4 anpp

Standard of acetylfentanyl (hydrochloride), acrylfentanyl (hydrochloride), alfentanil (hydrochloride), butyrylfentanyl, carfentanyl, (±)-cis-3-methylfentanyl (hydrochloride), (±)-cis-3-methylthiofentanyl (hydrochloride), fentanyl (hydrochloride), furanylfentanyl (hydrochloride), α-methylfentanyl (hydrochloride), α-methylthiofentanyl (hydrochloride), β-hydroxyfentanyl (hydrochloride), ortho-fluorofentanyl (hydrochloride), ocfentanyl, para-fluorofentanyl (hydrochloride), remifentanyl (hydrochloride), sufentanyl, 4-ANPP, norfentanyl, butyrylfentanyl carboxy metabolite, despropionylpara-fluorofentanyl, (±)-trans-3-methyl norfentanyl, methoxyacetyl norfentanyl (hydrochloride), valerylfentanyl carboxy metabolite, morphine, methadone, EDDP, 6-MAM, codeine, MT-45, fentanyl-d5 (hydrochloride) and nordentanyl-d5 were purchased from Cayman Chemical (Ann Arbor, MI, USA) in form of methanolic solution at the concentration of 1 mg mL⁻¹. Working solutions were prepared at the concentration of 1 μg mL⁻¹ and stored at −20 °C. Water, methanol, acetonitrile and 2-propanol were obtained from Carlo Erba (Milano, Italia) while sodium carbonate, sodium bicarbonate and ammonium formate were purchased from Sigma-Aldrich (Milwaukee, WI, USA).

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.