Carfentanil

For use in in vitro assays, drug stocks of DAMGO and fentanyl (Sigma‐Aldrich, Gillingham, Dorset, UK), morphine (MacFarlan Smith, Edinburgh, UK), alfentanil hydrochloride, sufentanil (Cayman Chemical, Ann Arbor, Michigan, USA) and β‐FNA (Tocris, Bristol, UK) were each dissolved in sterile de‐ionised water and stored at −20°C. Carfentanil (Cayman Chemical) was dissolved in a small amount of 100% DMSO before dilution in water. For cell signalling assays, drug stocks were further dissolved in phenol red‐free DMEM on the day of experiments. For electrophysiological experiments, all compounds or drugs were purchased from Sigma‐Aldrich except naloxone and Compound 101 (Hello Bio, Bristol, UK) and prazosin (Tocris). Drug stocks were prepared in de‐ionised water (or 100% DMSO for Compound 101) at 1000× the desired assay concentration and stored at −20°C. Drugs were diluted into the superfusing aCSF solution at 1:1000 ratio at the time of the experiment.

Working standards (acetylfentanyl, acetyl norfentanyl, alfentanil, butyrylfentanyl, butyrylfentanyl carboxy metabolite, butyryl norfentanyl, carfentanil, cyclopropylfentanyl, cyclopropyl norfentanyl, 4-ANPP, despropionyl para-fluorofentanyl, fentanyl, furanylfentanyl, furanyl norfentanyl, furanylethyl fentanyl, β-hydroxyfentanyl, β-hydroxythiofentanyl, methoxyacetylfentanyl, methoxyacetyl norfentanyl, norfentanyl, phenylacetyl fentanyl, sufentanil, and valerylfentanyl carboxy metabolite) and deuterated internal standards (IS; acetyl norfentanyl-D5 and fentanyl-D5) were purchased from Cayman Chemical (Ann Arbor, MI, USA) and stored at −20°C until use. LC-MS grade water, acetonitrile, methanol, and formic acid and LC grade acetone and dichloromethane were obtained from Sigma-Aldrich® (Milano, Italy). Ammonium acetate buffer was prepared with ≥97% purity ammonium acetate salt (Sigma-Aldrich®) dissolved in LC-MS water. VMA-T M3®, Washing Solution, and Multimatrix Eluent were acquired from Comedical® s.r.l. (Trento, Italy); their composition is not disclosed.