Ketamine d4

ketamine dosing solution was purchased from Putney, Inc. (Portland, MA) and either used as directed or aseptically diluted using sterile saline to a final concentration of 25 mg/mL. The concentration of the ketamine dosing formulations was verified using LC-MS as described below. Atropine was purchased from Sigma Aldrich (St. Louis, MO) and prepared at a concentration of 15 mg/mL in sterile saline. The pH of the vehicle was acidified with dilute hydrochloric acid to increase solubility before being neutralized to pH 7.2 with dilute sodium hydroxide. The 2-PAM used was purchased from Sigma Aldrich (St. Louis, MO) and prepared at 50 mg/mL in sterile saline and sterile filtered. Midazolam (5 mg/mL) was purchased from Hospira (Lake Forest, IL) and used without modification.
The analytical reference standards for ketamine, norketamine, ketamine-d4, and norketamine-d4 in methanol were obtained from Cerilliant (Round Rock, TX). Multiple lots of K₃EDTA rat plasma and brains were obtained from Bioreclamation (Westbury, NY). Purified water was collected from an in-house Labconco (Kansas City, MO) Water Pro Plus system. Glacial acetic acid was obtained from JT Baker (Center Valley, PA). Ammonium hydroxide was obtained from Sigma Aldrich (St. Louis, MO). LC-MS-grade water and acetonitrile were obtained from EMD (Billerica, MA). Formic acid was obtained from Thermo Scientific (Waltham, MA).