Examination of acetone findings in suspected drug-facilitated sexual assaults: A case series.

Acetone presence in human biological specimens can result from exogenous administration or endogenous production, resulting from diabetes, dietary composition, alcoholism, and stress response. Victims of drug-facilitated sexual assaults (DFSA) are understood to experience enhanced stress. At the Harris County Institute of Forensic Sciences (HCIFS), DFSA drug testing includes analysis of volatile compounds, ethanol, methanol, isopropanol, and acetone, by headspace gas chromatography/flame ionization detection. The prevalence of acetone-positive specimens in DFSA casework has been observed to exceed that of other human performance case types. In this report, DFSA cases received between 2019 and 2021 (n = 393) were reviewed and 41 acetone-positive cases were detailed. Overall, nearly 11% of the DFSA cases had acetone-positive blood or urine specimens, where 3% identified acetone only, 6% identified acetone and other drug(s), and 2% identified acetone, ethanol, and other drug(s). Acetone concentrations ranged from 0.010 to 0.147 g/100 mL in urine. Other drugs such as nor-carboxy-Δ9 -tetrahydrocannabinol, amphetamine, methamphetamine, ethanol, and benzoylecgonine were commonly detected. Elevated stress response encountered during DFSAs may facilitate the mechanism behind enhanced acetone production leading to increased identification. Limited availability of victim medical history precludes understanding the contribution of other disease states or physiological conditions. Nonetheless, the identification of acetone in DFSA specimens supports its potential as a biomarker of trauma in forensic toxicology casework and warrants future research within the community.

Review of analytical methods for screening and quantification of fentanyl analogs and novel synthetic opioids in biological specimens.

Fentanyl, fentanyl analogs, and other novel synthetic opioids (NSO), including nitazene analogs, prevail in forensic toxicology casework. Analytical methods for identifying these drugs in biological specimens need to be robust, sensitive, and specific. Isomers, new analogs, and slight differences in structural modifications necessitate the use of high-resolution mass spectrometry (HRMS), especially as a non-targeted screening method designed to detect newly emerging drugs. Traditional forensic toxicology workflows, such as immunoassay and gas chromatography mass spectrometry (GC-MS), are generally not sensitive enough for detection of NSOs due to observed low (sub-µg/L) concentrations. For this review, the authors tabulated, reviewed, and summarized analytical methods from 2010-2022 for screening and quantification of fentanyl analogs and other NSOs in biological specimens using a variety of different instruments and sample preparation approaches. Limits of detection or quantification for 105 methods were included and compared to published standards and guidelines for suggested scope and sensitivity in forensic toxicology casework. Methods were summarized by instrument for screening and quantitative methods for fentanyl analogs and for nitazenes and other NSO. Toxicological testing for fentanyl analogs and NSOs is increasingly and most commonly being conducted using a variety of liquid chromatography mass spectrometry (LC-MS)-based techniques. Most of the recent analytical methods reviewed exhibited limits of detection well below 1 µg/L to detect low concentrations of increasingly potent drugs. In addition, it was observed that most newly developed methods are now using smaller sample volumes which is achievable due to the sensitivity increase gained by new technology and new instrumentation.

Quantification of fentanyl analogs in oral fluid using LC-QTOF-MS.

Oral fluid is a valuable alternative matrix for forensic toxicologists due to ease of observed collection, limited biohazardous exposure, and indications of recent drug use. Limited information is available for fentanyl analog prevalence, interpretation, or analysis in oral fluid. With increasing numbers of fentanyl-related driving under the influence of drug (DUID) cases appearing in the United States, the development of detection methods is critical. The purpose of the present study was to develop and validate a quantitative method for fentanyl analogs in oral fluid (collected via Quantisal™) using liquid chromatography-quadrupole-time-of-flight-mass spectrometry (LC-QTOF-MS). Validation resulted in limits of detection and quantification ranging from 0.5 to 1 ng/mL. Established linear range was 1-100 ng/mL for all analytes, except acetyl fentanyl at 0.5-100 ng/mL (R2  > 0.994). Within- and between-run precision and bias were considered acceptable with maximum values of ±15.2%CV and ±14.1%, respectively. Matrix effects exhibited ionization enhancement for all analytes with intensified enhancement at a low concentration (9.3-47.4%). No interferences or carryover was observed. Fentanyl analogs were stable in processed extracts stored in the autosampler (4° C) for 48h. The validated method was used to quantify fentanyl analogs in authentic oral fluid samples (n=17) from probationers/parolees. Fentanyl and 4-ANPP concentrations were 1.0-104.5 ng/mL and 1.2-5.7 ng/mL, respectively.

Long-Term Stability of 13 Fentanyl Analogs in Blood.

Fentanyl analogs continue to play a major role in proliferating the opioid epidemic in the USA. With high rates of overdose deaths, forensic laboratories experience backlogs, which may lead to false-negative results due to drug instability. To address this issue, a quantitative method was validated for fentanyl analogs (3-methylfentanyl, 4-anilino-N-phenethylpiperidine (4-ANPP), 4-fluoro-isobutyrylfentanyl (4-FIBF), acetylfentanyl, acrylfentanyl, butyrylfentanyl, carfentanil, cyclopropylfentanyl, fentanyl, furanylfentanyl, methoxyacetylfentanyl, p-fluorofentanyl and valerylfentanyl) in blood using liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) and used to assess long-term stability under various temperature conditions (-20°C, 4°C, ∼25°C and 35°C) for 9 months. Authentic specimens were also analyzed 6 months apart for applicability to postmortem blood. Method validation resulted in calibration ranges of 1-100 ng/mL and limits of detection of 0.5 ng/mL. Precision and bias were acceptable (within ±7.2% coefficient of variation (CV) and ±15.2%, respectively). Matrix effects exhibited ion enhancement for all analytes, except carfentanil and 4-ANPP in low-quality control (>25%). For long-term stability, fentanyl analogs (except acrylfentanyl) remained stable under room temperature and refrigerated conditions at low and high concentrations (81.3-112.5% target) for 9 months. While most fentanyl analogs remained stable frozen, degradation was observed after 2 weeks (four freeze/thaw cycles). At elevated temperatures, most analytes were stable for 1 week (74.2-112.6% target). Acrylfentanyl was unstable after 24 h under elevated (70% loss) and room temperatures (53-60% loss), 48-72 h when refrigerated (28-40% loss) and 4 weeks when frozen (22% loss). In authentic bloods (n = 7), initial furanylfentanyl (FuF) and 4-ANPP concentrations were 1.1-3.6 and 1.4-6.4 ng/mL, respectively. Percentage loss of FuF and 4-ANPP over 6 months were 16.3-37.4% and 0.2-26.8%, respectively. Samples suspected to contain fentanyl analogs are recommended to be stored refrigerated or frozen with limited freeze/thaw cycles. Due to instability, in the event of an acrylfentanyl overdose, samples should be analyzed immediately or stored frozen with analysis within 1 month.

Quantification of Furanylfentanyl and its Metabolites in Human and Rat Plasma Using LC-MS-MS.

Fentanyl analogs (novel and traditional) continue to impact the ever-growing opioid epidemic. Furanylfentanyl (FuF) is one analog equipotent to fentanyl that has documented involvement in thousands of intoxication and fatality cases around the world. Due to its prevalence, toxicologists need to improve detection and understanding of this analog. A method for the quantification of FuF and its metabolites (4-ANPP, furanyl norfentanyl (FuNorF)) in a small volume (100 µL) of human plasma by LC-MS-MS was developed and validated according to ANSI/ASB Standard. The method was cross validated in rat plasma for a future pharmacokinetic (PK)/pharmacodynamic (PD) study. In human plasma, calibration ranges were 0.025-25 ng/mL (FuF and 4-ANPP) and 0.5-25 ng/mL (FuNorF). Limits of detection were 0.0125 ng/mL (FuF and 4-ANPP) and 0.25 ng/mL (FuNorF). Lower limits of quantification coincided with lowest calibrator concentrations of 0.025 ng/mL (FuF and 4-ANPP) and 0.5 ng/mL (FuNorF). Precision and bias values were determined to be acceptable for all analytes. Matrix effects were acceptable for all analytes (-8.6-25.0%), except FuNorF with suppression >25%. Extraction recoveries ranged from 84.5 to 98.1%. No carryover or endogenous interferences were observed. Qualitative interferences with 4-ANPP were observed from some n-acyl substituted fentanyl analogs predicted to be low-concentration standard impurities. Analytes were stable under all conditions and dilution integrity was sustained. The method was successfully cross validated in rat plasma with acceptable bias (-7.4-8.4%), precision (within-run < 19%CV and between-run < 12.6%CV), matrix effects (-9.3-17.2%, except FuNorF with >25% suppression), recoveries (79.2-94.5%) and dilution integrity (1/2 and 1/10).

Oral Fluid and Drug Impairment: Pairing Toxicology with Drug Recognition Expert Observations.

According to the Governors Highway Safety Association, drugs are detected more frequently in fatally injured drivers than alcohol. Due to the variety of drugs (prescribed and/or illicit) and their various physiological effects on the body, it is difficult for law enforcement to detect/prosecute drug impairment. While blood and urine are typical biological specimens used to test for drugs, oral fluid is an attractive alternative matrix. Drugs are incorporated into oral fluid by oral contamination (chewing or smoking) or from the bloodstream. Oral fluid is non-invasive and easy to collect without the need for a trained professional to obtain the sample, unlike urine or blood. This study analyzes paired oral fluid and urine with drug recognition expert (DRE) observations. Authentic oral fluid samples (n = 20) were collected via Quantisal™ devices from arrestees under an institutional review board-approved protocol. Urine samples (n = 18) were collected with EZ-SCREEN® cups that presumptively screened for Δ9-tetrahydrocannabinol (cannabinoids), opiates, methamphetamine, cocaine, methadone, phencyclidine, amphetamine, benzodiazepines and oxycodone. Impairment observations (n = 18) were recorded from officers undergoing DRE certification. Oral fluid samples were screened using an Agilent Technologies 1290 Infinity liquid chromatograph (LC) coupled to an Agilent Technologies 6530 Accurate Mass Time-of-Flight mass spectrometer (MS). Personal compound and database libraries were produced in-house containing 64 drugs of abuse. An Agilent 1290 Infinity LC system equipped with an Agilent 6470 Triple Quadrupole MS was used for quantification of buprenorphine, heroin markers (6-acetylmorphine, morphine) and synthetic opioids. Subjects were 23-54 years old; 11 (55%) were male and 9 (45%) were female. Evaluator opinion of drug class was confirmed in oral fluid 90% of time and in urine 85% of the time in reference to scope of testing by the LC-MS methods employed (excludes cannabis and central nervous system depressants). Data indicate that oral fluid may be a viable source for confirming driving under the influence of drugs.

Data-independent screening method for 14 fentanyl analogs in whole blood and oral fluid using LC-QTOF-MS.

Recently, fentanyl analogs account for significant number of opioid deaths in the United States. Routine forensic analyses are often unable to detect and differentiate these analogs due to low concentrations and presence of structural isomers. A data-independent screening method for 14 fentanyl analogs in whole blood and oral fluid was developed and validated using liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). Data were acquired using Time of Flight (TOF) and All Ions Fragmentation (AIF) modes. The limits of detection (LOD) in blood were 0.1-1.0 ng/mL and 0.1-1.0 ng/mL in TOF and AIF modes, respectively. In oral fluid, the LODs were 0.25 ng/mL and 0.25-2.5 ng/mL in TOF and AIF modes, respectively. Matrix effects in blood were acceptable for most analytes (1-14.4%), while the nor-metabolites exhibited ion suppression >25%. Matrix effects in oral fluid were -11.7 to 13.3%. Stability was assessed after 24 h in the autosampler (4 °C) and refrigerator (4 °C). Processed blood and oral fluid samples were considered stable with -14.6 to 4.6% and -10.1 to 2.3% bias, respectively. For refrigerated stability, bias was -23.3 to 8.2% (blood) and -20.1 to 20.0% (oral fluid). Remifentanil exhibited >20% loss in both matrices. For proof of applicability, postmortem blood (n = 30) and oral fluid samples (n = 20) were analyzed. As a result, six fentanyl analogs were detected in the blood samples with furanyl fentanyl and 4-ANPP being the most prevalent. No fentanyl analogs were detected in the oral fluid samples. This study presents a validated screening technique for fentanyl analogs in whole blood and oral fluid using LC-QTOF-MS with low limits of detection.