In July of 2016, carfentanil (CF) emerged in Northeast Ohio resulting in over 25 deaths within a 30-day period. A total of 125 deaths have occurred in Summit County and Cuyahoga County has reported 40 deaths, relating to the presence of CF either alone, or in combinations with heroin and fentanyl. Prior to this surge in CF cases, positive fentanyl enzyme-linked immunosorbent assay (ELISA) screening results were increasing in number. Many were negative for fentanyl confirmation by gas chromatography-mass spectrometry. Fentanyl analogs such as CF, acetyl fentanyl (AF), 2-furanyl fentanyl (2-Fu-F) and 3-methylfentanyl (3-MF) may be present in these cases. Some fentanyl analogs like CF and 3-MF do not cross-react with the Immunalysis ELISA fentanyl assay. With the emergence of potent synthetic fentanyl analogs, questions arose as to how to interpret their very low concentrations or absence in the blood in relation to cause of death. Driving under the influence of drugs (DUID) blood specimens had also tested positive for CF by reference laboratories. A liquid chromatography-tandem mass spectrometry method was developed to identify and quantify fentanyl, norfentanyl (NF) and four analogs: AF, 2-Fu-F, 3-MF and CF. The method has been utilized to quantify these fentanyl analogs in blood and vitreous humor in authentic antemortem and postmortem cases. Calibration curves were established between 0.10-4.0 ng/mL (NF, AF, 3-MF, 2-Fu-F and CF) and 1.0-40 ng/mL for fentanyl. In total, 98 postmortem cases analyzed produced the following blood concentration ranges: CF (0.11-0.88 ng/mL), 3-MF (0.15-1.7 ng/mL), 2-Fu-F (0.15-0.30 ng/mL), AF (0.14-0.16 ng/mL), fentanyl (1.1-15 ng/mL) and NF (0.10-3.7 ng/mL). Only CF, fentanyl and NF were detected in a statistically significant subset DUID population of 26 cases producing concentration ranges between 0.11 and 0.47 ng/mL, 1.0 and 9.8 ng/mL, and 0.11 and 3.5 ng/mL, respectively.

An analytical method for the detection of 40 benzodiazepines, (±)-zopiclone, zaleplon and zolpidem in blood and urine by solid-phase extraction liquid chromatography–tandem mass spectrometry was developed and validated. Twenty-nine of 43 analytes were quantified in 0.5 mL whole blood for investigating postmortem, drug-facilitated sexual assault (DFSA) and driving under the influence of drugs cases (DUID). The four different dynamic ranges of the seven-point, linear, 1/x weighted calibration curves with lower limits of quantification of 2, 5, 10 and 20 μg/L across the analytes encompassed the majority of our casework encountered in postmortem, DFSA and DUID samples. Reference materials were available for all analytes except α-hydroxyflualprazolam, a hydroxylated metabolite of flualprazolam. The fragmentation of α-hydroxyflualprazolam was predicted from the fragmentation pattern of α-hydroxyalprazolam, and the appropriate transitions were added to the method to enable monitoring for this analyte. Urine samples were hydrolyzed at 55°C for 30 min with a genetically modified β-glucuronidase enzyme, which resulted in >95% efficiency measured by oxazepam glucuronide. Extensive sample preparation included combining osmotic lysing and protein precipitation with methanol/acetonitrile mixture followed by freezing and centrifugation resulted in exceptionally high signal-to-noise ratios. Bias and between-and within-day imprecision for quality controls (QCs) were all within ±15%, except for clonazolam and etizolam that were within ±20%. All 29 of the 43 analytes tested for QC performance met quantitative reporting criteria within the dynamic ranges of the calibration curves, and 14 analytes, present only in the calibrator solution, were qualitatively reported. Twenty-five analytes met all quantitative reporting criteria including dilution integrity. The ability to analyze quantitative blood and qualitative urine samples in the same batch is one of the most useful elements of this procedure. This sensitive, specific and robust analytical method was routinely employed in the analysis of >300 samples in our laboratory over the last 6 months.

Since late 2014, fentanyl has become the major driver of opioid mortality in the United States. However, a descriptive analysis of fentanyl victims is limited. We studied the 2016 fentanyl and heroin overdose deaths and compared them to previously studied heroin-associated fatalities from 2012 over a wide range of demographic and investigative variables, including overdose scene findings, toxicology results, and prescription drug history. We observed a significant increase in fentanyl-related deaths (n = 421, 2016) versus heroin deaths (n = 160, 2012) but the baseline demographics between both cohorts remained similar. Victims were predominantly of ages 35-64 years (60%-64%), White (83%-85%), and male (73%-76%). 2016 fentanyl decedents were more likely to have naloxone administered upon overdose, and the majority still had a positive prescription history for a controlled substance. Toxicology data showed a decrease in mean morphine and 6-monoacetylmorphine concentrations when cointoxication with fentanyl occurred. Our study emphasizes the medical examiner's role as a public health data source and bridge between different stakeholders combating the opioid epidemic.

The presented analytical method enabled the Toxicology Department at the Cuyahoga County Medical Examiner’s Office to identify 26 and quantitatively report 24 compounds in 500 μL of whole blood, including fentanyl analogues (fentalogues) such as methoxyacetyl fentanyl (MeOAF) and cyclopropyl fentanyl (CPF). This second-generation method (FG2) was developed with the objective to improve the existing analysis (FG1) by decreasing sample size, lowering limits of detection (LOD) and lower limit of quantitation, minimizing ion suppression and resolving chromatographic interferences. Interferences may occur in the analysis of fentanyl, MeOAF, CPF, 3-methylfentanyl (3MF), butyryl fentanyl and isobutyryl fentanyl due to isobars and structural or geometric isomerism with another analogue or metabolite. The isomeric and isobaric fentalogues were grouped into three sets. The LOD established for Set 1 [MeOAF, para-methoxyacetyl fentanyl, para-fluoro acryl fentanyl (isobar), fentanyl carbamate], 2-furanyl fentanyl, Set 2 [CPF, (E)-crotonyl fentanyl] and carfentanil was 0.0125 ng/mL. The LOD established for N-methyl norfentanyl, norfentanyl, norcarfentanil, despropionyl fentanyl (4-ANPP), acetyl fentanyl, β-hydroxy fentanyl, benzyl fentanyl, acryl fentanyl, alfentanil, fentanyl, para-fluoro fentanyl, Set 3 [(±)-trans-3MF, (±)-cis-3MF, isobutyryl and butyryl fentanyl], para-fluoroisobutyryl fentanyl, sufentanil, phenyl fentanyl and cyclopentenyl fentanyl was 0.0625 ng/mL. Seven-point linear calibration curves were established between 0.025 and 4.0 ng/mL for the 8 analytes with the lower LOD and 0.125 and 20 ng/mL for the 18 analytes with the higher LOD. 4-ANPP and cyclopentenyl fentanyl met qualitative reporting criteria only. The results for five postmortem and two driving under the influence of drugs authentic case samples are presented. To the authors’ knowledge, FG2 is the first published method that achieved baseline resolution of the nine structural/stereo isomers and one isobar by ultra-high performance liquid chromatography–MS-MS and provided quantitative validation data for nine compounds. FG2 may be used as the new baseline for future isomers that need to be chromatographically separated.