6 × 250 mm, 5 μm; Phenomenex, Aschaffenburg, Germany), a LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), and a FC204 fraction collector (Gilson Inc., Middleton, WI, USA). The system was operated by Xcalibur 2.1 software (Thermo Fisher Scientific, Waltham, MA, USA). The post-column flow was split at a ratio of 1:5 between the mass spectrometer and the fraction collector. Fractions were sampled into 96-well plates, which were preconditioned with solid-phase scintillation material (Deepwell Luma Plates; PerkinElmer Life and Analytical Sciences, Shelton, CT, USA). The fraction collection interval was 0.15 min. After evaporation to dryness, the plates were analyzed by scintillation
counting using a microplate counter (TopCount NXT; Perkin Elmer Life and Analytical Sciences, Waltham, MA, USA). Radiochromatograms were reconstructed by conversion Tideglusib of raw data (counts per fraction vs. fraction number) into chromatographic data (counts per fraction vs. retention time) and processed by the Laura 4.0.3 (LabLogic Systems Limited, Sheffield, South Yorkshire, UK) software. Chromatographic peaks in the reconstructed radiochromatograms were manually integrated. Metabolites were quantified by calculating the percentage of each integrated radiopeak relative to the sum of all peaks in the radiochromatogram. The mass spectrometer was operated to collect full scan
and MS n data simultaneously if a predefined ion exceeded an intensity threshold. A radiochromatogram of each SHP099 chemical structure sample was reconstructed. All major radiopeaks were assigned and quantified considering an average background of 1 count per minute (CPM). The radiopeak areas were determined in CPM. Only radiopeaks with a signal-to-noise ratio >3 were EPZ5676 ic50 considered detectable and only radiopeaks with signal height of 9
enough CPM and above were accepted as quantifiable. Co-eluting metabolites were quantified together. Each quantifiable radiopeak area expressed in percent relative to the total radiochromatogram area was transformed into disintegrations per minute (DPM)/mL or DPM/g. The transformation was done considering the total DPM/mL or DPM/g value of each sample pool. The ng eq/mL values were determined for each quantifiable plasma metabolite. The transformation of the DPM/mL value into the ng eq/mL was carried out considering the specific activity (DPM/ng) of the radiolabeled parent compound [14C]setipiprant. 2.10 Structure Elucidation of Metabolites Structure assignments of the major metabolites were carried out by HPLC/MS n with an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), operated at a resolving power of 15,000 or higher. The capillary temperature of the instrument was kept at 275 °C and spectra in positive and negative ion mode were collected. The mass accuracy of the instrument was better than 2 ppm and unequivocally allowed the determination of the sum formula of the metabolites.