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

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2004-10, Vol.809 (2), p.199-203
• Main Authors: SVENSSON, Sophie, OLIN, Anna-Carin, LÄRSTAD, Mona, LJUNGKVIST, Göran, TOREN, Kjell
• Format: Article
• Language: English
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Asthma - metabolism; Biological and medical sciences; Breath Tests; Case-Control Studies; Flow Injection Analysis - methods; Fundamental and applied biological sciences. Psychology; General pharmacology; Humans; Hydrogen Peroxide - analysis; Medical sciences; Pharmacology. Drug treatments; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Fluorescence - methods
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/S1570-0232(04)00513-6

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 microl) 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).