A placebo-controlled study to assess Standardized Field Sobriety Tests performance during alcohol and cannabis intoxication in heavy cannabis users and accuracy of point of collection testing devices for detecting THC in oral fluid.

RATIONALE: Standardized Field Sobriety Tests (SFST) and oral fluid devices are used to screen for driving impairment and roadside drug detection, respectively. SFST have been validated for alcohol, but their sensitivity to impairment induced by other drugs is relatively unknown. The sensitivity and specificity for Δ(9)-tetrahydrocannabinol (THC) of most oral fluid devices have been low. OBJECTIVE: This study assessed the effects of smoking cannabis with and without alcohol on SFST performance. Presence of THC in oral fluid was examined with two devices (Dräger Drug Test® 5000 and Securetec Drugwipe® 5). METHODS: Twenty heavy cannabis users (15 males and 5 females; mean age, 24.3 years) participated in a double-blind, placebo-controlled study assessing percentage of impaired individuals on the SFST and the sensitivity of two oral fluid devices. Participants received alcohol doses or alcohol placebo in combination with 400 µg/kg body weight THC. We aimed to reach peak blood alcohol concentration values of 0.5 and 0.7 mg/mL. RESULTS: Cannabis was significantly related to performance on the one-leg stand (p = 0.037). Alcohol in combination with cannabis was significantly related to impairment on horizontal gaze nystagmus (p = 0.029). The Dräger Drug Test® 5000 demonstrated a high sensitivity for THC, whereas the sensitivity of the Securetec Drugwipe® 5 was low. CONCLUSIONS: SFST were mildly sensitive to impairment from cannabis in heavy users. Lack of sensitivity might be attributed to tolerance and time of testing. SFST were sensitive to both doses of alcohol. The Dräger Drug Test® 5000 appears to be a promising tool for detecting THC in oral fluid as far as correct THC detection is concerned.

Neurocognitive performance during acute THC intoxication in heavy and occasional cannabis users.

Performance impairment during Delta(9)-tetrahydrocannabinol (THC) intoxication has been well described in occasional cannabis users. It is less clear whether tolerance develops to the impairing effects of THC in heavy users of cannabis. The aim of the present study was to assess neurocognitive performance during acute THC intoxication in occasional and heavy users. Twenty-four subjects (12 occasional cannabis users and 12 heavy cannabis users) participated in a double-blind, placebo-controlled, two-way mixed model design. Both groups received single doses of THC placebo and 500 microg/kg THC by smoking. Performance tests were conducted at regular intervals between 0 and 8 h after smoking, and included measures of perceptual motor control (critical tracking task), dual task processing (divided attention task), motor inhibition (stop signal task) and cognition (Tower of London). THC significantly impaired performance of occasional cannabis users on critical tracking, divided attention and the stop signal task. THC did not affect the performance of heavy cannabis users except in the stop signal task, i.e. stop reaction time increased, particularly at high THC concentrations. Group comparisons of overall performance in occasional and heavy users did not reveal any persistent performance differences due to residual THC in heavy users. These data indicate that cannabis use history strongly determines the behavioural response to single doses of THC.

Cognition and motor control as a function of Delta9-THC concentration in serum and oral fluid: limits of impairment.

Cannabis use has been associated with increased risk of becoming involved in traffic accidents; however, the relation between THC concentration and driver impairment is relatively obscure. The present study was designed to define performance impairment as a function of THC in serum and oral fluid in order to provide a scientific framework to the development of per se limits for driving under the influence of cannabis. Twenty recreational users of cannabis participated in a double-blind, placebo-controlled, three-way cross-over study. Subjects were administered single doses of 0, 250 and 500 microg/kg THC by smoking. Performance tests measuring skills related to driving were conducted at regular intervals between 15 min and 6h post smoking and included measures of perceptual-motor control (Critical tracking task), motor impulsivity (Stop signal task) and cognitive function (Tower of London). Blood and oral fluid were collected throughout testing. Results showed a strong and linear relation between THC in serum and oral fluid. Linear relations between magnitude of performance impairment and THC in oral fluid and serum, however, were low. A more promising way to define threshold levels of impairment was found by comparing the proportion of observations showing impairment or no impairment as a function of THC concentration. The proportion of observations showing impairment progressively increased as a function of serum THC in every task. Binomial tests showed an initial and significant shift toward impairment in the Critical tracking task for serum THC concentrations between 2 and 5 ng/ml. At concentrations between 5 and 10 ng/ml approximately 75-90% of the observations were indicative of significant impairment in every performance test. At THC concentrations >30 ng/ml the proportion of observations indicative of significant impairment increased to a full 100% in every performance tests. It is concluded that serum THC concentrations between 2 and 5 ng/ml establish the lower and upper range of a THC limit for impairment.

Practical aspects of roadside tests for administrative traffic offences in Germany.

In the context of the European project ROSITA, the Institut of Legal Medicine Homburg/Saar has co-operated with the Saarland traffic police in order to assess different roadside drug tests for their functionality and reliability in traffic controls, and for their analytical force of evidence. In 254 cases within the time period from June 1999 to December 1999, police officers performed a (voluntary) roadside drug testing in saliva/sweat, or urine, to confirm or refute their initial suspicion that a driver had used drugs. Whereas in 45 cases the tests gave negative results (which were confirmed by lab urinalysis), in 209 cases the police officers ordered blood samples after a positive outcome of the tests. In 203 of the 209 positive cases, the results could be confirmed by GC/MS analysis. Regarding the prevalence of used drugs, a single consumption was found in 156 cases (113 cannabis, 38 amphetamines/methamphetamines, three opiates, two cocaine), and a consumption of two drugs was found in 44 cases (34 cannabis+amphetamines/methamphetamines, five cannabis+opiates, three cannabis + cocaine, two cocaine+amphetamines/methamphetamines). In three cases, multi-consumption was found. In six cases, the performed tests gave an incorrect prediction to the police officer at the roadside.The roadside tests gave 97.6% correct assistance to the police officers in the right direction (79.9% correct positive predictions and 17.7% correct negative predictions). As a consequence, the performed tests can be seen as a positive and needful tool for the police to get an immediate response to their initial suspicion and to take the right steps concerning a following legal action.

Frontline immunochromatographic device for on-site urine testing of amphetamines: laboratory validation using authentic specimens.

We evaluated a new test device for amphetamines and methamphetamines (Frontline, cut-off limit 300 ng/mL) using authentic clinical and forensic specimens. The device is based on immunochromatography and is dipped into urine and read visually by comparison with a colour scale after a few minutes. A total of 658 specimens were tested by comparing results of the screening procedure with established immunoassays. Discordant results were further investigated by gas chromatography mass spectrometry or gas chromatography (with flame ionization detector). The Frontline device had a sensitivity of 93% and a specificity of 98%. When specimens were classified by urine amphetamine concentration, close agreement was obtained at concentrations below 150 ng/mL and above 1000 ng/mL. A small number of specimens with amphetamine concentrations between 300 and 1000 ng/mL tested negative in the Frontline test. This finding could to some extent be explained by the enantioselectivity of the antibodies in the Frontline test to d-amphetamine. We conclude that the performance of the Frontline test device for amphetamines is adequate for presumptive clinical and forensic screening.

Specific attentional dysfunction in adults following early start of cannabis use.

RATIONALE AND OBJECTIVE: The present study tested the hypothesis that chronic interference by cannabis with endogenous cannabinoid systems during peripubertal development causes specific and persistent brain alterations in humans. As an index of cannabinoid action, visual scanning, along with other attentional functions, was chosen. Visual scanning undergoes a major maturation process around age 12-15 years and, in addition, the visual system is known to react specifically and sensitively to cannabinoids. METHODS: From 250 individuals consuming cannabis regularly, 99 healthy pure cannabis users were selected. They were free of any other past or present drug abuse, or history of neuropsychiatric disease. After an interview, physical examination, analysis of routine laboratory parameters, plasma/urine analyses for drugs, and MMPI testing, users and respective controls were subjected to a computer-assisted attention test battery comprising visual scanning, alertness, divided attention, flexibility, and working memory. RESULTS: Of the potential predictors of test performance within the user group, including present age, age of onset of cannabis use, degree of acute intoxication (THC+THCOH plasma levels), and cumulative toxicity (estimated total life dose), an early age of onset turned out to be the only predictor, predicting impaired reaction times exclusively in visual scanning. Early-onset users (onset before age 16; n = 48) showed a significant impairment in reaction times in this function, whereas late-onset users (onset after age 16; n = 51) did not differ from controls (n = 49). CONCLUSIONS: These data suggest that beginning cannabis use during early adolescence may lead to enduring effects on specific attentional functions in adulthood. Apparently, vulnerable periods during brain development exist that are subject to persistent alterations by interfering exogenous cannabinoids.

Determination of drugs of abuse in blood.

The detection and quantitation of drugs of abuse in blood is of growing interest in forensic and clinical toxicology. With the development of highly sensitive chromatographic methods, such as high-performance liquid chromatography (HPLC) with sensitive detectors and gas chromatography-mass spectrometry (GC-MS), more and more substances can be determined in blood. This review includes methods for the determination of the most commonly occurring illicit drugs and their metabolites, which are important for the assessment of drug abuse: Methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), N-ethyl-3,4-methylenedioxyamphetamine (MDEA), 3,4-methylenedioxy-amphetamine (MDA), cannabinoids (delta-9-tetrahydrocannabinol, 11-hydroxy-delta-9-tetrahydrocannabinol, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol), cocaine, benzoylecgonine, ecgonine methyl ester, cocaethylene and the opiates (heroin, 6-monoacetylmorphine, morphine, codeine and dihydrocodeine). A number of drugs/drug metabolites that are structurally close to these substances are included in the tables. Basic information about the biosample assayed, work-up, GC column or LC column and mobile phase, detection mode, reference data and validation data of each procedure is summarized in the tables. Examples of typical applications are presented.

Development and evaluation of immunochromatographic rapid tests for screening of cannabinoids, cocaine, and opiates in urine.

The test principle and the optimization of the reactive ingredients are described for the one-step dip and-read immunochromatographic FRONTLINE rapid tests for drugs-of-abuse testing in urine samples. In a multicenter evaluation the rapid tests were compared with FPIA and EMIT immunoassays. Discrepant results were further analyzed by gas chromatography-mass spectrometry methods. In the comparison of the cannabinoids rapid tests versus both immunoassays using clinical and forensic urine samples (399 versus FPIA and 755 versus EMIT), sensitivities and specificities were 97% or better for both comparisons. For cocaine, a sensitivity of 100% versus both routine technologies was obtained, whereas the specificity was reduced somewhat to 91% because of some cross-reactivity with metabolites of methadone and of clozapine. Specificity was very high for the cocaine rapid tests (98-100%) when applied to urine samples of persons not in a methadone maintenance program. Sensitivities and specificities for the opiates rapid tests were 99% or better at all sites when compared with the routine methods. In the screening of about 1200 clinical urine samples for cannabinoids, cocaine or opiates misuse only six samples would have stayed undetected by rapid test analyzes. These results show the FRONTLINE assays allow a reliable and immediate screening for drugs of abuse.

Influence of pigmentation on the codeine content of hair fibers in guinea pigs.

Tortoise shell guinea pigs (n = 7) were administered codeine (1 mg/mL codeine-base) in their drinking water for 3 weeks. Black, reddish-brown and white hair was collected separately from each animal before and after treatment. The hair samples were analyzed by GC/MS. The experiment showed positive results for all hair fibers with large individual variability of drug incorporation. Low drug intake resulted in small differences of the drug content in hair fibers different in color, whereas in cases of high drug intake a strong influence of hair pigmentation on the analytical results was observed. The highest drug content was always found in black hair samples, non-pigmented hair showed the lowest drug concentrations and the drug content in reddish-brown fibers was less than in black hair samples from the same animal. From the results it was concluded, that eumelanins rather than phenomelanins are the decisive factor for codeine-melanin binding in hair and the amount of drug intake was suggested to determine the relevance of hair pigmentation on the analytical results.

Biochemical approach on the conservation of drug molecules during hair fiber formation.

A biochemical concept for the endogenous incorporation of drug molecules into growing hair is presented. It is based on the principles of transport across biomembranes, on the principles of biotransformation and drug melanin affinity. The approach gives explanations for current observations in hair analysis, which up to date have not been understood sufficiently. Phenomena such as the ratio of parent drug to metabolite in hair, the dependence of incorporation on the physico-chemical properties of the drug, the independence of drug incorporation on active melanogenesis (incorporation into non-pigmented hair) as well as the dependence of drug content on hair pigmentation are elucidated.

On cosmetically treated hair--aspects and pitfalls of interpretation.

Popular hair cosmetic treatments like bleaching or permanent waving were found to affect the stability of incorporated drugs and to cause alterations of the fibers at an ultrastructural level. This may result in a partial or complete loss of drug substances, depending on the particular drug molecule and on its concentration prior to the cosmetic treatment. Moreover, from literature, there is some evidence that drug molecules are not only incorporated into the growing fiber by passive diffusion from blood into the matrix cells and melanocytes, but that the substances enter the hair also via perspiration such as sweat and sebum. Since permed and bleached hair shows an enhanced sorption capacity, the risk of false positives or an unusually high drug concentration in cosmetically treated hair was under investigation. Virgin, permed, mildly as well as severely bleached tresses were exposed to artificial sweat or sebum containing cocaine, benzoylecgonine, 6-acetylmorphine, morphine and codeine (500 ng/g). Except codeine, the concentrations measured by GC/MS were very small and quite close to the detection limit indicating a minor importance of drug uptake into hair fiber from the endogenous-exogenous shunt via sebum or sweat. From the results it is concluded that an increased risk of false positive results in hair analysis on bleached and permanent waved hair fibers does exist, but is not particularly severe.

Influence of sample preparation on analytical results: drug analysis [GC/MS] on hair snippets versus hair powder using various extraction methods.

The comparison of aqueous extraction methods and hair extraction by organic solvents performed on hair powder as well as on hair snippets of the same sample revealed different qualities of the procedures. Qualitative and quantitative results by the same derivatization step and GC/MS detection demonstrated, that the risk of missing a drug substance is higher using hair snippets than after drug extraction on pulverised hair. Drug recovery for opiates, cocaine and benzoylecgonine from hair was found to be best in aqueous solvents or in methanol extracts. The results are discussed under the aspects of solid-phase extraction, the hair sample representing an inhomogenous material. The localisation of drug molecules in hair, the hair swelling and penetration behavior of the particular extraction medium as well as the partition coefficient of solvent/hair phase for a particular drug substance are considered to influence the results.

Hair analysis as evidence in forensic cases.

The analysis of hair for drugs of abuse is a powerful tool useful in answering questions that cannot be solved by the detection of drugs in body fluids, e.g., blood, urine, or saliva. The most frequent forensic investigations occur in cases where narcotics laws are offended and in questions of criminal responsibility where the chronic use of a drug is an issue. In the list of drugs of abuse, heroin, cocaine, amphetamine, and cannabis are the drugs of abuse that are most frequently involved in judicial inquiries.

On pathways for small molecules into and out of human hair fibers.

This paper represents an experimental approach of histology of the human hair fiber in dyeing and diffusion phenomena and its contribution to the interpretation of hair analysis results for drug abuse. Rhodamine B was applied to human hair fibers from either aqueous solution or methanol/ethanol solvent. The experiments were performed on natural hair of different ethnic groups as well as on extensively bleached hair strands. The microscopical study of the pathway of diffusion of rhodamine B into the hair fibers indicated that the reagent had entered the unmodified fibers at the scale edges between the cuticle cells. At the beginning of the diffusion process intercellular diffusion was the preferred route predominantly along the nonkeratinous regions of the cell membrane complex (CMC) and intermacrofibrillar cement. Penetration into the high sulfur regions of the fiber occurred as dyeing in aqueous solution proceeded and resulted in evenly stained cross sections. The dye distribution pattern observed in natural hair exposed to nonaqueous solution showed that rhodamine B did not penetrate the cortex cells as easy as from aqueous solution and selectively stained nonkeratinous regions only. The determination of the amount of dye taken up by the fibers by spectrophotometric analysis demonstrated that samples diffusion generally increased by time and temperature. It also depended on the morphology of the hair sample. The penetration of rhodamine B from aqueous solution was much greater than from methanol/ethanol solvent.

The detection of 6-monoacetylmorphine in urine, serum and hair by GC/MS and RIA.

6-Monoacetylmorphine (6-MAM) is a good indicator for the intake of heroin and can be detected in blood, urine and hair of heroin users. A new radioimmunoassay (RIA) designed specifically for 6-monoacetylmorphine (6-MAM) was tested for its usefulness for the quantitation of the drug in urine, serum and hair. Its cross-reactivity with heroin and its metabolites, and related compounds was also determined. Eighty-nine hair, six serum and 25 urine samples where 6-MAM had been previously identified by GC/MS were analysed for 6-MAM with the new RIA kit. A good correlation existed between the GC/MS and RIA results for the hair samples. However, the amount of 6-MAM found in serum and urine differed considerably between the two methods. This difference could be explained by the cross-reactivity of the antibody with morphine and morphine-6-glucuronide, which are present in much larger amounts in serum and urine, than in hair. To evaluate a new rationalisation procedure, some hair samples were split into two portions after incubation. One part was analyzed for 6-MAM by RIA, and the other portion by GC/MS.

Simultaneous determination of drugs of abuse (opiates, cocaine and amphetamine) in human hair by GC/MS and its application to a methadone treatment program.

A new method was developed for the simultaneous detection and quantitation of 6-acetyl-morphine (MAM), amphetamine, benzoylecgonine (BZE), cocaine, codeine, dihydrocodeine, EDDP (methadone metabolite), methadone and morphine in hair. The hair samples were washed, cut into 2-cm segments, pulverized, incubated with phosphate buffer and beta-glucuronidase/aryl-sulfatase. After solid phase extraction and derivatization with pentafluoropropionic anhydride/pentafluoropropanol, the drugs were identified and measured by gas chromatography/mass spectrometry using their deuterated analogues as internal standards. The method is reproducible with detection limits under 0.1 ng/mg hair for almost all substances tested. Fifteen hair samples from five subjects of a methadone treatment program were collected in a 6-month period. The hair samples were segmented and examined for methadone, its main metabolite EDDP, and drugs of abuse. Of the 96 segments analysed, 95% were positive for methadone (mean value, 10.9 ng/mg), 76% for the metabolite EDDP (mean value, 1.2 ng/mg), 69% for opiates (mean values, MAM, 7.3 ng/mg; morphine, 2.9 ng/mg; codeine, 1.0 ng/mg) and 43% for cocaine (mean values, cocaine, 2.6 ng/mg; BZE, 1.1 ng/mg). A correlation of 0.63 was found between administered methadone dosages and concentrations measured by hair analysis. Further investigation is needed to clarify interindividual differences.

Hair analysis as evidence in forensic cases.

Because hair analysis can be used for the determination of drug use months after drug consumption, hair analysis data can often act as important and even decisive evidence in the courtroom. More recently developed GC/MS methods offer excellent sensitivity and can make the distinction between chronic heroin and codeine use, which was not possible earlier with radioimmunoassay techniques. From more than a thousand hair analyses, the morphine/codeine ratios necessary to determine heroin use were set at 5:1 for low morphine concentrations (< 1 ng/mg hair) and 2:1 for concentrations above 1 ng/mg hair. The distinction can be further focused with the additional analysis of the metabolite monoacetylmorphine (MAM). As can be seen from several case examples, hair analysis cannot pinpoint an exact date of opiate use, but it can be used to validate or invalidate a subject's statement concerning his/her drug consumption. Interpretations should always be made cautiously. Ranges, means and medians are also listed for amphetamine, cocaine and cannabis and work is under way to draw similar safety guidelines for these drugs.

Drug detection in hair by chromatographic procedures.

This article reviews the analysis of 31 drugs and drug metabolites in human hair by thin-layer chromatography, high-performance liquid chromatography, gas chromatography, gas chromatography-mass spectrometry and mass spectrometry. The most important detection method after chromatographic separation of the components is the mass spectrometry because of its sensitivity and specificity. Washing steps to exclude external contamination, extraction, derivatization, stationary phases, detection modes and detection limits of the mass spectrometric and gas chromatographic-mass spectrometric procedures are presented in five tables. Additionally, a method for a gas chromatographic-mass spectrometric screening procedure is presented.

Simultaneous quantitation of delta-9-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in serum by GC/MS using deuterated internal standards and its application to a smoking study and forensic cases.

A new procedure for the simultaneous detection of delta-9-tetrahydrocannabinol (THC) and its major metabolite, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in serum has been evaluated. The method combines rapid, efficient, solid-phase extraction and simple derivatization by methylation. Analysis and quantitation is performed by gas chromatography/mass spectrometry (GC/MS) using deuterated cannabinoids as internal standards (IS). Reproducibility and sensitivity of the method are good. The procedure is applied to serum specimens collected from a smoking study with 24 volunteers and 212 forensic cases. Results are interpreted based upon the current knowledge about THC metabolism and pharmacokinetics.