Optimising hydrogen peroxide measurement in exhaled breath condensate

BACKGROUND: Exhaled breath condensate (EBC) analysis has been proposed as a non-invasive method of assessing airway pathology. A number of substances, including hydrogen peroxide (H2O2), have been measured in EBC, without adequate published details of validation and optimisation. OBJECTIVES: To explore factors that affect accurate quantitation of H2O2 in EBC. MATERIALS AND METHODS: H2O2 was measured in EBC samples using fluorometry with 4-hydroxyphenylacetic acid. A number of factors that might alter quantitation were studied including pH and buffering conditions, reagent storage, and assay temperature. RESULTS: Standard curve slope was significantly altered by pH, leading to a potential difference in H2O2 quantification of up to 42%. These differences were resolved by increasing the buffering capacity of the reaction mix. H2O2 added to EBC remained stable for 1 h when stored on ice. The assay was unaffected by freezing assay reagents. The limit of detection for H2O2 ranged from 3.4 nM to 8.8 nM depending on the buffer used. CONCLUSIONS: The reagents required for this assay can be stored for several months allowing valuable consistency in longitudinal studies. The quantitation of H2O2 in EBC is pH-dependent but increasing assay buffering reduces this effect. Sensitive reproducible quantitation of H2O2 in EBC requires rigorous optimisation.