4 Oliceridine respiratory effects the subjects were coached to hyperventilate for 2 to 3 min while breathing a hyperoxic gas mixture (FIO2 ≈1.0), followed by normal breathing for 30 s of the hyperoxic gas mixture, after which rebreathing from a 6-l balloon containing 7% carbon dioxide in 93% oxygen was initiated. The rebreathing period lasted for 3 to 4 min. We obtained eight responses, one before any drug administration, and 30 min, 1, 2, 3, 4, 5, and 6 h after drug infusion. The following breathto-breath data were collected: minute ventilation, end-tidal oxygen and carbon dioxide concentration, and oxygen saturation. Blood Sampling and Analysis At the following time points, 2 ml blood was drawn from the arterial line for determination of oliceridine or morphine and morphine-6-glucuronide concentrations: 0 (predose) 2, 5, 10, 15, 30, and 45 min, and 1, 1.5, 2, 3, 4, 5, and 6 h (postdose). Plasma samples were shipped to Labcorp Bioanalytical Services LLC, Indianapolis, Indiana, for analysis. Oliceridine plasma concentrations were quantified using a validated high-performance liquid chromatography with tandem mass spectrometry bioanalytical assay.7,13 Oliceridine and the internal standard TRV0110813A:2 (tri-deutero 13C-labeled oliceridine) were extracted from human plasma containing K2EDTA by supported-liquid extraction. The lower limit of quantitation for oliceridine in human plasma was 0.05 ng/ml, with linearity demonstrable up to 50 ng/ml (upper limit of quantitation), using a 50-µl sample volume. Mean coefficient of variation among the various analytical runs ranged from 5.9 to 7.1% with bias ranging from 0.5 to 5.5% and accuracy from 100.5 to 105.5%. Oliceridine metabolites were not measured because none of them are pharmacologically active. Morphine and morphine-6-glucuronide concentrations were determined by a validated high-performance liquid chromatography with tandem mass spectrometry method, after solid-phase extraction of morphine and internal standard morphine-d3 and morphine-6-glucuronide and internal standard morphine-6-D-glucuronide-d3 from human plasma containing K2EDTA. The lower limits of quantitation for morphine and morphine-6-glucuronide in human plasma were both 0.5 ng/ml, with linearity demonstrable up to 250 ng/ml (upper limit of quantitation), using a 50-µl sample volume. For morphine, the mean coefficient of variations among the analytical runs ranged from 5.2 to 7.5% with bias ranging from 0.5 to 3.2% and accuracy from 100.5 to 103.2%. For morphine-6-glucuronide, the mean coefficient of variations ranged from 5.2 to 6.8% with bias ranging from 0.5 to 5.6% and accuracy from 100.5 to 105.6%. The assay has not been published previously, but see Dahan et al.13 To determine the drug metabolizer status of the participants, one additional blood sample was drawn for determination of the CYP2D6 genotype. Genotyping was performed by the ISO15189-accredited laboratory of the 69
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