic isotope effects associated with enzymatic and chemical reduction of dissolved O-2 illustrates how the proposed method can be applied for studying enzymatic O-2 activation mechanisms in a variety of (bio)chemical processes. Copyright (c) 2016 John Wiley & Sons, Ltd.