Development of a CD‐MEKC method for investigating the metabolism of tamoxifen by flavin‐containing monooxygenases and the inhibitory effects of methimazole, nicotine and DMXAA

Abstract

A selective and low-cost CD-MEKC method under acidic conditions was developed for investigating the N-oxygenation of tamoxifen (TAM) by flavin-containing monooxygenases (FMOs). The inhibitory effects of methimazole (MMI), nicotine and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on the given FMO reaction were also evaluated; 100 mM phosphate buffer (pH 8.6) was used for performing the enzymatic reaction and the separation of TAM and its metabolite tamoxifen N-oxide (TNO) was obtained with a BGE consisting of 100 mM phosphoric acid solution adjusted to pH 2.5 with triethanolamine containing 50 mM sodium taurodeoxycholate, 20 mM carboxymethyl β-CD and 20% ACN. The proposed method was applied for the kinetics study of FMO1 using TAM as a substrate probe. A Michaelis-Menten constant (K(m)) of 164.1 μM was estimated from the corrected peak area of the product, TNO. The calculated value of the maximum reaction velocity (V(max)) was 3.61 μmol/min/μmol FMO1; 50% inhibitory concentration and inhibition constant (K(i)) of MMI, the most common alternate substrate FMO inhibitor, were evaluated and the inhibitory effects of two other important FMO substrates, nicotine and DMXAA, a novel anti-tumour agent, were investigated.