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3 protocols using naloxone d5

1

Certified Reference Standards for Drugs

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Certified reference standards of 6-monoacetylmorphine, alprazolam, amphetamine, buprenorphine, benzoylecgonine, carisoprodol, chlordiazepoxide, clonazepam, cocaine, codeine, cotinine, dihydrocodeine, diazepam, EDDP, fentanyl, flurazepam, hydrocodone, hydromorphone, lorazepam, MDA, MDEA, MDMA, methamphetamine, meperidine, morphine, methylphenidate, methadone, naloxone, norbuprenorphine, nicotine, nordiazepam, norfentanyl, noroxycodone, alpha-hydroxyalprazolam, oxazepam, oxycodone, oxymorphone, tapentadol, temazepam, ll-nor-9-Carboxy-Δ9-THC, and cis-tramadol were purchased, each at a concentration of 1 mg/mL, from Cerilliant Corporation (Round Rock, TX). Isotopically labeled internal standards (IS) morphine-D3, codeine- D6, hydrocodone-D3, 6-monoacetylmorphine-D6, hydromorphone-D6, oxymorphone-D3, noroxycodone-D3, and naloxone-D5 were purchased, each at a concentration of 100 μg/mL, from Cerilliant Corporation (Round Rock, TX). Drug-free urine was obtained from Bio-Rad Laboratories (Irvine, CA). High-performance liquid chromatography (HPLC) grade water, methanol, and formic acid were purchased from Fisher Scientific (Pittsburgh, PA) and ammonium formate was purchased from Sigma Aldrich (St. Louis, MO).
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2

Quantification of Buprenorphine, Metabolites, and Naloxone

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All collected samples were centrifuged within 1 h and stored at −20°C. The concentration of BUP, norBUP and NLX were quantified using a validated high-performance liquid chromatography-tandem mass spectroscopy (HPLC-MS) method. The mobile phase for BUP and norBUP detection was 80% methanol (0.005  mol/L ammonium format and 0.1% formic acid), and for NLX, the mobile phase was 36% acetonitrile (0.005  mol/L ammonium format and 0.1% formic acid). The internal standards for mass spectrometry were BUP-D4 (Lot No. 6-JHY-131-5, Toronto Research Chemicals), norBUP-D3 (Lot No. FE102710-01, Cerilliant) and naloxone-D5 (Lot No. FN081810-02, Cerilliant). The lower limit of quantification (LLOQ) of BUP and norBUP was 50 ng/L and that of NLX was 20  ng/L .
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3

Opioid Compound Dosing in Mice and Rats

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U50,488H (U50,488 methanesulfonate), morphine sulfate, β-Funaltrexamine (β-FNA), Norbinaltorphimine (norBNI) and heroin HCl was provided by the National Institute on Drug Abuse Drug Supply Program (Bethesda, MD). U50,488H and morphine sulfate were dissolved in water. NCP was synthesized as described previously (Wang et al., 2021) and dissolved in saline or deionized water at 0.3-0.5 mg/ml as a stock solution, aliquoted and frozen and before use diluted with saline or deionized water, respectively. In mice, compounds were injected s.c. or i.p. at 10 µl/g body weight. Solutions for rat IV administration (heroin HCl and NCP) were passed through a 0.22-micron sterile filter (Millex GV, Millipore Sigma, Burlington, MA) before injection. Naloxone-d 5 , a reference standard, was purchased from Cerilliant Corp (Round Rock, TX). Paraformaldehyde and compound 48/80 were purchased from Sigma-Aldrich (St. Louis, MO). Formalin (5%) were prepared with Paraformaldehyde (1.85%). Other commonly used chemicals were obtained from Sigma-Aldrich or ThermoFisher Scientific.
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