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
F. Thies et Lh. Grimme, 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, Journal of chromatography, 684(1), 1994, pp. 168-173
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.