Perhydrolysis of acetyl resorufin (AR) was reported previously to work as a fluorometric indicator reaction for glucose determination using only glucose oxidase. However, hydrolysis of AR in blank solution rendered the working concentration range of this method less than two orders of magnitude. To exclude or at least significantly reduce this interference, acyl groups and reaction conditions in the competition between perhydrolysis and hydrolysis of various acyl resorufins were assessed. Fluorometric evaluation of reactions in the presence or absence of H2O2 in phosphate buffer (pH 7.5, 100 mM)–CH3CN at 25 °C demonstrated that in tert-butylacetyl, isobutyryl, cyclohexanecarbonyl and pivaloyl resorufins (TBAR, IBR, CHR and PVR, respectively) among 10 acyl resorufins examined here, the competitive situation was shifted in a much more favorable way to perhydrolysis than in AR, although fluorometric responses due to their H2O2-dependent deacylation were suppressed in comparison with AR. Examination of the effects of pH, components and concentrations of buffers as well as reaction temperature established reaction conditions that not only allowed perhydrolysis of each of these four compounds to prevail over hydrolysis more effectively, but also improved the H2O2-based fluorometric responses. Thus, perhydrolysis of TBAR, IBR, CHR and PVR in phosphate buffer (pH 8.0, 20 mM)–CH3CN at 25 °C worked effectively as fluorometric indicator reactions for H2O2 analysis, affording a calibration curve over a concentration range of three orders of magnitude. Taking sensitivity, reproducibility and the response for blank solution into consideration, PVR seemed to be the best choice as a fluorochromogen for H2O2 determination under these conditions. For H2O2 analysis at lower pH, perhydrolysis of IBR in phosphate buffer (pH 7.5, 20 mM)–CH3CN was shown to effectively function as an indicator reaction. Applicability of the fluorometric methods with PVR and IBR to blood glucose determination was also discussed, comparing with Trinder's method with phenol, 4-aminoantipyrine and peroxidase (POD).
2002 The Pharmaceutical Society of Japan