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Positron emission tomography (PET) is a sensitive non-invasive imaging technique. To reduce imaging measurements of defects, there is a demand for proper LC-ESI-MS/MS method to carry out with its specificity and sensitivity. This study describes a rapid and simple liquid chromatography electrospray ionization tandem-MS/MS (LC-ESI-MS/MS) method for determination of both PET tracers: N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]- N-(2-pyridyl)cyclohexanecarboxamide (WAY-100635) and 4-fluoro- N-[2-[4-(2-methoxylphenyl)-1-piperazino]ethyl]- N-(2-pyridyl)cyclohexanecarboxamide (FCWAY). Both target compounds were prepared by one-step protein precipitation with acetonitrile and methanol (1:1, v/v), and analyzed using a C column. This simple method has an excellent linearity, selectivity and sensitivity. Precision and accuracy values for the intra-day and inter-day validation were below 12 %. The limit of quantification (LOQ) for both target compounds was defined as 1 ng/mL in plasma and 5 ng/mL in brain homogenate. The stability of both compounds is considered stable under a various experimental conditions. The in vitro MDR-MDCK cell permeability showed the both compounds have high permeability (P ≥ 20 × 10cm/s) and low efflux ratio (≤2.0). Brain to blood (AUC/AUC) distribution ratios in rats were 3.15 ± 0.42 for WAY-100635 and 2.20 ± 0.34 for FCWAY, respectively, and these results suggest that LC-ESI-MS/MS method might be a supplementary way for the identifying and understanding of radiopharmaceuticals. [ABSTRACT FROM AUTHOR]

Abstract: Liquid chromatography/mass spectrometry (LC/MS), combined with commercially available hepatocytes, has become an indispensable tool in evaluating the presence of these metabolites in target tissues, especially in the brain. Results from in vitro metabolism studies using hepatocytes from different species can demonstrate species differences. Using these techniques, we evaluated the metabolic profile of 3-cis-FCWAY and 3-trans-FCWAY in rat, monkey and human hepatocytes. Hepatocytes were used to produce metabolites in vitro, and liquid chromatography/tandem mass spectrometry was used to identify these metabolites. We found that the metabolic profiles of rat, monkey and human hepatocytes differ dramatically. In rats, aromatic ring oxidation was the major metabolic pathway for both 3-cis-FCWAY and 3-trans-FCWAY; 3-trans-FCWAY had more metabolites (cyclohexane ring oxidation) than 3-cis-FCWAY. In humans, hydrolysis of amide linkage was the major metabolic pathway. In monkeys, both pathways (oxidation and amide hydrolysis) were found in the metabolites. We also found that 3-cis-FCWAY had the slowest defluorination of FCWAY analogues in all species. [Copyright &y& Elsevier]

In vivo imaging using positron emission tomography (PET) is important in the development of new radiopharmaceuticals in rodent animal models for use as biochemical probes, diagnostic agents, or in drug development. We have shown mathematically that, if small animal imaging studies in rodents are to have the same “quality” as human PET studies, the same number of coincidence events must be detected from a typical rodent imaging “voxel” as from the human imaging voxel. To achieve this using the same specific activity preparation, we show that roughly the same total amount of radiopharmaceutical must be given to a rodent as to a human subject. At high specific activities, the mass associated with human doses, when administered to a rodent, may not decrease the uptake of radioactivity at non saturable sites or sites where an enzyme has a high capacity for a substrate. However, in the case of binding sites of low density such as receptors, the increased mass injected could saturate the receptor and lead to physiologic effects and non-linear kinetics. Because of the importance of the mass injected for small animal PET imaging, we experimentally compared high and low mass preparations using ex vivo biodistribution and phosphorimaging of three compounds: 2-fluoro-2-deoxyglucose (FDG), 6-fluoro-L-metatyrosine (FMT) and one receptor-directed compound, the serotonin 5HT1A receptor ligand, trans-4-fluoro-N-{2-[4-(2-methoxylphenyl) piperazino]ethyl}-N-(2-pyridyl) cyclohexane- carboxamide (FCWAY). Changes in the mass injected per rat did not affect the distribution of FDG, FMT, and FCWAY in the range of 0.6–1.9 nmol per rat. Changes in the target to nontarget ratio were observed for injected masses of FCWAY in the range of ∼5–50 nmol per rat. If the specific activity of such compounds and/or the sensitivity of small animal scanners are not increased relative to human studies, small animal PET imaging will not correctly portray the “true” tracer distribution. These difficulties will only be exacerbated in animals smaller than the rat, e.g., mice. [Copyright &y& Elsevier]

Radiolabeled FCWAY (N-{2-[4-(2-methoxyphenyl)piperazino]}-N-(2-pyridinyl) trans-4-fluorocyclohexanecarboxamide) was prepared for human positron emission tomography (PET) studies by a simple one-step radiosynthesis. The LC–MS analysis of the products indicated that it contained impurities which may interfere with FCWAY uptake of 5-HT1A receptors and that these impurities were derived from an impurity originally present in the precursor preparation. Since preparative HPLC failed to resolve one of the impurities from the precursor, preparative-scale high-speed counter-current chromatography (HSCCC) was used for purification of this FCWAY precursor. A suitable two-phase solvent system composed of cyclohexane–ethyl acetate–methanol–water at a volume ratio of 1:1:1:1 or 4:5:4:5 was selected based on the partition coefficients of the precursor and impurity as determined by a LC–MS method. Using the second solvent ratio of 4:5:4:5 with the organic phase as a mobile phase, a 2.57 g amount of precursor preparation was successfully purified yielding 2.2 g of the pure precursor by a single run. [Copyright &y& Elsevier]

Liquid chromatography in combination with mass spectrometry (LC-MS) is key to the identification of metabolites in new molecular imaging probes. These metabolites can be generated using hepatocytes from various species in vitro. Metabolites produced from 4-fluoro-N-{2-[4-(2-methoxylphenyl)piperazino]ethyl}-N-(2-pyridyl)cyclohexane carboxamide (FCWAY) using rat and human hepatocytes are different, whereas metabolites from (3-(3-fluoropropylthio)-1,2,5-thiadiazol-4-yl)-tetrahydro-l-methyl-pyridine (FP-TZTP) using rat and human hepatocytes were similar. The combination of LC-MS and hepatocytes are an indispensable tool in the development of new molecular imaging probes. [Copyright &y& Elsevier]