Pharmacokinetic assessment

During each dose period, blood samples were taken from each subject before dosing and again at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 15, 24, 36, 48, and 72 hours after dosing. Samples were stored at or below −15°C until analysis. Bioanalytical assay of riociguat and its metabolite M1 in plasma was performed using a fully validated high-performance liquid chromatography–tandem mass spectrometry assay using [methoxycarbonyl-²H₃]-riociguat and [²H₃]M1 as internal standards. Quality control and calibration samples were analyzed concurrently with study samples. In study 1, the calibration range was 0.5–250 μg/L for both riociguat and M1. Quality control samples in the concentration range 1.5–200 μg/L were determined with an accuracy of 97.0%–98.3% for riociguat and 92.9%–99.4% for M1, with a precision of 4.3%–6.0% for riociguat and 4.6%–6.5% for M1. In study 2, the calibration range was 2.0–500 μg/L for riociguat and M1. Quality control samples in the concentration range 6–400 μg/L were determined with an accuracy of 92.0%–96.8% for riociguat and 93.2%–100% for M1, with a precision of 4.6%–5.3% for riociguat and 3.4%–4.6% for M1. Concentrations were calculated from the chromatographic raw data, and only values above the lower limit of quantitation were used to determine pharmacokinetic parameters. The bioanalyst was not blinded during analysis of study samples. Pharmacokinetic parameters were calculated using the model-independent (compartment-free) method and WinNonlin software (ver. 4.1; Certara, Princeton, NJ).