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.

Carbon monoxide-related deaths in a metropolitan county in the USA: an 11-year study.

Carbon monoxide (CO) poisoning as a cause of death is well documented in industrialized countries. The objective of this study was to compare demographic data in deaths due to accidents (in fires) and suicides in the same population between 1988 and 1998. Furthermore, the potential effect of a community wide education effort regarding safety in the home was assessed. Postmortem reports were reviewed for all deaths examined at the Office of the Cuyahoga County Coroner in Cleveland, OH, USA. During the study period, there were 209 accidental deaths due to fires in the home (6.5% of all accidents in the home) and 182 CO deaths by suicide (9.8% of all suicides). Demographic characteristics of the two groups differed: while males represented the majority of cases in both groups (55% of accidents, 70% suicides), race specific death rates were higher for whites than blacks (18/100,000 white, 3/100,000 black) in suicides compared with 29/100,000 deaths for blacks and 11/100,000 for whites in accidental cases. Fire deaths were prevalent in the young (0-9 years) and old (>60) whereas in the suicide group the age specific death rate was highest for those over 70 years. The majority of fire deaths occurred in the city of Cleveland but suicides were prevalent in the suburbs. More fire deaths occurred in December than any other month whereas more suicides occurred in April. In 1992, there was a community wide effort to provide free smoke detectors to residents in Cleveland. In 1992, there were 4.2/100,000 fire deaths in the city. This decreased to 0.6/100,000 in 1996, increased to 1.2/100,000 in 1997 followed by a decrease to 0.8/100,000 in 1998. This suggested that the program may have aided in decreasing these types of deaths. Deaths due to fires in the suburbs were <1/100,000 throughout the study period.

Evaluation of the Cedia heroin metabolite (6-AM) immunoassay with urine specimens from A criminal justice drug-testing program.

The ability to differentiate illicit from legitimate drug use in a drug-testing program would decrease costs by reducing the number of screening specimens requiring confirmation and also reduce the stigma attached to positive preliminary test results. Because many screening tests for drug detection use immunoassays, increasing the specificity of these tests has been a goal of manufacturers. In this study we evaluated the utility of one such assay, the Cedia heroin metabolite (6-acetylmorphine, 6-AM) assay to reliably detect heroin use. Specimens (N = 525) from a criminal justice drug-testing program were screened with this assay (cutoff concentration = 10 ng/mL 6-AM) and any positive samples were confirmed by gas chromatographic-mass spectrometric analysis (lower reporting limit for 6-AM = 5 ng/mL). The confirmation rate for the enzyme immunoassay (EIA) was 98% (517/525). Specimens contained 6-AM at concentrations ranging from 5 to 16,923 ng/mL (mean = 1251; median = 317). All confirmed specimens also contained morphine (range: 8-222,427 ng/mL; mean = 11,203 ; median = 4134). When challenged with standard drug solutions, the EIA correctly identified drug-free urine and produced positive results (lowest concentration, in ng/mL, that produced a positive result) with morphine at 10,000; oxycodone at 61,000; codeine at 60,000; hydromorphone at 10,000; hydrocodone at 60,000, 6-AM at 10, and pentazocine at 35,000 ng/mL. The Cedia heroin metabolite (6-AM) assay produced a high confirmation rate when challenged with urine specimens and therefore should be a useful tool in forensic toxicology. Potential users should be aware that high concentrations of other opioids (e.g., morphine, oxycodone) and structurally related compounds (e.g., pentazocine) may produce positive results.

Cannabis (marijuana) contamination of United States and foreign paper currency.

It is well known that United States paper currency in general circulation is contaminated with trace amounts of illicit substances such as cocaine, heroin and marijuana. As is the case with cocaine, differentiating "background levels" of the various cannabinoid constituents of Cannabis sativa L., namely, Delta(9)-tetrahydrocannabinol (THC), cannabinol (CBN), and cannabidiol (CBD) contaminating currency found in the general circulation from currency associated with illegal drug activity is imperative if a legal nexus is to be established with the latter. We analyzed 165 randomly collected paper currency notes from 12 U.S. cities (N = 125) and 4 foreign countries (N = 40) for THC, CBD, CBN, 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol, and 11-hydroxy-Delta(9)-tetrahydrocannabinol. Uncirculated US 1 dollar notes were added as negative controls. Drug residues were washed from individual bills, extracted using a liquid-liquid extraction protocol, derivatized, and quantitated by gas chromatography-mass spectrometry by selected ion monitoring. For the US 1 dollar currency, THC was present in 1.6% (2 notes), CBN 10.31% (13 notes), CBD 1.6% (2 notes). The following concentrations were determined: 0.085 microg/bill and 0.146 microg/bill for THC; 0.014-0.774 microg/bill (mean 0.166 microg/bill) for CBN; and 0.032 microg/bill and 0.086 microg/bill for CBD. For the foreign currency (Colombia, Qatar, India, and New Zealand), THC and CBN were present in 22.5% (9 notes). The following concentration ranges were determined: THC 0.026-0.065 microg/bill (mean 0.049 microg/bill), CBN 0.061-0.197 microg/bill (mean 0.115 microg/bill). All of the positive THC and CBN were found in the New Zealand polypropylene notes. This study demonstrated that marijuana (cannabinoids) may contaminate both paper and plastic currency.

'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.

Postmortem tissue distribution of MDPV following lethal intoxication by "bath salts".

3,4-Methylenedioxypyrovalerone (MDPV) is a psychoactive, synthetic analog of the central nervous system stimulant cathinone. Its recent popularity as a recreational drug in the United States has led to numerous reports to poison control centers across the country. As with other synthetic cathinones, the recreational use of MDPV has resulted in death. MDPV is thought to exert its pharmacologic effects by inhibiting the reuptake of dopamine and norepinephrine. This report describes the case of an exposure of a 39-year-old male to MDPV, which resulted in his death. Postmortem concentrations of MDPV in various tissues were measured. The detection of MDPV in tissues and fluids was accomplished using gas chromatography-mass spectrometry analysis after solid-phase extraction. Blood analysis also demonstrated therapeutic levels of lamotrigine, fluoxetine, risperidone, benztropine, pseudoephedrine and ibuprofen. The detection of cathinones in hair was conducted using high-performance liquid chromatography-tandem mass spectrometry after solid-phase extraction. MDPV was uniformly distributed among multiple tissues (blood, brain, muscle, cerebrospinal fluid and lung) at concentrations of approximately 0.4 to 0.6 µg/mL. Tissue and fluids responsible for detoxification/excretion had higher concentrations of MDPV (kidney, liver and bile > 0.8 µg/mL). A blood concentration ≥ 0.4 µg/mL was judged sufficient to cause death. The cause of death was ruled MDPV intoxication resulting in cardiac arrhythmia.

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.

Prevalence of dihydrocodeine in hydrocodone positive postmortem specimens.

Hydrocodone (HC) has received renewed interest in the US due to reported increases in opiate related deaths involving psychotherapeutic drugs. The relative contribution of dihydrocodeine (DHC) in these deaths is unknown since little testing of this compound is performed. The objective of the study was to determine the prevalence of DHC in HC positive decedents and report the range of concentrations detected in these cases in order to evaluate the potential role of DHC in the deaths and determine the usefulness of including this analyte in opioid testing protocols. Specimens were assayed by liquid-liquid or solid phase extraction followed by gas chromatography/mass spectrometry operated in the selected ion monitoring mode. A multipoint calibration was utilized in the linear range 2-600ng/mL. Accuracy for HC, DHC and hydromorphone (HM) was 101-106% and between day precision at 160ng/mL between 7% and 11%. One hundred and thirty six cases were identified with the majority male (62%) and white (83%). A search of HC positive cases identified 64 with DHC (47%). The range of HC concentrations was 9-3039ng/mL heart blood (n=43) and 42-12353ng/mL urine (n=21). DHC concentrations in these cases ranged 3-243ng/mL in heart blood and 5-1842ng/mL in urine. DHC/HC ratios ranged 0.00(7)-2.90 in blood (n=43), and 0.01-5.04 in urine (n=21) with 16% and 24% of these cases with ratios >0.50, respectively. HM was detected in only 9 HC cases with the majority positive in urine.