Doses of ketamine and psilocybin selected for PK evaluation were determined based on outcomes from the overt behavioral experiments and selected to include 1) dose(s) sub-threshold for eliciting behavioral signs, and 2) dose(s) that reliably induced ketamine therapy such signs. A total of 35 male Long Evans rats were used for this study.

Fifteen rats were randomly allocated to three groups (N = 5/group) and treated with ketamine at doses of 0.3, three or 30 mg/kg IP. At 0.25, 0.5, 0.75, 1, 1.5, 2, 3 h post treatment, ∼100 μL whole blood was collected via saphenous vein bleed into K2EDTA capillary tubes.

Twenty rats were allocated to four groups (N = 5/group) and treated with psilocybin at doses of 0.05, 0.1, one or 10 mg/kg SC. At 0.25, 0.5, 0.75, 1, 2, 4, and 6 h post-treatment, ∼100 μL whole blood was collected via saphenous vein bleed into K2EDTA capillary tubes.

All blood samples were kept on wet ice for a maximum of 5 min prior to centrifugation (3,300 rpm at 4°C, 5 min). Plasma was extracted and immediately frozen at −20°C before transfer to −80°C, where stored until shipment to the InterVivo Bioanalytical facility. for the determination of plasma concentrations of ketamine (ketamine study) or psilocybin and its primary metabolite psilocin.

Quantification of Ketamine, Psilocybin, and Psilocin by LC-MS/MS
Plasma samples were analyzed by an AB Sciex API4000 QTRAP liquid chromatography/mass spectrometric system equipped with an ESI source in positive ion mode and coupled to an Agilent 1,200 liquid chromatographic system.

Ketamine hydrochloride, ketamine-d4 hydrochloride, psilocin, psilocybin, psilocin-d10 and procainamide were purchased from Sigma-Aldrich.

For ketamine quantification, 50/50 methanol/acetonitrile containing internal standard (IS), ketamine-d4 was added to plasma sample (5 µL) to precipitate proteins.

The supernatant was diluted with mobile phase for injection prior to analysis. Chromatographic separation was performed on a Zorbax XDB-C18 column (2.1 × 30 mm, 3.5 µm) at 25°C using gradient elution (mobile phase: 10 mM ammonium formate, pH 3.0 (A) and 95% methanol/5% 10 mM ammonium formate, pH 3.0 (B)).

The multiple reaction monitoring (MRM) parameters for ketamine and IS were m/z 238.1 to 125.1 (quantitative)/220.2 (qualitative) and m/z 242.1 to 129.1, respectively. The calibration dynamic range was 0.5–5,000 ng/ml.

For psilocybin and psilocin quantification, 50/50 methanol/acetonitrile containing IS (procainamide and psilocin-d10 for psilocybin and psilocin, respectively) was added to plasma (20 µL). The supernatant was diluted with 0.1% formic acid in water prior to analysis.

Two serially connected Javelin Aquasil C18 columns (2.1 × 20 mm, 5 µm each) at 25°C were used with gradient elution (mobile phase: 0.1% formic ketamine therapy  acid in water (A) and 0.1% formic acid in methanol (B)).

The MRM parameters for psilocybin, psilocin, psilocin-d10 and procainamide were m/z 285.09 to 58.2, m/z 205.2 to 58.2, m/z 215.2 to 66.2 and m/z 236.1 to 163.0, respectively.

The calibration range was 0.5–200 ng/ml for psilocybin and 0.1–200 ng/ml for psilocin.