Detection and Characterization of the Effect of AB-FUBINACA and Its Metabolites in a Rat Model.

Synthetic cannabinoids were originally developed by academic and pharmaceutical laboratories with the hope of providing therapeutic relief from the pain of inflammatory and degenerative diseases. However, recreational drug enthusiasts have flushed the market with new strains of these potent drugs that evade detection yet endanger public health and safety. Although many of these drug derivatives were published in the medical literature, others were merely patented without further characterization. AB-FUBINACA is an example of one of the new indazole-carboxamide synthetic cannabinoids introduced in the past year. Even though AB-FUBINACA has become increasingly prominent in forensic drug and toxicology specimens analyses, little is known about the pharmacology of this substance. To study its metabolic fate, we utilized Wistar rats to study the oxidative products of AB-FUBINACA in urine and its effect on gene expressions in liver and heart. Rats were injected with 5 mg/kg of AB-FUBINACA each day for 5 days. Urine samples were collected every day at the same time. On day 5 after treatment, we collected the organs such as liver and heart. The urine samples were analyzed by mass spectrometry, which revealed several putative metabolites and positioning of the hydroxyl addition on the molecule. We used quantitative PCR gene expression array to analyze the hepatotoxicity and cardiotoxicity on these rats and confirmed by real-time quantitative RT-PCR. We identified three genes significantly associated with dysfunction of oxidation and inflammation. Our study reports in vivo metabolites of AB-FUBINACA in urine and its effect on the gene expressions in liver and heart.

Evaluation of the Compatibility of Ethyl Glucuronide and Ethyl Sulfate Levels to Assess Alcohol Consumption in Decomposed and Diabetic Postmortem Cases.

The aim of the study is to evaluate the contribution of ethanol metabolite detection in postmortem cases by showing the connection between the presence of ethanol metabolites, which are indicators of alcohol consumption, and the detection of potential postmortem ethanol formation in decomposed and diabetic cases. Determination of ethanol consumption before death is often one of the most important questions in death investigations. Postmortem ethanol formation or degradation products in the blood make it difficult to distinguish antemortem consumption or postmortem formation of ethanol and eventually may lead to misinterpretation. Decomposed bodies and diabetic cases are vulnerable to postmortem ethanol formation due to putrefaction, fermentation or other degradations. Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are two metabolites of ethanol produced only in the antemortem time interval. In this study, EtG and EtS levels in urine and vitreous humor samples of 27 postmortem cases, including diabetic and degraded bodies were compared to ethanol results of their blood, urine and vitreous humor samples. EtG and EtS in urine and vitreous humor were analyzed by liquid chromatography-tandem mass spectrometry, and ethanol was assayed by routine headspace gas chromatography-flame ionization detector. These cases were devoid of other influences from forensically relevant drugs, so ethanol and/or glucose were among the only positive findings in these cases. The results of this pilot study indicate the postmortem ethanol concentrations do not correlate with the measured EtG and EtS values but are beneficial in rulings of accidental or natural deaths. This preliminary study gives additional data to help distinguish between antemortem ethanol intake and postmortem formation. EtG and EtS were well correlated positively with antemortem ethanol use instead of forming spontaneously in samples from decedents who are decomposing or have a history of diabetic hyperglycemia.

A multiple drug fatality involving MK-801 (dizocilpine), a mimic of phencyclidine.

MK-801 (dizocilpine) is a non-competitive antagonist at the N-methyl-D-aspartate (NMDA) family of glutamate receptors in the central nervous system. It is an anticonvulsant and also shares several pharmacological properties with phencyclidine and ketamine. It is not observed routinely as a substance of abuse. The deceased, a 45-year-old white male, obtained MK-801 surreptitiously in an attempt to treat a self-diagnosed depression. He was discovered the next morning, unresponsive on the bathroom floor. An empty bottle, labeled to contain 25mg of MK-801, was found near the body. The autopsy was performed at the Joseph A Jachimczyk Forensic Center, Houston, TX. Body weight at autopsy was 88kg. Lungs were edematous and congested (right: 775g; left 700g). The heart had proportionate chambers and was otherwise unremarkable. The kidneys (right: 220g; left 225g) were smooth surfaced. The brain (1550g) was congested and without trauma. Microscopic evaluation of the heart, kidneys and lungs showed normal histology and confirmed pulmonary congestion and edema. Samples of heart blood, liver, bile, vitreous humor, stomach contents and urine were collected at autopsy. There were 550ml of stomach contents. Drugs in blood were screened by EMIT II Plus immunoassay procedures and by gas chromatography/mass spectrometry (GC/MS) of an organic solvent extract of basified blood. Alcohol was determined by gas chromatography with headspace injection. MK-801, benzodiazepines and alcohol were detected in blood. Amounts of MK-801 present in blood, bile, liver, vitreous humor and urine were 0.15, 0.29, 0.92, less than 0.1 and 0.36 mg/l (kg), respectively. The cause of death was benzodiazepine, dizocilpine and ethanol toxicity and the manner accidental.

Postmortem distribution of the novel antipsychotic drug quetiapine.

The objective of this research was to determine the concentrations and distribution of the atypical antipsychotic drug, quetiapine, in postmortem tissues from eight Medical Examiner cases. Quetiapine was isolated from liquid specimens and tissue homogenates by extraction at an alkaline pH into 1-chlorobutane. The 1-chlorobutane was decanted, and quetiapine, plus the internal standard (prochlorperazine), was back-extracted into 0.1N sulfuric acid. The acid layer was made basic, and quetiapine, plus the internal standard, was re-extracted into 1-chlorobutane. Quantitation was by gradient, high-pressure liquid chromatography on a C-8 ODS (2.1 x 150 mm, 5 mu) column with acetonitrile/0.1M ammonium hydroxide (pH 10) mobile phase and a photodiode array detector set at 258 nm. The apparent linear range of the assay was from 0.05 to 5.0 microg/mL. At known concentrations of 0.1 and 0.5, interday accuracy (n = 5) was 103.8 and 107.2%, respectively. Interday precision (% cv) at the same concentrations was 9.8 and 9.0, respectively. In the cases where quetiapine was not considered to have contributed to the death, the postmortem concentrations in blood, liver, and bile ranged between 0.15 and 2.7 mg/L (n = 6), 1.3 and 9.5 mg/kg (n = 8), and 10 and 46 mg/L (n = 5), respectively. In the one case involving a quetiapine overdose, concentrations in blood (19.8 mg/L), liver (12.6 mg/kg), and bile (161 mg/L) exceeded the ranges of concentrations determined in specimens from the quetiapine-unrelated deaths.

Validation of LC-TOF-MS screening for drugs, metabolites, and collateral compounds in forensic toxicology specimens.

Liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) analysis provides an expansive technique for identifying many known and unknown analytes. This study developed a screening method that utilizes automated solid-phase extraction to purify a wide array of analytes involving stimulants, benzodiazepines, opiates, muscle relaxants, hypnotics, antihistamines, antidepressants and newer synthetic "Spice/K2" cannabinoids and cathinone "bath salt" designer drugs. The extract was applied to LC-TOF-MS analysis, implementing a 13 min chromatography gradient with mobile phases of ammonium formate and methanol using positive mode electrospray. Several common drugs and metabolites can share the same mass and chemical formula among unrelated compounds, but they are structurally different. In this method, the LC-TOF-MS was able to resolve many isobaric compounds by accurate mass correlation within 15 ppm mass units and a narrow retention time interval of less than 10 s of separation. Drug recovery yields varied among spiked compounds, but resulted in overall robust area counts to deliver an average match score of 86 when compared to the retention time and mass of authentic standards. In summary, this method represents a rapid, enhanced screen for blood and urine specimens in postmortem, driving under the influence, and drug facilitated sexual assault forensic toxicology casework.

An overview of alcohol testing and interpretation in the 21st century.

Ethanol analysis is the most commonly carried out drug testing in a forensic toxicology laboratory. Determination of blood alcohol concentration (BAC) is needed in a multitude of situations, including in postmortem analysis, driving under the influence (DUI) and drug-facilitated sexual assault (DFSA) cases, workplace drug monitoring, and probation investigations. These analyses are carried out by direct measurement of ethanol concentrations as well as of metabolic by-products, such as ethyl glucuronide (EtG) and ethyl sulfate (EtS). This review article will discuss pharmacokinetics, including absorption, distribution, and elimination of ethanol, methods for the detection of ethanol, the effect of ethanol on human performance, the role of alcohol in injuries and fatalities, and information regarding the interactions that may occur between alcohol and other drugs. Finally, an explanation will be given on how to interpret alcohol levels as well as the extrapolation and calculation of blood alcohol levels at times prior to sample collection.

Analysis of alprazolam by DART-TOF mass spectrometry in counterfeit and routine drug identification cases.

The high prevalence of alprazolam abuse translates to an increased workload for crime laboratories in characterizing seized tablets. These tablets may originate as diverted pharmaceuticals or counterfeited mimics, so efficient analytical techniques should provide confirmatory data while minimizing destruction of evidence. We offer the first report of a validated forensic method for confirming alprazolam tablets by direct analysis in real time-time of flight (DART-TOF) mass spectrometric analysis. This technique provides rapid identification of target analytes with minimal sample preparation, allowing direct analysis in the atmospheric sample gap. Selectivity is achieved through high resolution and mass accuracy, unique ion fragments, and chlorine isotopic ratios. This method utilizes fragmentation in two separate voltage functions to observe the alprazolam pseudo molecular ion at 309.09070 using 40 V and major ion fragments of 281.07197 and 205.07657 at 120 V. These parameters allow our laboratory to confirm alprazolam tablets efficiently, without compromising quality forensic standards.

Methadone and methadone metabolites in postmortem specimens.

We have determined drug/metabolite concentrations and ratios of methadone (METH) to two of its metabolites (EDDP, 2-ethylidene-1, 5-dimethyl-3, 3-diphenylpyrrolidine; and EMDP, 2-ethyl-5-methyl-3,3-diphenylpyrroline) in postmortem peripheral blood and liver tissue by liquid chromatography/tandem mass spectrometry. The assays employed deuterated internal standards and multiple reaction monitoring (MRM) techniques. The assay linear range was 0.01-2.0 mg/l for each analyte. METH, EDDP, and EMDP were determined in liver and peripheral blood from 46 methadone-positive cases. METH and EDDP were detected in all specimens, whether blood or liver. EMDP was detected, only in liver, and only 17 cases, at concentrations much lower than those of EDDP. Concentrations of METH and EDDP in blood and liver from EMDP-positive cases were in ranges higher than, but overlapping with, concentrations in blood and liver from EMDP-negative cases. These data suggest that although METH is readily demethylated and cyclized to EDDP, in vivo, conversion to EMDP may be less efficient and its accumulation in postmortem tissues may be highly individual.

The estimation of blood alcohol concentration : Widmark revisited.

Expert witnesses and others involved in toxicology are frequently asked to perform retrograde extrapolation of blood alcohol concentration (BAC) or to estimate BAC based on a proposed drinking scenario. Although many individuals are reluctant to perform these calculations and some jurisdictions expressly prohibit them, a significant number of practitioners routinely estimate BAC based on this type of calculation, using as a basis the fundamental work of Widmark. Although improvements to the Widmark formula and other data pertaining to the pharmacology of alcohol have been published, these improvements are frequently ignored when estimating BAC. This article summarizes five published models for the estimation of BAC and proposes a sixth model that incorporates recent data on the rate of absorption of alcohol from the GI tract into the existing five models. The five improved models can be computerized and used to construct comparative snapshots of the BACs calculated by the different algorithms. This will allow practitioners to provide a more balanced picture of the variability in BAC calculations.