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

Rationale Oxygen isotope fractionation of molecular O₂ 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. Methods We developed a simple method to measure the δ¹⁸O values of dissolved O₂ in small samples using automated split injection for gas chromatography coupled to isotope ratio mass spectrometry (GC/IRMS). After creating a N₂ headspace, the dissolved O₂ partitions from aqueous solution to the headspace, from which it can be injected into the gas chromatograph. Results In aqueous samples of 10 mL and in diluted air samples, we quantified the δ¹⁸O values at O₂ concentrations of 16 μM and 86 μM, respectively. The chromatographic separation of O₂ and N₂ with a molecular sieve column made it possible to use N₂ as the headspace gas for the extraction of dissolved O₂ from water. We were therefore able to apply a rigorous δ¹⁸O blank correction for the quantification of ¹⁸O/¹⁶O ratios in 20 nmol of injected O₂. Conclusions The successful quantification of ¹⁸O-kinetic isotope effects associated with enzymatic and chemical reduction of dissolved O₂ illustrates how the proposed method can be applied for studying enzymatic O₂ activation mechanisms in a variety of (bio)chemical processes. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]