Metabolism and toxicological analysis of synthetic cannabinoids in biological fluids and tissues.

Synthetic cannabinoids, which began proliferating in the United States in 2009, have gone through numerous iterations of modification to their chemical structures. More recent generations of compounds have been associated with significant adverse outcomes following use, including cognitive and psychomotor impairment, seizures, psychosis, tissue injury and death. These effects increase the urgency for forensic and public health laboratories to develop methods for the detection and identification of novel substances, and apply these to the determination of their metabolism and disposition in biological samples. This comprehensive review describes the history of the appearance of the drugs in the United States, discusses the naming conventions emerging to designate new structures, and describes the most prominent new compounds linked to the adverse effects now associated with their use. We review in depth the metabolic pathways that have been elucidated for the major members of each of the prevalent synthetic cannabinoid drug subclasses, the enzyme systems responsible for their metabolism, and the use of in silico approaches to assist in predicting and identifying the metabolites of novel compounds and drug subclasses that will continue to appear. Finally, we review and critique analytical methods applied to the detection of the drugs and their metabolites, including immunoassay screening, and liquid chromatography mass spectrometry confirmatory techniques applied to urine, serum, whole blood, oral fluid, hair, and tissues.

Pharmacology, Toxicology, and Adverse Effects of Synthetic Cannabinoid Drugs.

Synthetic cannabinoid drugs have become an established part of the recreational drug landscape in the United States and internationally. These drugs are manufactured in clandestine laboratories internationally and distributed in the United States in smoking mixtures, use of which produces effects very similar to use of marijuana. The adverse-effect profile of the drugs has not been studied in humans and infrequently in animal models, so much of the information about their toxicity comes from emergency department and treatment reports and forensic case studies. This review considers the discovery and characterization of the endocannabinoid system, approaches to receptor-binding studies of various synthetic cannabinoids from the first wave of naphthoylindoles (e.g., JWH-018) to the emerging adamantoylindole drugs (e.g., AKB-48), and their analogs, to evaluate the potential activity of drugs in this class. Currently employed approaches to assessing functional activity of the drugs using in vitro and in vivo models is also described, and comparisons made to the effects of THC. The physiological effects of activation of the endocannabinoid system in humans are reviewed, and the physiological effects of cannabinoid use are described. Case reports of adverse events including emergency department admissions, mental health admissions, and clinical and forensic case reports are presented in detail and discussed to summarize the current state of knowledge of adverse effects, both clinical and forensic in humans, including effects on driving ability, and tissue injury and death. The greatest weight is accorded to those reports that include toxicological confirmation of use. Finally, we discuss the current status of attempts to schedule and control the distribution of synthetic cannabinoids and the relevance of receptor binding and functional activity in this context. There is growing toxicological and pharmacological evidence of impairment, psychosis, tissue injury, and isolated deaths attributable to this emerging class of drugs.

Analysis of Synthetic Cannabinoids in Botanical Material: A Review of Analytical Methods and Findings.

Synthetic cannabinoid analogs have gained a great deal of attention from the forensic community within the last four years. The compounds found to be of most interest to forensic practitioners include those of the following series: JWH, CP, HU, AM, WIN, RCS, and most recently, XLR and UR. Structurally the HU compounds are most similar in structure to Δ9-tetrahydrocannabinol (THC), the main psychoactive component of marijuana. The novel compounds include cyclohexylphenols, naphthoylindoles, naphthylmethylindoles, naphthylmethylindenes, benzoylindoles, naphthoylpyrroles, phenylacetylindoles, adamantoylindoles, and tetramethylcyclopropylindoles. Many of these compounds are cannabinoid receptor agonists and were originally synthesized for medical research purposes but have recently been appropriated into the illicit drug market. Their psychoactive effects, mimicking those of marijuana, as well as their indeterminate legal status, have made them popular for recreational use. Solutions of the compounds dissolved in organic solvents are sprayed onto botanical material and sold as "herbal incense" products via the Internet, and in smoke shops, convenience stores, and gas stations around the world. Many of the products are labeled "Not for human consumption" in an attempt to circumvent legislation that bans the sale and manufacture of certain compounds and their analogs for human use. The compounds that were first detected following forensic analysis of botanical materials included JWH-018, JWH-073, and CP 47,497 (C7 and C8 homologs). However, in the four years since their appearance the number of compounds has grown, and additional diverse classes of compounds have been detected. Governments worldwide have taken action in an attempt to control those compounds that have become widespread in their regions. This article discusses the history of synthetic cannabinoids and how they have been detected in the illicit drug market. It also discusses the analytical methods and techniques used by forensic scientists to analyze botanical products obtained via the Internet or from law enforcement investigations and arrests.

3,4-Methylenedioxymethamphetamine - Effects on Human Performance and Behavior.

3,4-Methylenedioxymethamphetamine (MDMA, "ecstasy") is a unique drug, sharing properties of hallucinogens and stimulants. The acute effects of empathy, euphoria, and excitement for which it is used recreationally can make it overwhelmingly distracting for the user in the context of driving. This review considers the chemistry, synthesis, analysis, pharmacology, pharmacokinetics, and documented effects of MDMA on cognitive and psychomotor skills important to driving. Laboratory studies show that users do experience cognitive impairments, and may also act more impulsively while under the influence of the drug's sympathomimetic effects. Psychomotor impairment may occur with elevated doses or after repeated administration, and residual psychomotor impairment during the "coming-down" phase may be compounded by fatigue, dehydration, combined drug use, or other confounding factors. There is growing anecdotal information providing evidence of MDMA-impaired driving, and it is evident that many users recognize and attempt to mitigate the effects by delaying driving until the acute affects have dissipated. The drug inevitably may affect a subject's judgment and ability to properly assess their fitness to drive also. Blood concentrations in MDMA-impaired drivers suggest that this impairment can be caused by normal patterns of recreational use, and MDMA use should be considered inconsistent with safe driving immediately following ingestion, and for up to a day or longer following use.

Methamphetamine - Effects on Human Performance and Behavior.

Methamphetamine is a popular recreational drug that has also had some historical use as a therapeutic agent. Its effect profile is complex, with stimulant, alerting effects during acute low-dose administration, progressively more disorienting effects on cognition, reasoning, and psychomotor ability with increased dosing and duration of use, and a depressant-like profile during withdrawal, often compounded by delusions and psychotic episodes, especially after high-dose or chronic use. This manuscript reviews the synthetic, structural, and analytical chemistry of the drug; the pharmacology of its central and peripheral effects; its pharmacokinetics following various routes of administration and dosage regimens; and its pharmacodynamics in both acute and chronic administration and therapeutic and recreational doses, noting in particular its effects on judgment, decision making, risk-taking, cognition and psychomotor performance, and violence. Finally, the review considers the issue of how these various effects can impact driving ability and can contribute to impairment. From the material reviewed it is concluded that the use of methamphetamine in anything other than low-dose, therapeutic administration with medical oversight raises the likelihood of some impairment of performance in complex psychomotor tasks such as driving.

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) and driving impairment.

3,4-Methylenedioxymethamphetamine, or MDMA, is increasing in popularity in the United States as a drug of abuse. It has stimulant and empathogenic mood altering properties with the potential to affect psychomotor skills and impact driving. This report reviews the literature relating to the relevant psychomotor effects of the drug, the relationship between dose and blood concentrations, and studies and case reports on specific effects of the drug on driving. The latter reports include both laboratory driving simulator studies and anecdotal reports, and case series. We also report details of eighteen cases of apparent MDMA impaired driving, including six drivers whose blood tested positive for MDMA alone. Most subjects displayed muscle twitching and body tremors, dilated pupils, slow pupillary reaction to light, elevated pulse and blood pressure, lack of balance and coordination, and most were perspiring profusely. Five of the six subjects were given field sobriety tests (one leg stand, walk and turn test), and all five performed poorly. There was no clear correlation between the blood concentration of MDMA and the specific demeanor of the subject. These findings are consistent with other reports, and lead to the conclusion that MDMA use is not consistent with safe driving, and that impairment of various types may persist for a considerable time after last use.

Amphetamines: an update on forensic issues.

Methamphetamine is currently enjoying a resurgence of popularity as a recreational drug. It presents a number of challenges to the forensic toxicologist both analytically and interpretively, and these latter interpretive issues are considered here. This review also discusses the current popular syntheses which account for the widespread domestic synthesis of the drug; the demographics of methamphetamine use in the United States as assessed from the Drug Abuse Warning Network (DAWN) data; developments in research of the neurotransporter pharmacology of the drug and its implications for interpretive forensic toxicology; the psychomotor effects of the drug and its potential for cognitive and functional impairment; interpretive issues related to postmortem blood drug concentrations and how these are impacted by evidence for incomplete distribution and the potential for postmortem redistribution; and, finally, concerns caused by designer methamphetamine analogues. All data indicate that methamphetamine and its analogues will present significant interpretive challenges to forensic toxicologists as the popularity of the drug continues to grow.

GHB and driving impairment.

Gamma hydroxybutyrate (GHB) was identified in the blood of 13 subjects arrested for impaired driving. GHB concentrations ranged from 26 to 155 mg/L (mean 87 mg/L, median 95 mg/L). In eight cases, GHB was the only drug detected, and signs of impairment were consistent with those of a CNS depressant, including erratic driving (weaving, swerving, ignoring road signs), confusion, incoherent speech, unresponsiveness, lack of balance, unsteady coordination, poor performances on field sobriety tests, and varying states of wakefulness. Given the ability of GHB to induce sleep and unconsciousness, it is evident from these cases that recreational use of the drug has the potential to impair a person's driving ability.

Zolpidem and driving impairment.

Zolpidem, a non-benzodiazepine hypnotic, was identified in the blood of 29 subjects arrested for impaired driving. Zolpidem concentrations ranged from 0.05 to 1.4 mg/L (mean 0.29 mg/L, median 0.19 mg/L). In the subjects whose cases we reviewed where zolpidem was present with other drugs and/or alcohol, symptoms reported were generally those of CNS depression. Symptoms included slow movements and reactions, slow and slurred speech, poor coordination, lack of balance, flaccid muscle tone, and horizontal and vertical gaze nystagmus. In five separate cases, where zolpidem was the only drug detected (0.08-1.40 mg/L, mean 0.65 mg/L, median 0.47 mg/L), signs of impairment included slow and slurred speech, slow reflexes, disorientation, lack of balance and coordination, and "blacking out." Although no quantitative relationship between blood concentrations and degree of driving impairment is currently possible, it is reasonable to conclude that because of its specific activity as a sleep inducer, blood concentrations consistent with therapeutic doses of zolpidem have the potential to affect driving in a negative way, and that concentrations above the normal therapeutic range would further impair a person's level of consciousness and driving ability.

Endogenous ethanol 'auto-brewery syndrome' as a drunk-driving defence challenge.

The concentration of ethanol in blood, breath or urine constitutes important evidence for prosecuting drunk drivers. For various reasons, the reliability of the results of forensic alcohol analysis are often challenged by the defence. One such argument for acquittal concerns the notion that alcohol could be produced naturally in the body, hence the term 'auto-brewery' syndrome. Although yeasts such as Candida albicans readily produce ethanol in-vitro, whether this happens to any measurable extent in healthy ambulatory subjects is an open question. Over the years, many determinations of endogenous ethanol have been made, and in a few rare instances (Japanese subjects with very serious yeast infections) an abnormally high ethanol concentration (> 80 mg/dl) has been reported. In these atypical individuals, endogenous ethanol appeared to have been produced after they had eaten carbohydrate-rich foods. A particular genetic polymorphism resulting in reduced activity of enzymes involved in hepatic metabolism of ethanol and a negligible first-pass metabolism might explain ethnic differences in rates of endogenous ethanol production and clearance. Other reports of finding abnormally high concentrations of ethanol in body fluids from ostensibly healthy subjects suffer from deficiencies in study design and lack suitable control experiments or used non-specific analytical methods. With reliable gas chromatographic methods of analysis, the concentrations of endogenous ethanol in peripheral venous blood of healthy individuals, as well as those suffering from various metabolic disorders (diabetes, hepatitis, cirrhosis) ranged from 0-0.08 mg/dl. These concentrations are far too low to have any forensic or medical significance. The notion that a motorist's state of intoxication was caused by endogenously produced ethanol lacks merit.

Postmortem forensic toxicology of trazodone.

Trazodone is a popular antidepressant medication that has been available for approximately 30 years. It has a reputation as a safe drug with relatively few reported fatalities attributed solely to it. We review the pharmacology and forensic toxicology of trazodone and report toxicology and cause and manner of death in a series of 37 deaths in which trazodone was detected. Although the normal upper therapeutic blood concentration for trazodone is about 2 mg/L, fatalities are rarely attributed solely to it at blood concentrations below 9 mg/L. Considering the pharmacology of the drug, potential interactions between other drugs with serotonin reuptake properties need to be considered, as does the increased susceptibility to the toxic effects in patients with pre-existing heart disease. In the cases reviewed, none were attributed solely to trazodone, although trazodone was frequently present together with other serotonergic drugs, such as the selective serotonin reuptake inhibitors like fluoxetine and sertraline. Ten cases had blood trazodone concentrations above 2 mg/L. Of these cases, trazodone played a primary role in the death of three subjects, with blood concentrations all greater than 9 mg/L. We confirm the conclusions of others that trazodone is a relatively safe drug except in massive overdose, although its toxicity may be influenced by the presence of other drugs and underlying pathophysiology.

Carisoprodol, meprobamate, and driving impairment.

This paper considers the pharmacology of the centrally acting muscle relaxant carisoprodol, and its metabolite meprobamate, which is also administered as an anxiolytic in its own right. Literature implicating these drugs in impaired driving is also reviewed. A series of 104 incidents in which these drugs were detected in the blood of drivers involved in accidents or arrested for impaired driving was considered, with respect to the analytical toxicology results, patterns of drug use in these subjects, the driving behaviors exhibited, and the symptoms observed in the drivers. Symptomatology and driving impairment were consistent with other CNS depressants, most notably alcohol. Reported driving behaviors included erratic lane travel, weaving, driving slowly, swerving, stopping in traffic, and hitting parked cars and other stationary objects. Drivers on contact by the police displayed poor balance and coordination, horizontal gaze nystagmus, bloodshot eyes, unsteadiness, slurred speech, slow responses, tendency to doze off or fall asleep, difficulty standing, walking or exiting their vehicles, and disorientation. Many of these cases had alcohol or other centrally acting drugs present also, making difficult the attribution of the documented impairment specifically to carisoprodol and meprobamate. In 21 cases, however, no other drugs were detected, and similar symptoms were present. Impairment appeared to be possible at any concentration of these two drugs; however, the most severe driving impairment and most overt symptoms of intoxication were noted when the combined concentration exceeded 10 mg/L, a level still within the normal therapeutic range.

Postmortem forensic toxicology of selective serotonin reuptake inhibitors: a review of pharmacology and report of 168 cases.

This paper reviews the complex pharmacology of the new class of antidepressant medications exhibiting selective inhibition of serotonin reuptake. The four selective serotonin reuptake inhibitors (SSRIs) considered--fluoxetine, fluvoxamine, sertraline and paroxetine--can result in toxicity and death through contributing to serotonergic excess resulting in serotonin syndrome, inhibiting the metabolism of other centrally acting drugs, leading to accumulation of toxic concentrations, and exerting complex vasoactive effects on the vascular smooth muscle. This latter feature is of particular concern in patients with preexisting heart disease. An analytical method involving isolation of the drugs by liquid/liquid extraction at alkaline pH into n-butyl chloride, and analysis by gas chromatography/mass spectrometry (GC/MS) is described, together with some of its limitations. Toxicologic and cause and manner of death data were examined in 60 deaths involving fluoxetine, 5 involving fluvoxamine, 75 involving sertraline, and 28 involving paroxetine. Deaths involving drug toxicity were generally a result of ingestion of multiple drugs, and in only a small number of the cases was death attributed principally to the SSRI involved. The potential for drug interactions between members of this class of drugs is discussed as well as their metabolites and a variety of other therapeutic and abused drugs which can contribute to their toxicity. In the absence of other risk factors, the lowest concentrations determined to have resulted in death were 0.63 mg/L for fluoxetine, 0.4 mg/L for paroxetine, and 1.5 mg/L for sertraline. We had insufficient data to make even this crude assessment for fluvoxamine. Drug-induced elevation of serotonin concentrations may be a significant risk factor for patients with atherosclerotic cardiovascular disease (ASCVD). Other factors including preexisting disease and the presence of other drugs and their pharmacology need to be carefully considered before determining the appropriate cause and manner of death in these cases.

Tabulation of alcohol content of beer and malt beverages.

We present data from the analysis of the alcohol content of 391 beers and malt beverages available for sale in the State of Washington. The beverages were tested by gas chromatography for their alcohol content. Considerable variability in the alcoholic strength was found, even within the same class. Overall, the range of concentrations was 2.92% (v/v) to 15.66% (v/v). The alcohol content of beverages consumed is a critical factor in Widmark or volume-of-distribution-type calculations used to estimate blood or breath alcohol content from patterns of alcohol consumption. Using the correct alcohol content for beer, when the brand is known, can make a significant difference in the reliability of the calculation, and the data presented here should assist with optimizing the accuracy of the calculation.

Determination of gamma-hydroxybutyrate (GHB) in biological specimens by gas chromatography--mass spectrometry.

A simple liquid-liquid extraction procedure for the analysis of gamma-hydroxybutyrate (GHB) in biological fluids without conversion to its lactone, gamma-butyrolactone, is described. Following derivatization to its di-TMS derivative, GHB was detected using gas chromatography-electron impact mass spectrometry. Diethylene glycol was used as the internal standard. The limit of quantitation in 1 mL of blood was 1 mg/L, and a linear response was observed over the concentration range 1 to 100 mg/L. Coefficients of variation for both intra-assay precision and interassay reproducibility ranged between 3.9 and 12.0%. GHB was detected in the blood of a sexual assault victim (3.2 mg/L), in the blood of two driving (DUI) cases (33 and 34 mg/L), and in the blood and urine of two nonfatal GHB-overdose cases (blood 130 and 221 mg/L; urine 1.6 and 2.2 g/L). The observed clinical symptoms ranged from confusion, disorientation, vomiting, and nystagmus to ataxia, sinus bradycardia, unconsciousness, and apnea.

Development of dosing guidelines for reaching selected target breath alcohol concentrations.

OBJECTIVE: This study evaluated gender-specific ethanol dosing protocols that were designed to result in one of two peak breath alcohol concentrations (BrACs)--0.07 or 0.10 g/2101. Inter- and intrasubject variability in BrAC were assessed and several possible methods for reducing variability in BrAC were evaluated. METHOD: Subjects (16 women, 16 men, ages 21-30 years) were studied after low (women 0.49 g/kg, men 0.53 g/kg consumed over 10 minutes) and high (women 0.81 g/kg, men 0.89 g/kg consumed over 20 minutes) ethanol doses, consumed following a 4-hour fast. All subjects were regular drinkers. RESULTS: Mean (+/-SD) peak BrACs actually achieved were 0.069+/-0.011 g/2101 after the low dose, and 0.105+/-0.014 g/2101 after the high dose. Mean values for peak BrAC, time to peak BrAC and area under the curve were not statistically significantly different between genders at either dose. BrACs varied by as much as twofold between subjects after equivalent gender and body weight adjusted doses. There was some reproducibility of ethanol pharmacokinetic parameters over dose and time in men, but not in women. CONCLUSIONS: The doses used resulted in equivalent mean ethanol exposures for women and men at each dose, with mean peak BrACs that closely approached the targets, but there was substantial inter- and intrasubject variability in ethanol pharmacokinetics.

Detection of the novel metabolite ethylphenidate after methylphenidate overdose with alcohol coingestion.

Methylphenidate is the most commonly prescribed psychostimulant in clinical use today. Known methylphenidate metabolites include ritalinic acid, corresponding lactams, and p-hydroxymethylphenidate. Recent in vitro work using rat liver preparations has indicated that the methylphenidate ethyl ester, ethylphenidate, is formed upon incubation with ethanol. This report describes the first detection of ethylphenidate in human blood and liver samples obtained from two suicide victims who had overdosed on methylphenidate and coingested ethanol. Amounts of ethylphenidate detected in whole blood specimens in these two cases (8 ng/mL and 1 ng/mL, respectively) were small relative to methylphenidate and ritalinic acid concentrations. Nonetheless, given the high likelihood that methylphenidate and ethanol coingestion frequently occurs, the detection of ethylphenidate in humans warrants further investigation into the extent of its formation as well as into any associated toxicity in nonoverdose situations.

Alcohol content of beer and malt beverages: forensic consideration.

Beer consumption is commonly an issue in a medico-legal setting, requiring estimates either of a likely blood alcohol concentration (BAC) for a given pattern of consumption or vice versa. Four hundred and four beers and malt beverages available for sale in the State of Washington were tested by gas chromatography for their alcohol content. Considerable variability in the alcoholic strength was found, even within the same class. Overall the range of concentrations was 2.92%v/v to 15.66%v/v. The mean alcohol concentration for ales was 5.51%v/v (SD 1.23%v/v), and for lagers, 5.32% (SD 1.43%v/v). Some specialty brews had characteristically higher or lower mean concentrations: ice beers 6.07%v/v, malt liquor 7.23%v/v, light beer 4.13%v/v, seasonal ales 6.30%v/v. Six brands of lager and four light beers account for the majority of all beer sales in the United States, and the mean alcohol concentration for these products was measured as 4.73%v/v and 4.10%v/v respectively. Few of the beers (17%) were labeled with respect to alcohol content, and in some cases, there was a significant disparity between the concentration listed on the label, and the measured alcohol concentration. Toxicologists need to exercise caution when performing Widmark type calculations, using all available information to select the most appropriate estimate for alcoholic strength of a beer or malt beverage.