Fluorimetric analysis of hydrogen peroxide with automated measurement

In the pathophysiology of chronic obstructive pulmonary disease (COPD) oxidative stress plays an important role, which can be determined by measuring hydrogen peroxide. Hydrogen peroxide can be measured fluorimetrically in exhaled breath condensate (EBC), however, not standardized. The objective of this study was to investigate the sensitivity and reproducibility of measuring the hydrogen peroxide concentration in EBC of COPD patients using an automated flow injection device with varying flow rates and measurements.

10 microl p-hydroxyphenylacetic acid (1.0 mmol/l) and 10 microl horseradish peroxidase (15 mU/l) were manually added to several hydrogen peroxide containing solutions and EBC of patients suffering from COPD. The fluorescence of the reaction product was measured with an automated sampler, flow injection and scanning fluorescence detector, excitation wave 295 nm, emission wave 405 nm, at different flow rates. The degree of fluorescence was expressed as either the area under the curve or the peak value.

A flow rate of 1 ml/min gave the best results. There were no significant differences in calibration curves or detection limits using area under the curve or peak value (respectively 0.007 and 0.005 micromol/l) (flow rate 1.0 ml/min). The mean volume of EBC was 2.8 ml, the mean hydrogen peroxide concentration in the patient group was 0.2 micromol/l and the standard deviation of duplication 0.009 micromol/.

The low detection limit may be explained by using flow injection, because it measures the fluorescence over a period of time. It is important to choose an appropriate flow rate. There is no difference in the detection limit between measuring the fluorescence as area under the curve or as peak value.