A computationally supported designer benzodiazepine strategy for public toxicology laboratories.

Public laboratories must balance innovative and existing methods to keep up with designer drug trends. This article presents a strategy for handling designer benzodiazepines (DBZDs) in casework from screening to interpretation. The cross-reactivity of 22 DBZDs and metabolites was tested against the Immunalysis™ Benzodiazepine Direct Enzyme-Linked Immunosorbent Assay kit. The kit had high intra-analyte precision (coefficients of variation < 15%). Inter-analyte performance varied, triggering confirmation testing at concentrations ranging from 35 to 460 µg/L. The CCRFSL implemented a 40-analyte benzodiazepine and Z-drug confirmation method in 2019. Ten additional analytes were later validated for qualitative reporting, and the limits of detection (LODs) for 13 analytes were lowered by 60%. The method of standard addition was also optimized for as-needed quantitation. Equal and 1/x weighting factors correlated well with target concentrations (coefficients of determination (r2) > 0.98), but 1/x weighting provided the most consistently accurate concentrations. Six computational models were developed to predict DBZD binding affinity to the γ-aminobutyric acid-A receptor to assist in case interpretation (r2 > 0.7 for cross-validation and test set prediction). These models were used to predict the binding affinity of analytes in the confirmation method. Other public laboratories can use this same practical strategy to adapt to any designer drug class (e.g., benzodiazepines, opioids, cannabinoids, and stimulants).

Development and Validation of an LC-MS-MS Method for the Detection of 40 Benzodiazepines and Three Z-Drugs in Blood and Urine by Solid-Phase Extraction.

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 ß-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.

Unique Structural/Stereo-Isomer and Isobar Analysis of Novel Fentanyl Analogues in Postmortem and DUID Whole Blood by UHPLC-MS-MS.

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, ß-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.

An LC-MS-MS Method for the Analysis of Carfentanil, 3-Methylfentanyl, 2-Furanyl Fentanyl, Acetyl Fentanyl, Fentanyl and Norfentanyl in Postmortem and Impaired-Driving Cases.

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.

In vitro formation of acetylmorphine from morphine and aspirin in postmortem gastric contents and deionized water.

Forensic toxicologists consider detection of 6-acetylmorphine (6-AM) definitive evidence of heroin abuse. This study investigated the possibility that aspirin, when in solution with morphine, may acetylate morphine to produce acetylmorphine (AM). Morphine sulfate-extended release tablets (15 mg) and aspirin (325 mg) tablets were incubated in 50 mL postmortem gastric contents or deionized water at 37°C. One-milliliter aliquots were taken at timed intervals, extracted by solid-phase extraction, derivatized and analyzed by the gas chromatograph with a mass selective detector. Both 3- and 6-AM were detected in samples containing morphine and aspirin in combination; no heroin was detected. Production of AM was pH dependent with optimal formation at pH ≥4. In gastric contents, concentrations of 3-AM exceeded that of 6-AM by ∼10-fold. Production of 3-AM in gastric contents was approximately twice as high as it was in water, while matrix did not appear to affect 6-AM production. Urine specimens (10,602) assayed at a pain management laboratory and postmortem cases (>6,000) were investigated for in vivo formation of AM. Three cases exhibited unexplained 6-AM results. These data indicate that in vivo formation of 6-AM from the co-administration of aspirin and morphine, if it happens, is quite rare. In instances where this is suspected, 3-AM should be monitored.

'Lingering' opiate deaths? Concentration of opiates in medulla and femoral blood.

'Lingering death' cases occur when the circumstances of death indicate an opiate overdose, but measured opiate blood levels are only in the therapeutic range; death results from cardiac and respiratory depression. This study examined the relative concentration of opiates in femoral blood and in the medulla oblongata (sites for cardiac and respiratory control) from 41 cases to determine whether a difference in opiate concentration might explain lingering deaths. Opiates from blood and medulla were analyzed using GC-EI-MS in selective ion monitoring mode. Results were correlated with gross and microscopic findings of the lungs and with cause and manner of death. Opiate concentrations for morphine, codeine and 6-acetylmorphine (6-AM) were higher in the medulla than in blood. The brain: blood ratio for the analytes demonstrated an increasing ratio from morphine, to codeine, to 6-AM (1.42, 2.48 and 4.86), which corresponds to the relative lipophilicity of these analytes. The average right and left lung weights were 762 and 668 g, respectively. Histologic examination showed edema, and/or polarizable microemboli, acute bronchopneumonia and acute bronchitis. The preferential distribution of opiates to medulla suggests that lingering opiate deaths may be explained, at least in part, because of higher relative concentrations of drug in brain, compared with femoral blood.