Evaluation of hydroxyl radicals production using 13CO2 gas chromatography-isotope ratio mass spectrometry.

A gas chromatography-isotope ratio mass spectrometry (GC-IRMS) technique for detecting the production of hydroxyl radicals is described. The decarboxylation of [7-13COOH]benzoic acid in the presence of a hydroxyl radicals source (a mixture of porphyrin and hydrogen peroxide) was followed by direct measurement of the 13CO2/12CO2 isotopic ratio. The production of hydroxyl radicals by the mixtures of porphyrin-hydrogen peroxide was proved by comparative study with electron spin resonance spectrometry and high-performance liquid chromatography analysis of hydroxylation products of benzoic acid. The water-soluble radical scavengers methanol, mannitol, and dimethyl sulfoxide led to the inhibition of 13CO2 production from [7-13COOH]benzoic acid. In contrast, high concentrations of the antioxidant ascorbate strongly increased [7-13COOH]benzoic acid decarboxylation. Finally, the use of anaerobic conditions showed that decarboxylation was independent of the presence of oxygen. The absence of the effect of superoxide dismutase could exclude a possible effect of the superoxide ion. This nonradioactive technique offers many advantages compared to the well-established method for detecting hydroxyl radicals based on the decarboxylation of [7-14COOH]benzoic acid. It is rapid and easy to perform as a simple tube test and is highly reliable for detecting hydroxyl radicals. This method provides an on-line analysis of carbon dioxide compared to the radiochemical method. In addition, 13CO2-enrichment measurements led to easy kinetic studies with high sensitivity and semiquantitative determinations.