6 mam

All the standards molecules (morphine 1 mg/mL in methanol and 6-MAM 1 mg/mL in acetonitrile) involved in this study were purchased from Sigma-Aldrich and stored at -20 °C. The Internal Standard (IS) SKF 525-A (Proadifen hydrochloride, analytical standard, > 95%, 100 mg) was purchased from Sigma-Aldrich as well.
Working solutions of each molecule and IS were prepared with methanol or acetonitrile at 100 µg/mL and 1 µg/mL, starting from standard solutions, and stored at -20 °C until use.
Solvents used in the extraction processes were purchased from Sigma-Aldrich (methanol) and from PanReac AppliChem ITW Reagents (phosphate buffer solution pH 4, phosphate buffer solution pH 9 and water for UV, HPLC, ACS).

Morphine, methadone, codeine, 6-MAM, EDDP standards, and Morphine-d3 and methadone-d3 internal standards (IS) were purchased from Sigma Aldrich (Milan, Italy). For the extraction procedure, distilled water (H₂O) for analysis, sodium hydroxide (NaOH), methanol (MeOH) for analysis (≥99.9%), hydrochloric acid for analysis (37%), dichloromethane for analysis stabilized with amylin (≥99.9%), 2-propanol (99.8%) and ammonium hydroxide (32%) of reagent grade were purchased from Carlo Erba (Cornaredo, Italy). For the UHPLC analysis, methanol for UHPLC-MS (99.99%), ultrapure water for UHPLC-MS and formic acid (FA) for LC-MS (≥99%) were purchased from Carlo Erba (Cornaredo, Italy). All reagents and laboratory consumables were stored according to the manufacturer’s instructions.