Recommendations for toxicological investigations of drug-facilitated sexual assaults.

The recent increase in reports of drug-facilitated sexual assaults has caused alarm in the general public and prompted forensic toxicologists from across North America to address the toxicological issues surrounding this matter. The authors have developed recommendations and guidelines to inform law enforcement, medical, and scientific personnel of the requirements for performing successful toxicological examinations in cases of drug-facilitated rape.

Medication development of ibogaine as a pharmacotherapy for drug dependence.

The potential for deriving new psychotherapeutic medications from natural sources has led to renewal interest in rain forest plants as a source of lead compounds for the development of antiaddiction medications. Ibogaine is an indole alkaloid found in the roots of Tabernanthe iboga (Apocynaceae family), a rain forest shrub that is native to equatorial Africa. Ibogaine is used by indigenous peoples in low doses to combat fatigue, hunger and in higher doses as a sacrament in religious rituals. Members of American and European addict self-help groups have claimed that ibogaine promotes long-term drug abstinence from addictive substances, including psychostimulants and cocaine. Anecdotal reports attest that a single dose of ibogaine eliminates withdrawal symptoms and reduces drug cravings for extended periods of time. The purported antiaddictive properties of ibogaine require rigorous validation in humans. We have initiated a rising tolerance study using single administration to assess the safety of ibogaine for treatment of cocaine dependency. The primary objectives of the study are to determine safety, pharmacokinetics and dose effects, and to identify relevant parameters of efficacy in cocaine-dependent patients. Pharmacokinetic and pharmacodynamic characteristics of ibogaine in humans are assessed by analyzing the concentration-time data of ibogaine and its desmethyl metabolite (noribogaine) from the Phase I trial, and by conducting in vitro experiments to elucidate the specific disposition processes involved in the metabolism of both parent drug and metabolite. The development of clinical safety studies of ibogaine in humans will help to determine whether there is a rationale for conducting efficacy trials in the future.

Xylazine toxicity--literature review and report of two cases.

Xylazine is a veterinary sedative, analgesic or general anesthetic. Its pharmaceutical action results in sympathetic discharge via stimulation of alpha-2-adrenoceptors. In humans, toxicity consists of central nervous system depression, bradycardia and hypotension. The dosages known to produce toxicity in humans vary from 40 mg up to 2400 mg. Because of decomposition, xylazine blood concentrations in two homicide victims were unknown; however, the concentrations in the brain, liver, and kidneys were much higher in the 23-year-old female versus the 33-year-old male victim. A bottle of xylazine found on the crime scene had a concentration of 100 mg/mL. This 50 mL bottle had 32 mL remaining. Therefore at some point in time 18 mL had been utilized. The amount of available milligrams of xylazine (1800 mg) were enough to cause toxicity in both the woman and the man. Of interest was the fact that the partially skeletonized heads were found remote from the torsos, however, the concentration of xylazine in the body tissues provided a toxicological match of which head belonged to which body. Xylazine toxicity in humans and its relationship to these homicides will be the focus of this report.

Fatal excited delirium following cocaine use: epidemiologic findings provide new evidence for mechanisms of cocaine toxicity.

We describe an outbreak of deaths from cocaine-induced excited delirium (EDDs) in Dade County, Florida between 1979 and 1990. From a registry of all cocaine-related deaths in Dade County, Florida, from 1969-1990, 58 EDDs were compared with 125 victims of accidental cocaine overdose without excited delirium. Compared with controls, EDDs were more frequently black, male, and younger. They were less likely to have a low body mass index, and more likely to have died in police custody, to have received medical treatment immediately before death, to have survived for a longer period, to have developed hyperthermia, and to have died in summer months. EDDs had concentrations of cocaine and benzoylecgonine in autopsy blood that were similar to those for controls. The epidemiologic findings are most consistent with the hypothesis that chronic cocaine use disrupts dopaminergic function and, when coupled with recent cocaine use, may precipitate agitation, delirium, aberrant thermoregulation, rhabdomyolysis, and sudden death.

Quantitation of N,N-dimethyltryptamine and harmala alkaloids in human plasma after oral dosing with ayahuasca.

Harmine, harmaline, tetrahydroharmine (THH), and N,N-dimethyltryptamine (DMT) were quantitated in plasma from 15 healthy male volunteers after the ingestion of ayahuasca, a beverage that has been used for religious purposes in Brazil since pre-Columbian times. A growing awareness of the interest in this ancient shamanistic practice in modern urban cultures and the widespread popular dissemination of the inebriant effects and type and sources of the plant admixtures used to prepare the beverage have provided additional impetus for this study. The three harmala alkaloids were quantitated from protein-precipitated plasma by high-performance liquid chromatography using fluorescence detection. Recovery from blank human plasma was quantitative, and the limit of quantitation (LOQ) was below 2 ng/mL of plasma for each of the harmala alkaloids. Standard concentrations ranged from 10 to 250 ng/mL for harmine and THH and from 1.0 to 25.0 ng/mL for harmaline, respectively. Linearity was observed for harmine, harmaline, and THH within these respective ranges. The highest concentrations of harmala alkaloids in human plasma were found to be 222.3 ng/mL for harmine, 134.5 ng/mL for THH, and 9.4 ng/mL for harmaline. DMT was quantitated by gas chromatography using nitrogen-phosphorus detection after liquid-liquid extraction with diphenhydramine as an internal standard. DMT recovery was quantitative, and the limit of detection and LOQ were 0.5 and 5 ng/mL, respectively. Linearity for DMT was observed from 5 to 1000 ng/mL. The one-step extraction method for DMT and the protein precipitation method for the three harmala alkaloids afford rapid, sensitive, and quantitative analyses of these alkaloids with minimal analyte loss. The analytical methods also may be applicable to other matrices, including whole blood and urine samples and homogenized tissue specimens. These are the first reported observations of DMT and harmala alkaloids in plasma after ritual ingestion of ayahuasca.

Pharmacological screen for activities of 12-hydroxyibogamine: a primary metabolite of the indole alkaloid ibogaine.

The purported efficacy of ibogaine for the treatment of drug dependence may be due in part to an active metabolite. Ibogaine undergoes first pass metabolism and is O-demethylated to 12-hydroxyibogamine (12-OH ibogamine). Radioligand binding assays were conducted to identify the potency and selectivity profiles for ibogaine and 12-OH ibogamine. A comparison of 12-OH ibogamine to the primary molecular targets identified previously for ibogaine demonstrates that the metabolite has a binding profile that is similar, but not identical to the parent drug. Both ibogaine and 12-OH ibogamine demonstrated the highest potency values at the cocaine recognition site on the 5-HT transporter. The same rank order (12-OH ibogamine > ibogaine), but lower potencies were observed for the [3H]paroxetine binding sites on the 5-HT transporter. Ibogaine and 12-OH ibogamine were equipotent at vesicular monoamine and dopamine transporters. The metabolite demonstrated higher affinity at the kappa-1 receptor and lower affinity at the NMDA receptor complex compared to the parent drug. Quantitation of the regional brain levels of ibogaine and 12-OH ibogamine demonstrated micromolar concentrations of both the parent drug and metabolite in rat brain. Drug dependence results from distinct, but inter-related neurochemical adaptations, which underlie tolerance, sensitization and withdrawal. Ibogaine's ability to alter drug-seeking behavior may be due to combined actions of the parent drug and metabolite at key pharmacological targets that modulate the activity of drug reward circuits.

Identification and quantitation of ibogaine and an o-demethylated metabolite in brain and biological fluids using gas chromatography-mass spectrometry.

This report describes a sensitive method for quantitating ibogaine and a single major metabolite in biological fluids and brain tissue. We identified the metabolite as 12-hydroxy-ibogamine (12-OH-ibogamine or noribogaine) by full-scan, electron-impact gas chromatography-mass spectrometry (GC-MS). Ibogaine, 12-OH-ibogamine, and o-(methyl)-ibogaine-d3 (ibogaine-d3) internal standard were isolated by solvent extraction under basic conditions. The resulting organic extract was evaporated to dryness, and the residue was derivatized at room temperature with ethyl iodide in the presence of trimethyl anilinium hydroxide in dimethyl sulfoxide. The reaction was terminated by acidification and washed with organic solvents to remove impurities. The aqueous phase was then alkalinized and reextracted. The organic extract was concentrated and analyzed by GC-MS. Quantitation was based upon the ratios of the molecular ions at m/z 310 for ibogaine, m/z 313 for ibogaine-d3, and m/z 324 for 12-OH-ibogamine ethyl ether. The limit of detection was 5 ng/mL for both ibogaine and derivatized 12-OH-ibogamine, and limits of quantitation were between 5 and 10 ng/mL for all matrices tested. Calibration curves were linear in the range of 3-1000 ng/mL or ng/g for both analytes.

Identification of a primary metabolite of ibogaine that targets serotonin transporters and elevates serotonin.

Ibogaine is a hallucinogenic indole with putative efficacy for the treatment of cocaine, stimulant and opiate abuse. The purported efficacy of ibogaine following single dose administrations has led to the suggestion that a long-acting metabolite of ibogaine may explain in part how the drug reduces craving for psychostimulants and opiates. We report here that 12-hydroxyibogamine, a primary metabolite of ibogaine, displays high affinity for the 5-HT transporter and elevates extracellular 5-HT. In radioligand binding assays, 12-hydroxyibogamine was 50-fold more potent at displacing radioligand binding at the 5-HT transporter than at the DA transporter. Ibogaine and 12-hydroxyibogamine were equipotent at the dopamine transporter. In vivo microdialysis was used to evaluate the acute actions of ibogaine and 12-hydroxyibogamine on the levels of DA and 5-HT. Administration of 12-hydroxyibogamine produced a marked dose-related elevation of extracellular 5-HT. Ibogaine and 12-hydroxyibogamine failed to elevate DA levels in the nucleus accumbens over the dose range tested. The elevation in synaptic levels of 5-HT by 12-hydroxyibogamine may heighten mood and attenuate drug craving. The effects of the active metabolite on 5-HT transmission may account in part for the potential of ibogaine to interrupt drug-seeking behavior in humans.

High affinity cocaine recognition sites on the dopamine transporter are elevated in fatal cocaine overdose victims.

Cocaine mediates its powerful reinforcement by binding to recognition sites on the dopamine (DA) transporter. The pharmacological identity of cocaine recognition sites and their relevance to dopamine transport function has remained unclear. Ligand binding studies with transport inhibitors and cocaine congeners have provided evidence for multiple sites or "states" of the DA transporter. The potent cocaine congener [3H]WIN 35,428 ((CFT), 2B-carbomethoxy-3 beta-(4-fluorophenyl)-tropane) has been shown to recognize high and low affinity binding sites on the DA transporter. We have used [3H]WIN 35,428 to map and quantify the high affinity cocaine recognition site on the DA transporter in victims of fatal cocaine overdose. Region-of-interest densitometric analysis of the autoradiograms demonstrated a 2- to 3-fold elevation in the apparent density of [3H]WIN 35,428 binding in particular sectors of the striatum from victims of cocaine overdose as compared to age-matched and drug-free control subjects. The most marked increase in [3H]WIN 35,428 binding was seen in the nucleus accumbens. The apparent increase in the density of high affinity sites was confirmed by saturation binding analysis of [3H]WIN 35,428 to putamen membranes. Saturation analysis revealed high and low affinity binding components with affinities (KD values) of 4.3 +/- 1.2 and 84.7 +/- 19.7 nM (mean +/- S.E.) and densities of 9.9 +/- 4.0 and 193.0 +/- 28.6 pmol/g of tissue, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of cocaine and metabolites in brain using solid phase extraction and full-scanning gas chromatography/ion trap mass spectrometry.

A method is described for the determination of cocaine, benzoylecgonine and cocaethylene in the human brain using Clean Screen solid phase extraction cartridges and gas chromatography/ion trap mass spectrometry with electron impact and full scan analysis. The procedure uses deuterated internal standards. Run-to-run and within-run coefficients of variation are < 7% and the sensitivity proved to be 50 ng/g from 1 g of sample. The procedure has been applied to a number of forensic cases involving cocaine intoxication. Cocaine was relatively unstable in brain tissue stored at 4 degrees C when compared to storage at -80 degrees C.

m-Hydroxybenzoylecgonine: an important contributor to the immunoreactivity in assays for benzoylecgonine in meconium.

Meconium has been reported to be a more suitable specimen than maternal or neonatal urine for detecting fetal exposure to cocaine. In a study comparing various immunoassays with gas chromatography/mass spectrometry (GC/MS), several unexplained discrepancies among the assays were noted. Using methanol extracts of meconium samples, an immunoreactive spot that was more polar than benzoylecgonine was detected by thin-layer chromatography (TLC). An extract of this spot analyzed by GC/MS yielded a fragmentation pattern indicative of an aryl hydroxylated benzoylecgonine. Standards of m-hydroxybenzoylecgonine, o-hydroxybenzoylecgonine, and p-hydroxybenzoylecgonine were synthesized; it was determined that m-hydroxybenzoylecgonine had the same retention time and ion ratios as the TLC immunoreactive spot. Furthermore, m-hydroxybenzoylecgonine proved to be immunoreactive. Ten meconium samples immunoreactive for benzoylecgonine were analyzed by GC/MS. Results before and after hydrolysis with beta-glucuronidase (type IX) showed free m-hydroxybenzoylecgonine comprising 59 to 94% of the total m-hydroxybenzoylecgonine and showed total m-hydroxybenzoylecgonine values ranging from 0.2 to 6.3 times as high as benzoylecgonine. Therefore, m-hydroxybenzoylecgonine appears to be a quantitatively important cocaine metabolite in meconium, which is responsible for a significant portion of the discrepancy between benzoylecgonine concentrations in meconium extracts as measured by immunoassay and GC/MS.

A solid phase extraction technique for the isolation and identification of opiates in urine.

A quantitative method was developed for the simultaneous analysis of morphine, codeine, hydromorphone, hydrocodone, and oxycodone in urine by gas chromatography/mass spectrometry. Samples were hydrolyzed with beta-glucuronidase and then extracted by solid phase extraction on Bond Elute Certify cartridges at pH 6.8. Nalorphine was used as the internal standard. The opiates were analyzed by full-scan electron impact GC/MS after derivatization with acetic anhydride-pyridine. The standard curves for all five drugs were linear between 50 and 1000 ng/mL, with correlation coefficients exceeding 0.99. Coefficients of variation were less than 7%. The method was applied to the analysis of postmortem urines positive by EMIT opiate assay, and the effect of the hydrolysis procedure on recovery of each drug was measured. The results indicate that the hydrolysis procedure is effective in increasing the recovery of all five drugs from urine. The described method enables the laboratory to identify the five opiates most commonly encountered in forensic and clinical laboratories. Its sensitivity for all five drugs is well below GC/MS cutoffs for codeine and morphine employed in NIDA laboratories, and it provides for conclusive full-scan drug identification.

Analysis of cocaine and cocaethylene in blood and tissues by GC-NPD and GC-ion trap mass spectrometry.

Cocaethylene, the ethyl homolog of cocaine, is an active cocaine metabolite found in cocaine users who simultaneously consume cocaine and ethanol. To study the combined incidence of cocaine and cocaethylene, an analytical method was devised that would simultaneously quantify both drugs in whole blood or tissues. The method includes a quantitative procedure by GC-NPD with a confirmation by ion trap mass spectrometry. Propylbenzoylecgonine, the propyl homolog of cocaine, was used as the internal standard. The quantitative method was linear from 0.05 to 10.0 mg/L with a limit of detection of 0.02 mg/L. Cocaethylene and propylbenzoylecgonine were synthesized in the laboratory. The complete analytical procedure and the method of synthesis are presented, along with brief descriptions of medical examiner cases where both cocaine and cocaethylene were quantified and where cocaethylene may have played a part in the cause of death. In the five cases presented, the cocaine concentration ranged from 0.03 to 1.4 mg/L and cocaethylene ranged from 0.03 to 0.53 mg/L in the blood.

The use of hair analysis to document a cocaine overdose following a sustained survival period before death.

Hair analysis for cocaine was used in the investigation of a case of accidental cocaine poisoning. A 25-year-old man consumed an entire bottle of Pony Malta, an imported Colombian soft drink. Unbeknownst to him, the 6.2-oz bottle contained a large quantity of cocaine. It was subsequently discovered that this bottle was one of 45 recovered that contained cocaine as part of a smuggling scheme. The subject was hospitalized for acute cocaine intoxication but died 24 days later. The admission blood contained 2.3 mg/L of cocaine and 4.5 mg/L of benzoylecgonine. Subsequent segmental analysis by MS/MS of hair samples taken at the autopsy revealed a peak cocaine concentration in the segment corresponding to the time he ingested the tainted beverage. The use of hair analysis as an adjunct to traditional analytical techniques may help document drug use history and is especially useful in situations have blood and urine specimens have not been collected in a timely fashion.

Cocaethylene is more potent than cocaine in mediating lethality.

Cocaethylene is a pharmacologically active cocaine metabolite that is formed in the presence of ethanol by the activity of liver enzymes. The pharmacology of cocaethylene has not been extensively investigated and its acute toxicity is unknown. The acute toxicity of cocaethylene was compared to cocaine in Swiss-Webster mice. The LD50 of cocaethylene was 60.7 mg/kg and 63.8 mg/kg in female and male mice, respectively. In comparison, the LD50 of cocaine was 93.0 mg/kg in both female and male mice. These studies demonstrate that the cocaine-alcohol metabolite, cocathylene, is more potent in mediating lethality than the parent drug.

Site-dependent postmortem changes in blood cocaine concentrations.

When a forensic toxicologist interprets postmortem blood cocaine findings he usually must make assumptions regarding perimortem drug concentrations. In-vitro studies have shown that cocaine rapidly hydrolyzes in unpreserved blood, particularly at elevated temperatures. However, other studies have demonstrated site-dependent postmortem release of some drugs from tissue stores accompanied by increases in drug concentrations in the blood. This study was undertaken to investigate whether blood cocaine concentrations change in the body during the postmortem interval and, if so, to measure the direction and magnitude of the changes. In medical examiner cases in which scene investigation suggested that the decreased was a cocaine user, blood samples were collected as soon after death as possible. At autopsy, a second set of samples was collected. Analysis of paired samples by gas chromatography/mass spectrometry (GC/MS) revealed dramatic differences in the cocaine concentration. The magnitude and direction of the change appears to be site dependent. Usually, but not invariably, cocaine concentration in subclavian vein blood decreases while that in heart, aorta, and femoral vein blood increases during the interval between death and autopsy. The findings emphasize the danger inherent in attempting to estimate the concentration of cocaine in blood at the time of death from postmortem data.

Cocaethylene: a unique cocaine metabolite displays high affinity for the dopamine transporter.

Concurrent cocaine and alcohol use is common practice in the general population, as indicated by recent prevalence studies. In the presence of ethyl alcohol, cocaine is metabolized to its ethyl homolog, cocaethylene. The transesterification of cocaine and ethanol to cocaethylene takes place in the liver and represents a novel metabolic reaction. Cocaethylene was detected in postmortem blood, liver, and neurological tissues in concentrations equal to and sometimes exceeding those of cocaine. In vitro binding studies demonstrate that cocaethylene has a pharmacological profile similar but not identical to that of cocaine at monoamine transport sites assayed in the human brain. Cocaethylene was equipotent to cocaine at inhibiting [3H]mazindol binding to the dopamine transporter. The blockade of dopamine reuptake in the synaptic cleft by cocaethylene may account for the enhanced euphoria associated with combined alcohol and cocaine abuse.