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Over the last decade, the United States has experienced increasing numbers of overdose deaths attributed to opioid abuse. While the opioid crisis originated with prescription opioids, recent impacts are credited to synthetic novel psychoactive substances (NPS). Synthetic opioids, a category of NPS, can be divided into two categories: fentanyl- and non-fentanyl derivatives. Fentanyl analogs, the focus of this research, not only pose a significant threat to public safety, but also challenges to forensic laboratories due to their high potency, low concentrations, similar molecular structures, and progressive prevalence. To address detection issues faced by forensic toxicologists, it is necessary to develop highly sensitive analytical methods for detecting fentanyl analogs in traditional and alternative biological matrices.

The goals of this study were to 1) develop and validate a data-independent screening method for fentanyl analogs in whole blood and oral fluid for application to postmortem specimens and antemortem oral fluid collected from detainees, respectively; 2) develop and validate a quantitative method for furanyl fentanyl and its metabolites in human and rat plasma for future pharmacological assessment; 3) develop and validate a quantitative method for prevalent fentanyl analogs in whole blood and perform a long-term stability study; and 4) develop and validate a quantitative method for fentanyl analogs in oral fluid for application to antemortem oral fluid samples collected from probationers/parolees.

A data-independent screening method was developed and validated for fentanyl analogs (n=14) in whole blood and oral fluid using liquid chromatography-quadrupole-time-of-flight-mass spectrometry (LC-QTOF-MS). Data were acquired in time of flight (TOF) and All Ions fragmentation (AIF) modes and low limits of detection were achieved. A personal compound database and library (PCDL) was developed for targeted and exogenous compound identification. Postmortem blood samples (n=30) received from National Medical Services (NMS) Labs and oral fluid samples (n=20) collected from detainees in Texas detention centers were screened for fentanyl analogs. In the blood samples, analogs of furanylfentanyl (n=16), 4-ANPP (n=16), cis-3-methylfentanyl (n=4), fentanyl (n=4), norfentanyl (n=2), and valerylfentanyl (n=1) were detected. No fentanyl analogs were detected in the oral fluid samples.

A quantitative method was developed and validated for furanylfentanyl and its metabolites (4-ANPP and furanyl norfentanyl) in human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using American National Standards Institute/American Standards Board (ANSI/ASB) Standard 036: Standard Practices for Method Validation in Forensic Toxicology. Low limits of detection and small sample volumes (100 μL) were achieved. The method was cross validated in rat plasma for potential application to a pre-clinical pharmacodynamic/pharmacokinetic (PD/PK) study.

A method was developed and validated for the quantification of prevalent fentanyl analogs (n=13) in blood using targeted data acquisition on an LC-QTOF-MS. The method was validated according to ANSI/ASB Standard 036. The method was applied to a long-term stability study assessing fentanyl analog degradation over 9 months at four temperature conditions (-20⁰C, 4⁰C, 25⁰C, and 35⁰C). Results described minimal instability under room temperature and refrigerated storage, degradation after 4 freeze/thaw cycles, and instability after 1 week of elevated exposure. Acrylfentanyl had a high degree of instability under most temperature conditions and breakdown mechanism remains undetermined. Authentic forensic blood specimens stored under refrigeration were analyzed 6 months apart to assess stability in postmortem samples. Furanylfentanyl (n=4) and 4-ANPP (n=7) were quantifiable and exhibited percent loss of 0.2-26.8% and 16.3-37.4%, respectively. Loss was attributed to sample source, age, and composition.

The aforementioned data acquisition was utilized to develop and validate a quantification method for fentanyl analogs (n=13) in oral fluid using LC-QTOF-MS by ANSI/ASB Standard 036. The method was applied to authentic oral fluid samples (n=16) received from Redwood Toxicology obtained from probationers/parolees. Oral fluid samples were positive for fentanyl (n=16) and 4-ANPP (n=3) at concentrations of 1.0-104.5 ng/mL and 1.2-5.7 ng/mL, respectively. No fentanyl analogs were detected.

The present work describes sensitive analytical methods for the detection and quantification of fentanyl analogs with proven applicability to forensically relevant samples. In addition, challenges associated with analyte detection, compound differentiation, and drug instability have been addressed. With the constant emergence of novel fentanyl analogs, forensic toxicologists must be proactive with advancement of analytical analyses and sample treatment.



Chemistry, Analytical