Advances in forensic toxicology.

Over the last 15 years there have been many changes in the practice of forensic toxicology. One of the most noteworthy has been the recognition of the need for good laboratory practices in the forensic toxicology laboratory. This has resulted in the development of an accreditation program for laboratories. Increasingly, forensic toxicologists are asked to interpret results in driving under the influence of drug cases. These interpretations are also difficult because of the lack of data correlating blood (or plasma) concentrations with impairment. The development of newer immunoassays and hyphenated mass spectrometric techniques now allow the forensic toxicologist to assay a large number of drugs (both traditional and products of the biotechnology revolution) with increasing sensitivity. This article focuses on these changes and some of the challenges facing the forensic toxicologists of the 21st century.

Financial viability of screening for drugs of abuse.

Urine drug testing is now a common practice in the American workplace; a recent survey indicated that > 90% of companies with > 5000 employees have some type of testing program. These programs have indeed reduced the rate of drug-positive test results; for example, recent data from the Federal Aviation Agency show that the rate for 1993 was 0.82% compared with 0.95% for 1992. Many corporations have stated that urine drug testing, as a component of a substance abuse policy, results in significant savings, e.g., from decreased absenteeism and turnover. The United States Postal Service recently completed a longitudinal study on the economic benefits and found that, over the average tenure of an annual intake of employees, there were savings of more than $100 million. Although this study clearly demonstrates the financial benefits of preemployment drug testing, the decision to test is not based solely on this but also on the regulatory environment and on the potential impact of a major accident attributable to the use of drugs or alcohol in the workplace.

Analytical and technical aspects of testing for drug abuse: confirmatory procedures.

Many laboratories are now performing urine drug testing for employers, governmental agencies, and other institutions. It is now recognized that presumptive positive screening results have to be confirmed by an analytical procedure based on a different chemical technique with greater than or equal sensitivity to the screening test. Thin-layer chromatography has been widely used for this; however, it is relatively insensitive for certain drugs, and it cannot satisfy the accuracy and precision requirements needed to determine threshold concentrations reliably. Gas chromatography-mass spectrometry is able to satisfy these threshold requirements and has become the method of choice for confirming initial immunoassay results.

Toxicological findings in a fatal overdose of verapamil.

Presented is a case where the death was attributed to the deliberate ingestion of an overdose of verapamil (V). Blood, urine, and gastric concentrations of the drug were determined by gas chromatography with nitrogen phosphorus detection (GC-NPD). Identification of norverapamil (NV) was made. A presumptive identification of o-demethylnorverapamil (DNV) was also made.

Profiles of delta 9-tetrahydrocannabinol metabolites in urine of marijuana users: preliminary observations by high performance liquid chromatography-radioimmunoassay.

Metabolic profiles of 11-nor-9-carboxylic acid-delta 9-tetrahydrocannabinol (COOH-THC) and other THC metabolites were determined in an infrequent and a frequent marijuana user by high performance liquid chromatography-radioimmunoassay (HPLC-RIA). In the infrequent user, no unconjugated COOH-THC was detected in urine samples for the first 8 h following smoking, whereas this metabolite was detected in the urine samples from a frequent user. A metabolite was also detected in the frequent user, which was not present in the urine sample from the infrequent user.

The acute effects of methamphetamine, amphetamine and p-chloroamphetamine on the cortical serotonergic system of the rat brain: evidence for differences in the effects of methamphetamine and amphetamine.

Cortical tryptophan hydroxylase (TPH) activity was reduced 3 h after a 10 or 15 mg/kg i.p. dose of either amphetamine (AMP), methamphetamine (METH), or p-chloroamphetamine (PCA). These injections of METH or PCA also decreased cortical serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) concentrations; none of the four doses of AMP decreased indoleamine concentrations. The time course of the effects following a 15 mg/kg dose of each amine was also different. Cortical TPH activity was reduced by all three amines for periods up to 24 h, whereas only METH and PCA significantly decreased 5-HT and 5-HIAA concentrations for long periods. These data suggest that each of the amphetamines may inhibit TPH activity, whereas only METH and PCA produced long-lasting decreases in indoleamine concentrations, reflecting either varying degrees of toxicity or differential effects of AMP on enzyme activity and neurotransmitter concentrations.

Concentrations of lidocaine and monoethylglycylxylidide (MEGX) in lidocaine associated deaths.

Concentrations of lidocaine and MEGX were determined in a variety of tissues and other samples collected at autopsy. In 13 of the cases examined in which lidocaine was associated with death, tissue concentrations were greater than 15 mg/kg. Tissue concentrations in other patients treated with lidocaine were significantly lower.

Studies on the mechanism of tolerance to methamphetamine.

We have reported that the ability of high doses of methamphetamine to impair dopamine and serotonin synthesis in the rat brain is attenuated when animals are pretreated with gradually increasing doses of methamphetamine. To examine the mechanism of this tolerance phenomenon, the effect of methamphetamine on several neurochemical parameters was determined in naive and methamphetamine-pretreated rats. The elevation of nigral substance P concentrations by methamphetamine was attenuated in pretreated compared to naive rats. The methamphetamine-induced reduction in [3H]sulpiride binding in the rat neostriatum and nucleus accumbens was similarly attenuated in animals pretreated with methamphetamine. Determination of brain concentrations of methamphetamine and amphetamine revealed significantly lower concentrations of both compounds in the brains of pretreated compared to naive animals. The results indicate a reduction in the ability of methamphetamine to increase dopamine transmission in the brains of methamphetamine-pretreated rats. Furthermore, this effect appears to be due, at least in part, to a change in the disposition of methamphetamine in pretreated animals.

Effect of cimetidine on verapamil disposition.

The effects of multiple doses of cimetidine on single-dose verapamil kinetics were studied in nine healthy men. Baseline hepatic blood flow was estimated by indocyanine green elimination on day 1. On day 2, the subjects received verapamil, 10 mg iv, after which the plasma concentration-time profile was determined. After a 2-day washout, cimetidine, 300 mg, was taken by mouth four times a day for 5 days. The indocyanine green study was repeated on day 9 and verapamil was taken on day 10. Cimetidine reduced verapamil clearance by 21% and increased the elimination t1/2 by 50%. The volume of distribution at steady state did not change. Cimetidine increased hepatic blood flow in some subjects, while decreasing it in others. There was no correlation between individual changes in verapamil clearance and hepatic blood flow. These data indicate that cimetidine reduces verapamil clearance by mechanism(s) other than a change in hepatic blood flow or volume of distribution.

Methamphetamine-induced depression of monoamine synthesis in the rat: development of tolerance.

Animals treated with high doses of amphetamines have been used as a model of schizophrenia due to the similarities between the psychosis associated with this mental disorder and that induced by chronic amphetamine abuse. When administered to naive rats in high doses, the amphetamine-like CNS stimulant methamphetamine produces drastic alterations in the neurochemical parameters of the neostriatal monoaminergic systems. These alterations are characterized by a decrease in the activities of the rate-limiting enzymes for dopamine and serotonin synthesis, as well as a decrease in the concentrations of both neurotransmitters and their metabolites. However, tolerance develops to these neurochemical effects when drug administration occurs in a pattern similar to that encountered during chronic amphetamine abuse. The results indicate that the neurochemical alterations produced by amphetamines in naive and tolerant animals differ widely. This suggests that the administration of high doses of amphetamine-like central stimulants to naive rats may not be an appropriate model for studying the neurochemical changes associated with psychosis and amphetamine abuse.

Drugs in fatally injured young male drivers.

One or more drugs were detected in 81 percent of 440 male drivers, aged 15-34, killed in motor vehicle crashes in California; two or more drugs were detected in 43 percent. Alcohol, the most frequently found drug, was detected in 70 percent of the drivers, marijuana in 37 percent, and cocaine in 11 percent. Each of 24 other drugs was detected in fewer than 5 percent. Except for alcohol, drugs were infrequently found alone; typically, they were found in combination with high blood alcohol concentrations. The causal role of drugs in crashes was assessed by comparing drivers with and without drugs in terms of their responsibility for the crash. Alcohol was associated with increased crash responsibility; the role of other drugs could not be adequately determined.

Stability of delta 9-tetrahydrocannabinol (THC), 11-hydroxy-THC, and 11-nor-9-carboxy-THC in blood and plasma.

The stabilities of delta 9-tetrahydrocannabinol (THC) and two of its metabolites, 11-hydroxy-delta 9-tetrahydrocannabinol (HO-THC) and 11-nor-9-carboxy-delta 9-tetrahydrocannabinol (COOH-THC), were determined in blood and plasma stored at -10 degrees C, 4 degrees C, and room temperature. Each of the cannabinoids was added to freshly-drawn blood and plasma to give concentrations of 20 ng/mL. Two-mL aliquots were stored in silanized tubes and the cannabinoid concentrations were monitored by gas chromatography/mass spectrometry over a 6-month period. No significant changes were observed in the concentrations of the cannabinoids for the first month of storage. However, the concentrations of THC and HO-THC in blood stored at room temperature had decreased significantly at 2 months. No statistically significant changes were detected in cannabinoid concentrations in plasma or blood stored at 4 degrees or -10 degrees C for up to 4 months. After 6 months at room temperature, the blood concentrations of THC and HO-THC had decreased by 90 and 44%, respectively, whereas the concentration of COOH-THC was not significantly different from the control. The possibility of loss of cannabinoids from blood due to adsorption onto the grey stoppers used in Venoject tubes was also investigated. Over a 24-hr period, no significant differences were detected in any of the cannabinoid concentrations regardless of sample size (1.3 or 8 mL), differences in temperature (-10 degrees C, 4 degrees C, or room temperature), or extent of contact with the tube's stoppers.

The effects of a single dose of amphetamine and iprindole on the serotonergic system of the rat brain.

Rats treated with iprindole (IPR) (10 mg/kg, i.p.) were given a single dose (15 mg/kg, i.p.) of amphetamine (AMP). Marked decreases in the activity of tryptophan hydroxylase (TPH) were observed in both the cerebral cortex and neostriatum after 6 hr, with maximum depression observed at 24 hr. Enzyme activity had returned to control levels in neostriatum after 3 days and in cerebral cortex after 7 days. Levels of serotonin (5-HT) in both the cerebral cortex and neostriatum were significantly lowered at 24 hr but had recovered by 72 hr. Levels of tryptophan (TRP) in the cortex were significantly elevated after 6 hr, recovering by 24 hr. In the neostriatum, the activity of tyrosine hydroxylase (TH) was significantly depressed by 24 hr and remained so for 7 days. Concentrations of dopamine (DA) were decreased at all times examined. This study clarifies the differences previously observed in the response of the serotonergic system to amphetamine or methamphetamine (METH) in iprindole-treated rats.

Quantitative analysis of emetine and cephaeline by reversed-phase high performance liquid chromatography with fluorescence detection.

A versatile method for the quantitation of emetine and cephaeline in biological samples is described. Two milliliters of samples containing N-propylprocainamide as the internal standard are buffered to pH 9 and extracted with n-butyl chloride. After subsequent back extraction into 0.01 M hydrochloric acid, a portion of the acid layer is analyzed by reversed-phase high performance liquid chromatography with fluorescence detection. Routinely, the minimum level of detection for both drugs is 5 ng/mL and linearity is demonstrated from 5 to 2500 ng/mL.

High performance liquid chromatography-immunoassay of delta 9-tetrahydrocannabinol and its metabolites in urine.

High performance liquid chromatographic-immunoassay (HPLC-IA) profiles of cannabinoid metabolites in urine samples were obtained using four different antisera. The urines were chromatographed on a reverse phase system using a gradient of acetonitrile in water (pH 3.3) and fractions collected every 30 s. Some urine samples were hydrolyzed with methanolic sodium hydroxide before fractionation. Peaks of immunoreactivity were detected at a fraction corresponding to 11-nor-9-carboxy-delta 9-tetrahydrocannabinol (COOH-THC) and at an early eluting fraction; however, the profiles depended upon the specificity of the antisera used.

Drugs and driving: a systematic analytical approach.

To collect useful epidemiological data about drug involvement in highway safety, it is essential that sensitive and specific analytical procedures be used to establish the presence of and to determine the concentrations of drugs and metabolites in samples collected from drivers. This paper describes a comprehensive and systematic screening procedure requiring 6 mL of blood, which has been used for the analysis of samples collected from injured and fatally injured drivers. The procedure uses radioimmunoassay, gas chromatography with selective detectors, and high performance liquid chromatography. Drugs and metabolites presumptively identified are then confirmed primarily using gas chromatography--chemical ionization mass spectrometry.

Effects of a single dose of methamphetamine and iprindole on the serotonergic and dopaminergic system of the rat brain.

A single dose (17.5 mg/kg i.p.) of methamphetamine was administered to iprindole-treated (10 mg/kg i.p.) rats. Forebrain concentrations of methamphetamine and amphetamine were significantly increased in iprindole-treated rats 1 and 6 hr after injection; in contrast to rats pretreated with saline, both amines were also detected after 18 hr. Three and 7 days after injection, significant decreases were seen in tryptophan hydroxylase (TPH) activity and serotonin concentrations in the cerebral cortex, neostriatum and hypothalamus. Hypothalamic TPH activity had recovered by 14 days. Neostriatal tyrosine hydroxylase activity and dopamine concentrations were significantly depressed at all time points examined. Iprindole alone produced a significant increase in cortical TPH activity after 1 day. After 3 days, TPH activity was significantly decreased when compared with control, whereas serotonin and 5-hydroxyindoleacetic acid concentrations were significantly increased. This study demonstrates that persistence of methamphetamine and/or amphetamine at the site of action is important for neurotoxicity.

The effects of phencyclidine on glutamic acid decarboxylase activity in several regions of the rat brain.

Glutamic acid decarboxylase (GAD) activity in several regions of the rat brain were monitored after administration of phencyclidine. Sub-acute (4 injections over 12 h) treatment decreased cerebellar GAD activity 6 and 12 h after the last dose; recovery was noticed by 24 h. This effect occurred with doses of 5 and 10 mg/kg. GAD activity in other brain regions was not affected by this treatment. Acute and chronic treatments with phencyclidine caused no change in GAD activity in any of the brain regions examined.