IN-VIVO O-DEALKYLATION OF RESORUFIN AND COUMARIN ETHERS BY THE GREEN-ALGA CHLORELLA-FUSCA ANALYZED BY A RAPID AND SENSITIVE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ASSAY

The O-dealkylation of resorufin or coumarin ethers is known to be a cy tochrome P450-mediated reaction. It is used as a reference to characte rize the corresponding enzyme activity and its state of induceability. In adopting this approach, an in vivo O-dealkylase assay was develope d (a) using the unicellular green alga Chlorella as the biotransformin g system and (b) applying a rapid and sensitive high-performance liqui d chromatography (HPLC) assay which makes use of the wide pH stability of modified aluminium oxide packings. Separation was achieved isocrat ically at pH 9.8 using an Aluspher RP select B analytical column. The eluate was monitored by fluorescence detection with a detection limit of less than or equal to 3.5 pg for both resorufin and umbelliferone. The time needed for HPLC analysis is about 2 min, indicating that the method is suitable for automation.