Methanol adducts leading to the identification of a reactive aldehyde metabolite of CPAQOP in human liver microsomes by ultra-high-performance liquid chromatography/mass spectrometry.

The incubation of compound AZX (1-[(2R)-2-[[4-[3-chloro-4-(2-pyridyloxy)anilino] quinazolin-5-yl]oxymethyl]-1-piperi-dyl]-2-hydroxy) with human liver microsomes generated several metabolites that highlighted the hydroxyacetamide side chain was a major site of metabolism for the molecule. The metabolites were derived predominantly from oxidative biotransformations, however two unexpected products were detected by LC-UV-MS and identified as methanol adducts. It became apparent that a metabolite formed in the microsomal incubation reacted with methanol in the mobile phase when no methanol adducts were detected in the analysis where acetonitrile was used. This observation prompted further investigations into the metabolic modification of the parent. Although this reactive metabolite could not be isolated or structurally characterised by LC-MS, several metabolic indications enabled the proposal of a reactive aldehyde. Experiments using methoxyamine post-incubation showed the disappearance of the two methanol adducts and appearance of a methoxyamine adduct, confirming the presence of an aldehyde group. The proposed structure of the reactive aldehyde derived from oxidation of the terminal hydroxyl group on the hydroxyacetamide side chain, leading to the formation of the diastereoisomeric methanol adducts detectable by LC-UV-MS.