Alpha-methylfentanyl and beta-hydroxyfentanyl LC–MS-MS quantification in rat plasma after long-term ethanol exposure

Fentanyl and its analogues are highly abused drugs that dominate the illicit drug trade. alphaMethylfentanyl (A-F) and beta-hydroxyfentanyl (B-F) are two fentanyl analogues that require the development of rapid detection technologies. The current study established and validated a rapid and high-sensitivity liquid chromatography–tandem mass spectrometry (LC–MS-MS) method to measure A-F and B-F concentrations in rat plasma following intravenous drug administration (20 µg/kg). Because fentanyl is primarily metabolized by the liver, we evaluated the concentrations of A-F and B-F in vivo in rats, in a control group and a group with liver damage induced by 55 days of oral ethanol gavage (6.5 g/kg, 22.5% v/v). Liquid–liquid extraction and LC–MS-MS operating in the positive ion multiple reaction monitoring mode were used. A C₁₈ column was used, and the mobile phase consisted of 0.1% formic acid aqueous and acetonitrile. The limit of detection was 3 pg/mL (S/N > 5) for A-F and B-F. The calibration curves were linear within the concentration range of 0.01–5 ng/mL (R² = 0.9991) and 0.005–20 ng/mL (R² = 0.9999) for A-F and B-F, respectively. Extraction recoveries were 91.3%–97.6% with RSD ≤11.2% and 90.5%–94.3% with RSD ≤10.5% for A-F and B-F, respectively. Plasma matrix effects were 80.61%–84.58% for A-F and 80.67%–81.33% for B-F with RSD ≤13.9%. The validated assay indicated no significant differences in pharmacokinetic parameters (AUC_0-t, Cmax and T_1/2) derived from the assessment of A-F and B-F plasma concentrations between control and ethanol-exposed rats. This assay, for which the LOD was 3 pg/mL for A-F and B-F may help the forensic science field to determine fentanyl analogue-related causes of death and identify illicit drug tampering.