Untargeted screening of phase I metabolism of combretastatin A4 by multi-tool analysis

The aim of the current study was to apply different strategies for generation of metabolites of combretastatin A4 (CA4) and subsequent identification of the unknown products of phase I metabolism. CA4 is a potent anti-tubulin agent currently undergoing clinical trials. The multi-tool analytical approach was based on electrochemistry (EC), in silico predictions, and in vitro studies with the use of rat liver microsomes. With the latter, two different analytical sample preparation methods were applied: protein precipitation and solid phase microextraction, both hyphenated to the liquid chromatography-high-resolution mass spectrometry platform (LC-HRMS). The EC was coupled directly to HRMS. Conventional techniques using enzyme fractions pooled from human or animals remain a method of choice for determinations of phase I of drug metabolism, EC and in silico methods, which enable determinations of metabolism patterns, are in turn considered to have great potential as fast alternatives to in vitro assays. While individual findings attained via employment of these four methods showed high similarity in relation to generated metabolic pathways for CA4, each method was found to provide unique features not identified with other approaches. In this paper, these differences are reviewed in view of potential artifacts and true metabolite production via various metabolism patterns under different experimental conditions. In addition, the reliability, applicability, MS compatibility issues, and potential of each of these technologies are discussed.