Determination of hydrogen peroxide in exhaled breath condensate by flow injection analysis with fluorescence detection

A method for the determination of hydrogen peroxide in exhaled breath condensate (EBC) by automated flow injection analysis (FIA) with fluorescence detection was developed and validated. In the enzymatic assay a fluorescent dimer of para-hydroxyphenyl acetic acid (HPAA) was formed by the redox coupling reaction between hydrogen peroxide and horseradish peroxidase (HRP). The calibration curve of hydrogen peroxide was linear over a range of 40–5000 nM. The coefficient of variation (CV) for within-day precision was 1–3%; for between-day precision, it was 2–5% over the validated range. The assay requires a small sample aliquot (150 μl) and no incubation time, and has an analytical runtime of <2 min. It is therefore suitable for larger studies. The method was used to detect hydrogen peroxide in EBC of asthmatic patients and healthy volunteers. A statistically significant difference was found between patients with asthma (n = 19) and control subjects without asthma (n = 19), 780 nM versus 480 nM (P = 0.03).