title: Determination of metabolic stability of positron emission tomography tracers by LC-MS/MS: an example in WAY-100635 and two analogues.
abstract: 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.
authors.affiliation: Department of Analytical Chemistry, Institute of Chemistry, Uppsala University, Box 599, 751 24 Uppsala, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Lavén
authors.forename: O
authors.initials: O
authors.lastname: Itsenko
authors.forename: K
authors.initials: K
authors.lastname: Markides
authors.forename: B
authors.initials: B
authors.lastname: Långström
mesh_terms: D000818:Animals; D001711:Biotransformation; D002250:Carbon Radioisotopes; D002853:Chromatography, Liquid / Q000379:methods*; D004355:Drug Stability; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000378:metabolism*; D010879:Piperazines / Q000737:chemistry / Q000378:metabolism*; D049268:Positron-Emission Tomography / Q000379:methods; D011725:Pyridines / Q000737:chemistry / Q000378:metabolism*; D011849:Radioactive Tracers; D019275:Radiopharmaceuticals / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results; D012702:Serotonin Antagonists / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002250:Carbon Radioisotopes; D010879:Piperazines; D011725:Pyridines; D011849:Radioactive Tracers; D019275:Radiopharmaceuticals; D012702:Serotonin Antagonists; C090413:N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide