Determination of metabolic stability of positron emission tomography tracers by LC-MS/MS: an example in WAY-100635 and two analogues.

A method is presented for determination of microsomal metabolic stability of potential positron emission tomography (PET) tracers by LC-MS/MS in the lower nm range. The PET tracers used for the study were the serotonin receptor antagonist WAY-100635 and two potential tracer analogues. The sensitivity permitted the substrates to be directly collected from PET radiolabelling batches, containing very low amounts of substance (0.3-7 microg), for subsequent metabolic stability incubations. Sample preparation was minimal, with addition of internal standard, acetonitrile and a fast centrifugation step, as a result of the low protein concentration of the microsome solutions. Linearity (R2 > or = 0.99), precision (inter-assay R.S.D. < 7%) and accuracy (bias < or = 8%) for the tested concentration range 0.5-5 nM proved to be well within accepted limits. No significant differences in metabolic rates were detected using substrates from cold (non-labelling) chemistry syntheses and PET labelling batches, indicating the validity of using substrates from the latter source. A para-methoxy-benzamide analogue (MeO-WAY) displayed a significantly lower rate of metabolism compared to WAY-100635, whereas a para-iodo-benzamide analogue was more susceptible to metabolic transformation. LC-MS/MS Analysis of formed metabolites from WAY-100635 and MeO-WAY suggested similar metabolic pathways, with hydroxylation, demethylation and dearylation reactions. The main metabolic route in humans, amide hydrolysis, was not observed with the rat liver microsome assay.