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Since lethal insulin injection has been used in murder and suicide cases, its non-ambiguous detection in postmortem, mostly hemolytic blood samples is still a problem. In the present study the stability of insulin and reasons for its loss in those blood samples were examined. When incubated with buffer, serum or with intact blood cell suspensions insulin concentrations were found to remain stable over time, but a significant loss of insulin was observed in hemolyzed blood samples. This was not due to an enzymatic cleavage, but predominantly to the presence of hemoglobin. Incubation of insulin with a hemoglobin solution containing the same hemoglobin content as hemolyzed blood caused a dramatic decrease of the insulin concentration. Degradation of insulin reached its maximum after 23 h of incubation. The charge state of the ferric ion of hemoglobin could not be held accountable for the insulin-loss, but rather the protein part of hemoglobin. Alkylation experiments using iodoacetamide suggested that the thiol groups of the globin molecule are involved in the insulin loss preventing degradation at least partially. The same was observed by lowering the pH to 2.7 in the incubation mixture. Two degradation products of insulin were identified by mass spectrometry such as modified insulin A and B chains with 4 (A chain) and 2 Da (B chain) lower masses. These results suggest that thiol groups of hemoglobin cause splitting of the disulfide bonds of insulin which immediately leads to the formation of new intramolecular disulfide bridges, a reaction which occurs in hemolytic blood and may explain the gradual loss of insulin in postmortem blood samples.

Oral fluid (OF) tests aid in identifying drivers under the influence of drugs. In this study, 17 heavy cannabis users consumed alcohol to achieve steady blood alcohol concentrations of 0 to 0.7 g/L and smoked cannabis 3 h afterward. OF samples were obtained before and up to 4 h after smoking and on-site tests were performed (Drager DrugTest 5000 and Securetec DrugWipe 51). Maximum concentrations of tetrahydrocannabinol (THC) immedi- ately after smoking (up to 44,412 ng/g) were below 4,300 (median 377) ng/g 1 h after smoking and less than 312 (median 88) ng/g 3 h later with 5 of 49 samples negative, suggesting that recent can- nabis use might occasionally not be detectable. An influence of alcohol was not observed. Drinking 300 mL variably influenced THC concentrations (median only -29.6%), which suggests that drinking does not markedly affect on-site test performance. Many (92%) Drager tests performed 4 h after smoking were still positive, indi- cating sufficient sensitivity for recent cannabis use. Differences in the results of a roadside study with DrugTest 5000 (sensitivity 84.8%, specificity 96.0%, accuracy 84.3%) could be explained by a higher number of true negatives, differences between OF and serum and differences between occasional and chronic users.

RATIONALE: Experimental research has shown that 3,4-methylenedioxymethamphetamine (MDMA) can improve some psychomotor driving skills when administered during the day. In real life, however, MDMA is taken during the night, and driving may likely occur early in the morning after a night of "raving" and sleep loss. OBJECTIVES: The present study assessed the effects of MDMA on road-tracking and car-following performance in on-the-road driving tests in normal traffic. METHODS: Sixteen recreational MDMA users participated in a randomized double-blind placebo-controlled four-way cross-over design. They received single, evening doses of 0, 25, 50, and 100 mg MDMA on separate occasions. Actual driving tests were conducted in the evening when MDMA serum concentrations were maximal and in the morning after a night of sleep loss. RESULTS: The primary measure of driving, i.e., standard deviation of lateral position (SDLP, a measure of weaving) was significantly increased during driving tests in the morning in all treatment conditions, irrespective of MDMA dose and concentration. The increments in SDLP were of high clinical relevance and comparable to those observed for alcohol at blood alcohol concentrations >0.8 mg/mL. These impairments were primarily caused by sleep loss. CONCLUSIONS: In general, MDMA did not affect driving performance nor did it change the impairing effects of sleep loss. It is concluded that MDMA cannot compensate for the impairing effects of sleep loss and that drivers who are under the influence of MDMA and sleep deprived are unfit to drive.

In view of the widespread application of methylphenidate for attention-deficit/ hyperactivity disorder (ADHD) therapy its interaction with alcohol was investigated in an in-vitro assay and in a study involving 9 male volunteers. The study conditions were: methylphenidate (20 mg) only, methylphenidate followed by ethanol (0.8 g/kg body weight) and ethanol followed by methylphenidate. Methylphenidate (CAS 113-45-1), ritalinic acid (CAS 19395-41-6) and ethylphenidate (CAS 57413-43-1) were assayed in blood samples collected up to 7 h after ingestion using liquid chromatography-mass spectrometry (LC/MS). It was found that methylphenidate is hydrolyzed to ritalinic acid by the same esterase that degrades cocaine. In the presence of ethanol this is inhibited and the active metabolite ethylphenidate is formed. The pharmacokinetic evaluation showed that methylphenidate concentrations were not markedly affected by ethanol, but ritalinic acid concentrations were lower, especially if ethanol was ingested first. Ethylphenidate concentrations were low with only about 10% of methylphenidate concentrations suggesting that concurrent ethanol use does not impair methylphenidate's therapeutic efficacy. Unexpectedly one subject exhibited a methylphenidate hydrolysis defect yielding very high methylphenidate and low ritalinic acid concentrations in all study conditions.

Cannabis is not only the most widely used illicit drug worldwide but is also regularly consumed along with ethanol. In previous studies, it was assumed that cannabis users develop cross-tolerance to ethanol effects. The present study was designed to compare the effects of ethanol in comparison to and in combination with a cannabis joint and investigate changes in pharmacokinetics. In this study, 19 heavy cannabis users participated and received three alcohol dosing conditions that were calculated to achieve steady blood alcohol concentrations (BAC) of about 0, 0.5 and 0.7 g/l during a 5-h time window. Subjects smoked a Delta(9)-tetrahydrocannabinol (THC) cigarette (400 mu g/kg) 3 h post-onset of alcohol dosing. Blood samples were taken between 0 and 4 h after smoking. During the first hour, samples were collected every 15 min and every 30 min thereafter. Mean steady-state BACs reached 0, 0.36 and 0.5 g/l. The apparent elimination half-life of THC was slightly prolonged (1.59 vs. 1.93 h, p

Saliva is of continuing interest in detecting the influence of drugs while driving. Commercial tests are currently available where cannabis detectability is a major challenge. The present study aids in the understanding of tetrahydrocannabinol (THC) pharmacokinetics in oral fluid. The oral fluid analyses exhibited no significant differences between 12 occasional users and 12 chronic users smoking a standardized cannabis joint, except for the maximum concentrations in the first samples (occasional users, 397- 6438 ng/g; chronic users 387-71,747 ng/g). THC was detectable in all samples with medians in the last samples (8 h) of 6.3 and 11.3 ng/g in occasional and chronic users, respectively. The elimination half-life in both groups was 1.6 +/- 0.4 h. A series of samples was obtained over a period of 8 h without actual drug use representing a later elimination phase. Of these oral fluid samples, only 4.3% were negative for THC despite positive serum, and 24.1% of serum samples were negative despite positive oral fluid. This confirms that THC is detectable for longer in oral fluid than in serum. The oral fluid-to-serum ratios were 0.3 to 425 (median 16.5) with no difference between chronic and occasional users. The large inter- and intraindividual variability observed precludes a reliable estimation of THC serum concentrations from oral fluid data using this collection device.

Es wird uber den Fall eines 56 Jahre alt gewordenen Mannes berichtet, der durch eine in suizidaler Absicht erfolgte Monointoxikation mit Rohypnol-Filmtabletten (Flunitrazepam) zu Tode kam. Toxikologische Untersuchungen erbrachten den Nachweis einer nur kurze Zeit uberlebten Flunitrazepamintoxikation. Weitere Drogen, Medikamente oder Alkohol konnten nicht nachgewiesen werden. Bei der gerichtlichen Leichenoffnung fand sich eine Durchsetzung des rechten Lungenunterlappens mit weislichen Herden, fraglich wie bei einer Geschwulstbildung oder Lungenentzundung. Zunachst wurde dieser Befund als mittelbare Folge der Flunitrazepamintoxikation und der damit verbundenen Bewusstseinseinschrankung vermutet. Die weitere histologische Aufarbeitung ergab jedoch eine ausgedehnte, frische, aber mindestens 2–3 Tage alte Bronchopneumonie. Die Lungen zeigten daruber hinaus Zeichen einer akuten und einer chronischen Lungenstauung mit lokaler Fibrosierung sowie ein deutliches Lungenemphysem als weitere Hinweise auf vorbestehende Lungenerkrankungen. Todesursachlich war die Kombination aus vorbestehender Erkrankung (Bronchopneumonie) und Nebenwirkung des Flunitrazepams (Atemdepression). Dieser Fall unterstreicht einmal mehr, dass die Feststellung von Monointoxikationen mit Benzodiazepinen als alleinige Todesursache sehr sorgfaltiger und alle Untersuchungsmoglichkeiten ausschopfender Masnahmen bedarf, um im Ergebnis haltbar zu sein.

Travel-related poisoning is an emerging social and public health emergency in Bangladesh but its cause and significance have not been determined. To investigate this syndrome we performed a prospective clinical study and retrospective analysis of hospital records in a general medicine unit of a public tertiary care teaching hospital in Dhaka, Bangladesh, using toxicological analysis by fluorescence polarization immunoassay (FPIA) and liquid chromatography coupled to time-of-flight mass spectrometry (LC–TOF MS). The participants of the prospective study were 130 consecutive patients aged 16–80 years who were admitted with central nervous system depression (Glasgow Coma Score 3–14) after using public transportation, in the absence of other abnormalities, from January through June 2004, and a convenience sample of 15 such patients admitted during 3 days in May 2006. In 2004–2006, travel-related poisoning increased from 6.1 to 9.5% of all admissions (210–309 of 3266–3843 per year), representing 46.6–55.7% of all admitted poisoning cases. Incidents were associated with bus (76%), taxi, train, and air travel, or local markets; 98% of patients remembered buying or accepting food or drinks before losing consciousness. Direct financial damage (missing property) was diverse and frequently existential. Among 94 urine samples analyzed by FPIA, 74% tested positive for benzodiazepines. Among 15 urine samples analyzed by LC–TOF MS, lorazepam was detected in all; five also contained diazepam or metabolites; nitrazepam was present in three. FPIA results obtained for these 15 samples were below the recommended cut-off in eight (53%; lorazepam only). Our findings show that the massive medicosocial emergency of travel-related poisoning in Bangladesh is the result of drug-facilitated organized crime and that benzodiazepine drugs are used to commit these crimes, suggesting modifications to the local emergency management of the victims of this type of poisoning. They also highlight the need for more research in the neglected field of acute poisoning in Bangladesh, and for criminal investigations of the use of benzodiazepine drugs in this country.

Grundproblematik und Fragestellung: Die Engelstrompete (Brugmansia sp.) ist aufgrund ihrer einfachen Haltung und uppigen Blutenpracht als Gartenpflanze weit verbreitet. Sie gehort zur Familie der Nachtschattengewachse (Solanaceae) und enthalt erhebliche Mengen an Alkaloiden (Parasympatholytika). Aufgrund ihrer halluzinatorischen Wirkung werden Blatter und Bluten von Jugendlichen zunehmend als »LSD-Ersatz« konsumiert. Im Sommer 1997 ereignete sich ein Todesfall, nachdem eine Gruppe Jugendlicher die aus Vorgarten gesammelten Bluten verzehrt hatte. Zur Identifikation und Konzentrationsbestimmung der Alkaloide in den einzelnen Pflanzenbestandteilen wurde eine Versuchsreihe durchgefuhrt. Methodik: 4 jungere und eine 8 Jahre alte Pflanze wurden von Mai bis Oktober im Freien aufgestellt und in wochentlichen Abstanden Bluten und Blatter geerntet. Alle Proben wurden tiefgefroren (-20° C), zeitgleich aufgetaut, gewogen und in Methanol extrahiert. Die Bestimmung der Alkaloide erfolgte mittels HPLC (Flussigkeitschromatographie) Diodenarray Detektor, die Auftrennung mittels Hypersil HyPurity Kartusche, die quantitative Auswertung bei einer Wellenlange von 220 nm. Ergebnisse: Alle 66 Bluten, 32 Blatter und 2 Samenkapseln enthielten Tropanalkaloide. Hauptalkaloid war Scopolamin. Die hochsten Konzentrationen konnten in den Samenkapseln festgestellt werden, gefolgt von den Bluten. Die Blatter enthielten nur geringe Konzentrationen. Der durchschnittliche Gesamtalkaloidgehalt je Blute der jungeren Pflanzen lag bei circa einem Milligramm (0,94 mg), der alteren Pflanze bei circa 2 Milligramm (1,81 mg). Die Bluten der alten Pflanze enthielten bis zu 3 mg Scopolamin. Folgerung: Schon die Aufnahme einzelner Bluten kann relevante Vergiftungserscheinungen hervorrufen, daher ist die einfache Verfugbarkeit der Pflanze als bedenklich einzustufen. Vor dem Hintergrund der zunehmenden, gewollten Aufnahme durch Jugendliche sollte gerade in den Sommermonaten differentialdiagnostisch bei unklaren Verwirrtheitszustanden und Halluzinationen auch an eine Vergiftung durch die Engelstrompete gedacht werden. Background and objektive: Angel's trumpet (Species Brugmansia) is widely used as a garden plant because it is easily kept and the luxuriance of its flowering. Belonging to the Family Solanacea it contains a large amount of alkaloids (parasympatholytics). Because of its hallucinogenic action, its leaves and flowers are increasingly used by young people as a substitute for the hallucinogen LSD (lysergic acid diethylamide). In the summer of 1997, one of a group of youths died after they had ingested its flowers which they had gathered from front gardens. An investigation was undertaken to identify the alkaloids and measure their concentration in the various parts of the plant. Methods: Four young and one eight-year old plant were kept outdoors from May until October, and its flowers and leaves were removed for analysis weekly. All samples were deep-frozen at -20° C and later, at the same time, thawed out, weighed and extracted in methanol. The alkaloids were identified by high pressure liquid chromatography (HPLC), diode array detector, separated by means of a Hypersil HyPurity cartridge, and measured at a wave-length of 220 nm. Results: All 66 flowers, 32 leaves and 2 speed capsules contained tropane alkaloids, mainly scopolamine. The highest concentrations were found in the seed capsules, lower ones in the flowers, while the leaves contained only small amounts. Total alkaloid content per flower of the younger plants averaged 0.94 mg, of the younger ones 1.81 mg. The flowers of the old plant contained up to 3 mg scopolamine. Conclusion: The ingestion of even a few flowers of Angel's trumpet can cause symptoms of poisoning. Easy availability of the plant thus presents a danger. Because of the increasing incidence of deliberate ingestion by young people, poisoning by Angel's trumpet should be included in the differential diagnosis in patients with confusion and hallucinations of uncertain origin, especially during the summer months.

Cannabinoid pharmacokinetics in occasional users is well studied, but the interpretation of data from heavy users is difficult. In the present study, blood pharmacokinetic properties were investigated in occasional and heavy users in cannabis and placebo conditions. The results obtained with occasional users were in contrast to those of the heavy users who admitted cannabis use on 4-25 occasions during the previous week. Of the 12 heavy users, 10 exhibited up to 12.3 microg/L Delta(9)-tetrahydrocannabinol (THC) prior to smoking. During the 8 h after smoking, the distribution and elimination patterns were comparable to those of the occasional users and the concentrations returned to 68-196% (median 110%) of the initial values. However, the maximal concentration and the areas under the curves were significantly higher with marked interindividual variation. In contrast to the cannabis conditions, the THC concentrations in the placebo phase decreased more slowly (elimination half-life 17.5-43.5 h vs. 1.0-5.9 h) in accordance with a late elimination phase. The elimination half-lives of 11-hydroxy-THC and 11-nor-9-carboxy-THC in th cannabis conditions (medians 3.1 h and 6.2 h, respectively) were longer than those of THC, which was different in the placebo phase (medians 7.2 h and 13.0 h, respectively). From the results, it must be cautioned that cannabinoid blood concentrations from heavy users in a late elimination phase may be difficult to distinguish from concentrations measured in occasional users after acute cannabis use.

The South-American red bellied toads (Melanophryniscus spp.) belonging to the Bufonidae family contain toxic alkaloids in their skin, predominantly of the pumiliotoxin group. Whole animal methanolic extracts of individual specimens of three species (Melanophryniscus atroluteus, M. devincenzii, and M. montevidensis) were analyzed for the presence of toad specific bufadienolides and indolalkylamines (serotonin derivatives) by HPLC-electrospray (ESI)-MS-TOF. No bufadienolides, but few bufotenines, mainly dehydrobufotenine, were detected in the extracts in variable amounts. The concentration of the dehydrobufotenine in the extracts seems to be species specific. Whereas M. atroluteus and M. montevidensis contain very low or trace amounts, M. devincenzii specimens exhibit high concentrations of this indolalkylamine. In comparison, analysis of extracts from Bufo arenarum (Uruguay) and from B. bufo (Germany) confirmed the presence of bufadienolides as well as of bufotenine derivatives. Tadpoles of both species exhibited a different pattern: extracts from B. arenarum tadpoles contained only dehydrobufotenine, but those from B. bufo tadpoles bufotoxin and two alkylamines. Melanophryniscus toads appear not to be able to compensate the high variability of toxic skin alkaloids by producing defensive bufadienolides.

Oral fluid is considered to be an alternative to urine testing for the detection of acute ingestion of drugs. The OraSure Intercept DOA Oral Specimen Collection Device (OSCD) has been used in studies for the quantitation of Delta9-tetrahydrocannabinol (THC), but concerns have been raised. In the present study, we investigated whether the volume of oral fluid can be determined and how much THC remains adsorbed on the device. It was found that THC is markedly adsorbed onto the absorptive pad. The recovery using the standard elution procedure was only 37.8 +/- 9.4% for 10 ng/mL and 55.6 +/- 1.0% for 100 ng/mL of THC in oral fluid (n = 5 each). With an additional methanol wash, a further 25% could be eluted. Therefore, a modification of the procedure was evaluated, consisting of the addition of 2 mL of methanol to the elution buffer. THC could be completely recovered over a range of concentrations (1 to 1000 ng/mL). For the determination of the amount of oral fluid absorbed, a gravimetric approach was evaluated as the weights of the devices vary only by 0.6% relative standard deviation. After application of 0.5 mL oral fluid to pads and evaluation of the weight differences, the applied amount could be estimated with a precision of 7.5% (n = 8) and an accuracy of 6.1%. From these results it can be concluded that the OraSure OSCD is useful to collect oral fluid for reliable quantitative THC assay applying a modified elution procedure and gravimetric determination of the amount of oral fluid.

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 mu g/kg THC by smoking. Performance tests measuring skills related to driving were conducted at regular intervals between 15 min and 6 h 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.

A study was performed to acquire urine, serum and oral fluid samples in cases of suspected driving under the influence of drugs of abuse. Oral fluid was collected using a novel sampling/testing device (Drager DrugTest ® System). The aim of the study was to evaluate oral fluid and urine as a predictor of blood samples positive for drugs and impairment symptoms. Analysis for cannabinoids, amphetamine and its derivatives, opiates and cocaine was performed in urine using the Mahsan Kombi/DOA4-test, in serum using immunoassay and gas chromatography–mass spectrometry (GC–MS) confirmation and in oral fluid by GC–MS. Police and medical officer observations of impairment symptoms were rated and evaluated using a threshold value for the classification of driving inability. Accuracy in correlating drug detection in oral fluid and serum were >90% for all substances and also >90% in urine and serum except for THC (71.0%). Of the cases with oral fluid positive for any drug 97.1% of corresponding serum samples were also positive for at least one drug; of drug-positive urine samples this were only 82.4%. In 119 of 146 cases, impairment symptoms above threshold were observed (81.5%). Of the cases with drugs detected in serum, 19.1% appeared not impaired which were the same with drug-positive oral fluid while more persons with drug-positive urine samples appeared uninfluenced (32.7%). The data demonstrate that oral fluid is superior to urine in correlating with serum analytical data and impairment symptoms of drivers under the influence of drugs of abuse.

The testing of saliva or oral fluid at the roadside could be a powerful tool to detect drivers under the influence of drugs and has several advantages over urine screening. In 177 cases of individuals suspected of driving under the influence of drugs, oral fluid was collected at the roadside and analyzed in parallel to serum samples. The study was performed to investigate the variability of oral fluid analysis results in relation to blood/serum. In 45% of the cases single-drug use was found, and in 50% poly-drug use was found. Cannabis was most prevalent (78%), and 70% of these individuals were also positive for tetrahydrocannabinol in serum. Overall, 97% of oral fluid samples positive for any substance were also positive in serum. Comparing data of oral fluid and serum for amphetamine, MDMA, morphine, benzoylecgonine, and tetrahydrocannabinol, the sensitivities were 100%, 97%, 87%, 87%, and 92%, respectively. Overall specificity and accuracy were in the range of 91-98%. Discrepancies between a negative oral fluid sample and a positive serum sample could be explained by analytical insensitivity in the lower volume of oral fluid analyzed (estimated for 0.1 mL confirmation vs. 1 mL of serum) or a shorter detection window in oral fluid. The low prevalence of discrepancies with positive oral fluid and negative serum results (2-9% of the cases) may be explained by persistent oral contamination especially for orally consumed drugs, like MDMA and cannabis. It is concluded that the detection of a psychoactive substance in oral fluid taken at the roadside is highly predictive for the detection of the corresponding drug or its metabolite in serum. Oral fluid testing is therefore suitable for the efficient confirmation of drug use of drivers suspected of being under the influence of drugs.

The detection of the pyrolysis product anhydroecgonine methyl ester (AEME, methylecgonidine) after cocaine smoking using gas chromatography-mass spectrometry is hampered by the artifactual production of AEME. The amount of AEME increases with the amount of cocaine used producing false positive values in authentic samples. A method for the correction of quantitative values was established using calibration of pyrolysis and estimation of the artifactual AEME. Authentic AEME in serum was differentiated from the artifact above 3.5 microg/l, 99% prediction limits of the quantitation were +/-3.1 microg/l. In 16 serum samples and five postmortem blood samples, cocaine and AEME were detected, but after application of the correction method only ten were truly positive for AEME.

Aims The stimulating herbal drug khat is habitually used in East Africa and the Arabian peninsula but is also imported into other countries. The aim was to study the pharmacokinetics of its alkaloids cathinone, cathine and norephedrine.

Introduction: The stimulating herbal drug kath is uncommon in most countries, and information on its detection and interpretation of analytical results is limited. Therefore, a study with kath was carried out to compare the efficiencies of different analytical techniques used to detect drug use. Methods: Four volunteers chewed kath leaves for 1 h; urine samples were collected up to 80 h afterward and analyzed by the Abbott fluorescence polarization immunoassay (FPIA), the Mahsan-AMP300 on-site immunoassay, the Bio-Rad Remedi HS HPLC system with photodiode array detection (DAD), and gas chromatography–mass spectrometry (GC-MS). Results: FPIA gave negative results, whereas positive results were obtained with the Mahsan test during the first day. With HPLC, one peak could be observed up to 50 h, but its DAD spectrum could not be identified by the system. Further investigations indicated that the kath alkaloids coeluted and produced a mixed DAD spectrum. With GC-MS, the specific kath ingredient cathinone was detected up to 26 h, whereas cathine and norephedrine were still detectable in the last samples. Maximum concentrations of cathinone, cathine, and norephedrine in urine samples from the study were 2.5, 20, and 30 mg/L, respectively, whereas in authentic cases the concentrations were much higher. Conclusion: GC-MS is superior to the screening techniques Mahsan-AMP300 and Remedi with respect to specificity and sensitivity for the detection of kath use in urine.

Cocaine is transformed into hepatotoxic metabolites through oxidative pathways. For anhydroecgonine methyl ester (AEME), the main constituent in crack smoke, the oxidative metabolism has not been studied. Therefore, incubation of AEME with rat liver microsomes was performed and a metabolite of AEME, anhydroecgonine methyl ester N-oxide (AEMENO), was identified. The chemical structure of this new metabolite was confirmed by synthesis and by comparative interpretation of electrospray multiple-stage mass spectra, which were obtained in the positive ion mode. This metabolite was also detected in whole blood, serum and urine samples from crack users. The application of liquid chromatography/electrospray mass spectrometry or nanoelectrospray mass spectrometry was necessary because AEMENO is susceptible to thermal degradation during gas chromatographic/mass spectrometric analysis. This study demonstrated that AEMENO is produced by rat hepatic microsomal metabolism in vitro and is present in body fluids from crack users.

The enzymatic degradation of cocaine in blood samples, even during transport to a forensic laboratory, is a common problem in toxicological analysis. This can be avoided by the use of blood-sampling devices such as gray-top Vacutainers containing the cholinesterase inhibitor sodium fluoride. In the present study, which included 147 authentic cases, blood samples were collected into two different tubes, one containing fluoride/oxalate and one without stabilizing agents. In all cases, both samples were analyzed for drugs of abuse using Abbott FPIA immunoassays after precipitation and gas chromatography-mass spectrometry (GC-MS) for quantitative analysis. The cannabinoid immunoassay showed markedly lower values in the fluoride-containing samples; this was investigated further and could be explained by hemolysis of these samples. In addition, the concentrations of 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) were lower in these samples. A stability study with the THCCOOH acyl glucuronide showed that it is unstable in unpreserved serum, which could explain our observation. GC-MS quantitative data for amphetamine and derivatives, opiates, delta9-tetrahydrocannabinol, and 11-hydroxy-delta9-tetrahydrocannabinol were essentially identical; however, they also differed substantially for cocaine, cocaethylene, ecgonine methylester, and benzoylecgonine. Unexpectedly, the concentrations of benzoylecgonine in unpreserved serum were almost half as high as in the fluoride-containing samples.

Bei der Begutachtung einer drogenbedingten Fahruntuchtigkeit sind auch rauschmittelbedingte Folgeerscheinungen zu beachten. Anhand von 37 Fallen des Fahrens unter dem Einflus von Amphetamin bzw. Amphetamin-Derivaten aus den Jahren 1995–1998 wurde die Erschopfungsreaktion nach Wirkende untersucht. Hierzu wurden die von Polizei und/oder untersuchendem Arzt festgestellten Auffalligkeiten im Leistungsbild ausgewertet und einer Erschopfungsreaktion bzw. einer Stimulationsphase zugeordnet und schlieslich mit den Analysebefunden verglichen. Es zeigt sich, das bei niedrigem Amphetamin- bzw. Amphetamin-Derivat-Blutspiegel Symptome einer Erschopfungsreaktion im Leistungsbild beschrieben wurden. Auch zeigten die Fahrzeuglenker in der Erschopfungsreaktion tendenziell haufiger Fahrunsicherheiten.

Mit der Erweiterung des §24 a StVG auf Drogenkonsum bei Kraftfahrern besteht ein zunehmender Bedarf fur zuverlassige immunologische Vortests im Blut. Der neue Mahsan Mikrotiterplatten-Enzymimmunoassay (MTP-EIA) soll nach Herstellerangaben matrixunabhangig und empfindlich sein. Es wurde eine Vergleichsstudie der Cannabinoid- und Opiat-Assays von Mahsan und Abbott (Fluoreszenzpolarisationsimmunoassay, FPIA) an Serumproben aus dem Untersuchungsgut durchgefuhrt. Als Cut-offs wurden fur die Mahsan MTP-EIAs die Herstellerangaben ubernommen (Cannabinoide 2 ng/¶mL, Opiate 10ng/mL) und fur die Abbott FPIAs nach Proteinfallung Werte aus eigenen Voruntersuchungen (Cannabinoide 20 ng/¶mL, Opiate 30 ng/mL). Von 136 auf Cannabinoide untersuchten Proben waren 42% und von 123 auf Opiate untersuchten Proben 18% mit GC-MS positiv. Der Abbott FPIA lieferte falsch negative Ergebnisse (23% bei Cannabinoiden, 9% bei Opiaten, bezogen auf die GC-MS-Positiven), der Mahsan MTP-EIA keine. Falsch positiv waren je 13% der Cannabinoidbefunde beider Immunoassays und 2% der Mahsan Opiatbefunde. Die Ergebnisse unterstutzen die empfohlenen Cut-Off-Werte fur eine qualifizierte Differenzierung zwischen Positiven und Negativen.

In two species of meloid beetles, Hycleus oculatus and Hycleus tinctus, from southern Africa, cantharidin and demethylcantharidin (palasonin) were assayed quantitatively. For cantharidin the mean value per specimen was about 1 mg for H. oculatus and 0.2 mg for H. tinctus, the mean palasonin concentration was 20 (H. oculatus) and 12 times (H. tinctus) lower, respectively. However, considerable individual variation in the cantharidin concentration was observed and values of more than 6 mg of this compound per beetle were measured pointing to the high risk of severe and even fatal poisoning when ingesting these insects.

The discrimination between smoking of crack and other routes of cocaine application has forensic implications. The pyrolysis product anhydroecgonine methyl ester (AEME, methylecgonidine) has been found to be a marker for smoked cocaine. An improved method for the determination of AEME in serum was developed, consisting of mixed phase solid-phase extraction and GC-MS. Special care was taken for the volatility of AEME and tert.-butyldimethylsilylation was used for derivatization. Thus AEME could be determined for the first time in 13 serum samples from living subjects. The concentrations found were in a range of 3 to 34 ng/ml, a correlation with the storage time of the samples or with benzoylecgonine concentrations could not be found.

Der Keto-Diabur Test 5000 (Boehringer Mannheim) ist ein Schnelltest zur Bestimmung von Glucose- und Ketonkorpern im Urin. Mit diesem routinemasig durchgefuhrten Test wurden bei der Obduktion von drei eindeutig infolge Erfrierens bzw. Unterkuhlung verstorbener Personen stark positive Resultate fur Ketone erhalten, ohne das gleichzeitig die Glucose im Harn erhoht war. Mittels Headspace-Gaschromatographie konnten in allen drei Urinproben uberhohte Aceton-Konzentrationen von 140, 1004 und 2703 mg/L festgestellt werden. Ein stark positiver Keto-Diabur-Test 5000 bei normalem Glucose-Befund im Urin kann somit als diagnostische Hilfe zur Erkennung eines Todes infolge Unterkuhlung dienen.

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 Delta(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 (Drager Drug Test(R) 5000 and Securetec Drugwipe(R) 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 mug/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 Drager Drug Test(R) 5000 demonstrated a high sensitivity for THC, whereas the sensitivity of the Securetec Drugwipe(R) 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 Drager Drug Test(R) 5000 appears to be a promising tool for detecting THC in oral fluid as far as correct THC detection is concerned.

Oral ingestion of allyl alcohol by a 55-year-old man resulted in death within 100 min. At autopsy, bloody, reddish fluid was found in mouth, larynx, esophagus, and trachea. The mucous membranes of the trachea, stomach, and duodenum were congested and inflamed. The stomach contained a pungent green-black fluid, and all internal organs exhibited a strong pungent odor. Toxicological analysis of blood identified allyl alcohol using solid-phase microextraction and gas chromatography-mass spectrometry. Quantitative determination of allyl alcohol and its toxic metabolite, acrolein, was performed using headspace gas chromatography with flame-ionization detection. Total amounts of allyl alcohol in gastric content, bile, and urine were 3.6 g, 15 mg, and 0.5 mg, respectively. The concentration in blood was 309 mg/L. Acrolein was not detected in gastric contents and only in small amounts in bile and urine. The concentration of acrolein in blood was 7.2 mg/L. Death was attributed to acrolein-induced acute cardiotoxicity, similar to that previously documented in animal experiments.

Acetaldehyde adducts of hemoglobin have been regarded as potential biochemical markers of alcohol exposure. In this study a novel sensitive method using liquid chromatography coupled to time-of-flight mass spectrometry (LC–TOF MS) has been used to investigate changes in adduct levels in alcohol detoxification patients. Hemoglobin and authentic blood samples from 66 adults with an alcohol-dependence syndrome and from 12 children were analyzed for acetaldehyde modifications with and without trypsin digestion using LC–TOF MS. After in-vitro incubation of hemoglobin with increasing concentrations of acetaldehyde, followed by tryptic digestion, 21 modified peptide fragments could be identified from their accurate mass and retention time shift. Eight of these could also be detected in authentic human blood samples. Trace amounts in children’s blood were indicative of an endogenous source. Modified peptide levels in patients’ samples with and without ethanol were significantly different, as also were levels in samples from admission and from five days later. Samples obtained 5, 10, or 15 days after admission did not differ in adduct levels. The LC–TOF MS method was sensitive enough to detect acetaldehyde-modified hemoglobin peptides in blood samples from patients with an alcohol-dependence syndrome. However, elevated levels were only observed after recent ethanol consumption and decreased during five days of abstinence, suggesting that acetaldehyde-modified tryptic peptides of hemoglobin are potential biomarkers only for short-term ethanol ingestion.

The venom of cone snails (Conus spp.) is a rich source of peptides exhibiting a wide variety of biological activities. Several of these conopeptides are neuronal nicotinic acetylcholine receptor (nAChR) antagonists and belong to the A-, M-, S-, C and the recently described D-superfamily (alphaD-conopeptides). Here we describe the discovery and characterization of two alphaD-conopeptides isolated from the venom of Conus mustelinus and Conus capitaneus. Their primary structure was determined by Edman degradation, MS/MS analysis and by a PCR based approach. These peptides show close structural homology to the alphaD-VxXIIA, -B and -C conopeptides from the venom of Conus vexillum and are dimers (about 11kDa) of similar or identical peptides with 49 amino acid residues and a characteristic arrangement of ten conserved cysteine residues. These novel types of conopeptides specifically block neuronal nAChRs of the alpha7, alpha3beta2 and alpha4beta2 subtypes in nanomolar concentrations. Due to their high affinity, these new ligands may provide a tool to decipher the localisation and function of the various neuronal nAChRs.

Abstract Performance impairment during Δ9-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 μg/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.

This study was performed to determine whether analysis of clopidogrel and its main carboxylic acid metabolite in plasma provides additional information about the wide variability of platelet aggregation inhibition in clopidogrel-treated patients with peripheral arterial occlusive disease. Consecutive outpatients (n = 56) with stable peripheral arterial occlusive disease treated with 75 mg clopidogrel daily, without co-administration of aspirin, were investigated. With use of a standardized questionnaire, the time of drug intake was documented. Blood sampling was performed within 24 hours after the most recent drug intake. Platelet function was measured by optical aggregometry using adenosine diphosphate (ADP) (2 mumol/L) as the agonist. Plasma concentrations of clopidogrel and its main metabolite, clopidogrel carboxylic acid, were quantitated using high-performance liquid chromatography analysis coupled to mass spectrometry. In 95% (53/56) of patients, clopidogrel carboxylic acid was detected. In 40% (22/56) of patients, the ADP-induced aggregation response was within the normal range despite clopidogrel treatment. In 14% (3/22) of these patients, neither clopidogrel nor its main metabolite could be detected. Two of these patients agreed to ingest 75 mg/d clopidogrel under observation and to undergo blood sampling after 2, 12, and 24 hours. Clopidogrel carboxylic acid and a significant inhibition of platelet aggregation were detected even after 24 hours in both patients, confirming noncompliance as the reason for the lack of inhibition of ADP-induced platelet aggregation observed in the initial measurements. In the subgroup of patients who had taken clopidogrel within 4 hours before blood sampling, a large range of carboxylic acid concentrations was detected, indicating a high variability of drug metabolism among patients. In conclusion, determining clopidogrel metabolite plasma concentrations could be a useful tool for identifying poor compliance and variable metabolism in clopidogrel-treated patients. Nevertheless, in the majority of clopidogrel-treated patients, the variability of platelet response is not caused by noncompliance.

Rhythm generation within the mammalian circadian system is achieved by clock genes and their protein products. As an integral part of this system, the pineal gland serves the need to tune the body to the temporal environment by the rhythmic synthesis and release of melatonin. A number of human disorders and syndromes are associated with alterations in circadian rhythms of clock genes and their protein products and/or a dysfunction in melatonin synthesis. In the human, little is known about the molecular signature of time management. Pineal tissue from regular autopsies was allocated to asserted time-of-death groups (dawn, day, dusk, night), and analyzed by RT-PCR, immunoblotting, immunohistochemistry, and confocal laser scanning microscopy for expression of clock genes. Despite the observed diurnal rhythms in activity of the arylalkylamine N-acetyltransferase and in melatonin content, mRNA levels for the clock genes Period1, Cryptochrome1, Clock, and Bmal1, and also amounts of corresponding clock gene proteins showed no differences between time- of-death groups. In contrast, a time-of-day-dependent nucleocytoplasmic shuttling of clock gene proteins was detected. These data confirm the minor importance of a transcriptional regulation for dynamics in the human pineal gland, and offer a novel twist in the molecular competence of clock gene proteins.

The neurobiological basis of suicidal behaviour is multifactorial and complex. Several lines of evidence indicate that environmental factors as well as multiple genes and interactions of both are implicated in its aetiology. The aim of this study was to establish the transcriptomic expression profile of post-mortem brain tissue of suicide victims in order to identify new candidate genes and biological patterns for suicidal behaviour. Post-mortem orbitofrontal cortex tissue was derived from 11 suicide victims and 10 non-psychiatric controls carefully selected from a brain bank of over 150 brains, and the expression of more than 23000 messenger RNAs was assessed in this case-control study. Validation experiments were carried out using quantitative RT-PCR as an independent method. A classification of the differentially expressed genes according to their biological function and statistical analyses of the data were performed in order to identify biological pathways that are over-represented in the suicide group. In total, 124 transcripts demonstrated significant changes (fold changes > or = 1.3, p value < or = 0.01), with 59 showing under-, and 65 over-expression in the suicide group. The results could be validated for nine particularly interesting transcripts (CDCA7L, CDH12, EFEMP1, MLC1, PCDHB5, PTPRR, S100A13, SCN2B, and ZFP36). The pathway analysis showed that the Gene Ontology categories 'central nervous system development', 'homophilic cell adhesion', 'regulation of cell proliferation' and 'transmission of nerve impulse' were significantly enriched. The differentially expressed genes and significant biological processes might be involved in the pathophysiology of suicide and warrant further attention.

Poster presentation: The mammalian pineal organ is a peripheral oscillator, depending on afferent information from the so-called master clock in the suprachiasmatic nuclei of the hypothalamus. One of the best studied outputs of the pineal gland is the small and hydrophobic molecule melatonin. In all vertebrates, melatonin is synthesized rhythmically with high levels at night, signalling the body the duration of the dark period. Changes or disruptions of melatonin rhythms in humans are related to a number of pathophysiological disorders, like Alzheimer's disease, seasonal affective disorder or the Smith-Magenis-Syndrome. To use melatonin in preventive or curative interferences with the human circadian system, a complete understanding of the generation of the rhythmic melatonin signal in the human pineal gland is essential. Melatonin biosynthesis is best studied in the rodent pineal gland, where the activity of the penultimate and rate-limiting enzyme, the arylalkylamine N-acetyltransferase (AA-NAT), is regulated on the transcriptional level, whereas the regulatory role of the ultimate enzymatic step, achieved by the hydroxyindole O-methyltransferase (HIOMT), is still under debate. In rodents, Aa-nat mRNA is about 100-fold elevated during the night in response to adrenergic stimulation of the cAMP-signalling pathway, with AA-NAT protein levels closely following this dynamics. In contrast, in all ungulates studied so far (cow, sheep), a post-transcriptional regulation of the AA-NAT is central to determine rhythmic melatonin synthesis. AA-NAT mRNA levels are constantly elevated, and lead to a constitutive up-regulation of AA-NAT protein, which is, however, rapidly degraded via proteasomal proteolysis during the day. AA-NAT proteolysis is only terminated upon the nocturnal increase in cAMP levels. Similar to ungulates, a post-transcriptional control of this enzyme seems evident in the pineal gland of the primate Macaca mulatta. Studies on the molecular basis of melatonin synthesis in the human being are sparse and almost exclusively based on phenomenological data, derived from non-invasive investigations. Yet the molecular mechanisms underlying the generation of the hormonal message of darkness can currently only be deciphered using autoptic material. We therefore analyzed in human post-mortem pineal tissue Aa-nat and Hiomt mRNA levels, AA-NAT and HIOMT enzyme activity, and melatonin levels for the first time simultaneously within tissue samples of the same specimen. Here presented data show the feasibility of this approach. Our results depict a clear diurnal rhythm in AA-NAT activity and melatonin content, despite constant values for Aa-nat and Hiomt mRNA, and for HIOMT activity. Notably, the here elevated AA-NAT activity during the dusk period does not correspond to a simultaneous elevation in melatonin content. It is currently unclear whether this finding may suggest a more important role of the ultimate enzyme in melatonin synthesis, the HIOMT, for rate-limiting the melatonin rhythm, as reported recently for the rodent pineal gland. Thus, our data support for the first time experimentally that post-transcriptional mechanisms are responsible for the generation of rhythmic melatonin synthesis in the human pineal gland.

In a study on the effects of smoked cannabis (18.2 +/- 2.8 mg as low and 36.5 +/- 5.6 mg as high dose) paired blood and oral fluid samples were collected from 10 study participants up to 6 h after smoking and analyzed for the cannabinoids Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-THC (THC-OH) and 11-nor-9-carboxy-THC (THCA) using gas chromatography-mass spectrometry. Highest concentrations in serum were 47.8 +/- 35.0 and 79.1 +/- 42.5 microg/L at the end of smoking (low and high dose, respectively) and decreased to less than 1 microg/L during 6 h with elimination half-lives of 1.4 +/- 0.1 h calculated from 1 to 6 h, which is shorter than reported previously. The elimination half-lives of THC-OH (2.0 +/- 0.3 h) and THCA (3.4 +/- 0.9 h) were significantly higher. The THC concentrations in oral fluid were highest with 900 +/- 589 and 1041 +/- 652 microg/L (low and high dose, respectively) in the first sample collected at 0.25 h and decreased to 18 +/- 12 microg/L over 6 h with elimination half-lives of 1.5 +/- 0.6 h. The elimination half-life of THC in serum and oral fluid and between the two doses did not significantly differ. Oral fluid/serum ratios were 46 +/- 27 and 36 +/- 20 (low and high dose, respectively), which are higher than previously reported and might be based on sample collection and/or analytical issues. In conclusion, despite similar elimination rates of THC in serum and oral fluid, which appear incidental, the high differences in oral fluid/serum ratios are not a reliable basis for correlating THC concentrations in oral fluid and serum. The oral compartment and its kinetics for drugs, particularly THC, are not yet satisfactorily understood.

The mammalian pineal gland synthesizes rhythmically the hormone melatonin, which provides the body with a signal coding the duration of the night period. The ultimate enzymatic step in melatonin synthesis is achieved by the hydroxyindole O-methyltransferase (HIOMT); the rate-limiting enzyme is, however, the arylalkylamine N-acetyltransferase (AA-NAT). In contrast to the central importance of a transcriptional regulation of the Aa-nat gene for rodent melatonin synthesis, mechanisms in the human pineal gland are elusive. Therefore, pineal tissue, taken from regular autopsies (n = 69; postmortem intervals ranging from 9 to 147 h) was analyzed simultaneously for Aa-nat and Hiomt mRNA levels by PCR, AA-NAT activity using (14)C-acetyl-coenzyme A, HIOMT activity using S-adenosyl-l-[(14)C]-methionine, and melatonin content using an ELISA. Results were allocated to asserted time-of-death groups (day, 1,000 to 1,630 h; dusk, 1,630 to 2,200 h; night, 2,200 to 0730 h; dawn, 0730 to 1,000 h). RNA degradation rates of genes of interest ran in parallel, and, therefore, data normalization could be established, regardless of postmortem delay in tissue sampling. Aa-nat and Hiomt mRNA and HIOMT activity showed no diurnal rhythm. In contrast, a significant rhythm was found for the correlation between time of death and both AA-NAT activity and melatonin content, with elevated values during dusk and night. Presented data demonstrate that postmortem brain tissue can be used to detect the remnant of premortem adaptive changes in neuronal activity. In particular, our results give strong experimental support for the idea that transcriptional mechanisms are not dominant for the generation of rhythmic melatonin synthesis in the human pineal gland.

The use of the herbal stimulant khat (Catha edulis FORSK) is maintained by immigrants from countries where it is part of their cultural life (Arabian Peninsula and eastern Africa). In western countries the drug and its effects are largely unknown and no experience in evaluating impairment symptoms due to the khat-alkaloids, e.g. cathinone, cathine and norephedrine exists. Blood and urine samples from khat users involved in 19 cases of suspected driving under the influence of drugs were analysed and correlated with the results of medical examination and police officer reports. In 3 cases impaired driving and in 10 cases marked impairment of psychophysical functions was observed such as effects on the nervous system (slow pupil reaction to light, dry mouth, increased heart-rate), trembling, restlessness/nervousness, daze/apathy/dullness, impairment of attention, walking and standing on one leg. However, the alkaloid concentrations assayed in blood did not correlate with the impairment symptoms. Apart from an acute phase of indirect sympathomimetic action the development of habituation and withdrawal symptoms must also be considered in explaining the diversity of effects observed. From these results it can be concluded that chewing khat may severely impair driving ability, but may also be without noticeable effects.

An alternative to detoxification by oral therapy with the narcotic antagonist naltrexone is the subcutaneous implantation of naltrexone pellets. From detoxified patients with naltrexone implants (1g) 26 blood samples were collected up to 73 days after implantation. The assay for naltrexone and 6-beta-naltrexol in plasma was developed using automated mixed-mode solid-phase extraction, catalysed trimethylsilylation and gas chromatography-mass spectrometry in single ion monitoring mode with naloxone as internal standard. The analytical method was very sensitive with limits of detection of 0.1 ng/ml and was linear up to 60 ng/ml for naltrexone and 200 ng/ml for naltrexol. Intra-day precision for naltrexone and naltrexol were 24.3 and 22.9%, respectively, at the LLOQ (accuracy 1.4 and 0.4%, respectively) and less than 10% (2.0, 6.0 and 20.0 ng/ml, n = 6 each) above. Inter-day precision was 7.9% (accuracy -0.6%) for naltrexone and 10.9% (accuracy 1.6%) for naltrexol (20 ng/ml, n = 10). Extraction recoveries were 83% for both analytes (10 ng/ml, n = 6). The concentrations of naltrexone and naltrexol in the plasma samples were in the range of 0.7-13.7 and 0.9-17.0 ng/ml, respectively. The simple analytical procedure described provided good sensitivity for the assay of naltrexone and naltrexol in plasma after naltrexone pellet implantation.

Cocaine metabolism has been studied previously with respect to the formation of predominant hydrolytic or hepatotoxic metabolites via oxidative pathways. In the present study, cocaine and eight of its metabolites (norcocaine, ecgonine methyl ester, benzoylecgonine, benzoylnorecgonine, 3-hydroxy-benzoylecgonine, cocaethylene, norcocaethylene, and ecgonine ethyl ester) were incubated with microsomes from rat liver, kidney, lung, and brain. Qualitative analysis of the metabolites produced was performed using solid phase extraction (SPE), trimethylsilylation, and GC/MS. It was found that the metabolites with a free carboxylic group (e.g., benzoylecgonine) were not further oxidized by microsomal enzymes and their presence in urine or blood may therefore be due to hydrolysis of the respective alkylated entities. Although microsomes from all organs exhibited oxidative metabolism, significant differences were noted. Kidney microsomes produced essentially the same results as liver, but aryl hydroxylated metabolites were not found in incubations with lung and brain microsomes. N-Hydroxy-norcocaine was found only in traces with brain microsomes. It appears that cocaine is converted to N-hydroxy-norcocaine (which is the precursor of toxic metabolites) not only in the liver but also in other organs of rat. This might be relevant in the development of lung toxicity observed in smokers of cocaine ("crack").

The presence of the cocaine pyrolysis product anhydroecgonine methyl ester (AEME, methylecgonidine) in plasma indicates the smoking of cocaine. The stability of this analyte in human plasma has not been studied. In the present investigation AEME and its hydrolysis product anhydroecgonine (AE, ecgonidine) were assayed in plasma and buffers using gas chromatography-mass spectrometry. It was found that 50% of AEME in human plasma was hydrolyzed to AE within 5 days at room temperature and within 13 days at 4 degrees C. Addition of esterase inhibitors such as sodium fluoride or echothiophate iodide reduced the hydrolysis significantly. Hydrolysis of AEME also occurred in buffers with pH values above 5, but the hydrolysis rate was significantly lower when compared with plasma and could be markedly increased by the addition of butyrylcholine esterase. The data suggest that AEME is degraded via two mechanisms: chemical hydrolysis at basic pH and enzymatic hydrolysis by butyrylcholine esterase. Therefore, proper storage conditions must be provided for the assay of AEME in plasma.

The stimulating herbal drug kath is uncommon in most countries, and information on its detection and interpretation of analytical results is limited. Therefore, a study with kath was carried out to compare the efficiencies of different analytical techniques used to detect drug use.Four volunteers chewed kath leaves for 1 h; urine samples were collected up to 80 h afterward and analyzed by the Abbott fluorescence polarization immunoassay (FPIA), the Mahsan-AMP(300) on-site immunoassay, the Bio-Rad Remedi HS HPLC system with photodiode array detection (DAD), and gas chromatography-mass spectrometry (GC-MS).FPIA gave negative results, whereas positive results were obtained with the Mahsan test during the first day. With HPLC, one peak could be observed up to 50 h, but its DAD spectrum could not be identified by the system. Further investigations indicated that the kath alkaloids coeluted and produced a mixed DAD spectrum. With GC-MS, the specific kath ingredient cathinone was detected up to 26 h, whereas cathine and norephedrine were still detectable in the last samples. Maximum concentrations of cathinone, cathine, and norephedrine in urine samples from the study were 2.5, 20, and 30 mg/L, respectively, whereas in authentic cases the concentrations were much higher.GC-MS is superior to the screening techniques Mahsan-AMP(300) and Remedi with respect to specificity and sensitivity for the detection of kath use in urine.

Wenn Kinder zum ersten Mal in ihrem Leben mit alkoholhaltigen Getranken in Kontakt kommen, z.B. durch Nippen am Glaschen eines suslich aromatischen Eierlikors, reagieren die Erwachsenen i.d.R. mit einem amusanten Lacheln uber die „Heldentat“ der Kleinen oder aber bisweilen betroffen ob ihrer Befurchtungen, nun sei der Grundstein fur das Entstehen des Alkoholismus gelegt.

SirWe thank Drs Nordstrom and Hart (2006) for theircomments, because it gives an opportunity to addressan issue of growing importance in experimental cannabisresearch: the comparability of THC dosing across experi-mental performance studies. In general, researchers haveemployed two types of units for reporting the mean amountof THC delivered to subjects when smoking an experimentalcannabis cigarette: (1) %THC contained in a cannabiscigarette and (2) absolute mass of THC (mg). The formerprovides only a relative measure of mass THC that cannotbe used for comparison between studies that usually employdifferent sizes/weights of cannabis cigarettes. The latterprovides an absolute measure of total amount of THCcontained in a cigarette and is much to be preferred overrelative units, because it does allow comparison of THCdose across studies. However, even comparison betweenabsolute units of THC doses can be misleading becauseof variations in smoking procedures that are beingemployed across studies. It has been amply shown thatvariations in puff volume, number of puffs, and breathholdduration produce dose-related changes in plasma levels ofTHC (Azorlosa et al, 1995). Therefore, the best approach forincreasing comparability between studies is to report THCdose as well as plasma THC concentrations. Unfortunately,assessment of plasma THC concentration is not standardpractice in experimental performance research. However, ifit were, it would have helped to evaluate the claims byDrs Nordstrom and Hart that THC doses in the studiesby Hart et al (2001) and Ramaekers et al (2006) werecomparable and that experienced cannabis users aretolerant to the impairing effects of THC on cognition.We will illustrate this point.In their original publication, Hart et al (2001) did notindicate the total amount of THC that was delivered to theirsubjects by smoking. They merely indicated that theirhigh concentration cigarettes contained 3.9% THC of whicheach subject smoked three standardized puffs. Each puffconsisted of 5s of inhalation, 10s of breathhold, and 40s ofexhalation and rest. They failed to report the total massof the cannabis cigarette. Nordstrom and Hart (2006)have now corrected this omission by stating that eachcigarette weighed 1g, on average of which subjects smokedthree-quarters, within a 3min period. This, they argue, isapproximately 30mg of THC which is close to the largestdose (35mg) in the study by Ramaekers et al (2006).It is difficult to see, however, how subjects in the study byHart et al (2001) could have smoked three-quarters of acigarette in only three standardized puffs, when subjectsin the study by Ramaekers et al (2006) needed about 25standardized puffs, that is, 4s of inhalation, 10s ofbreathhold, and 15s of exhalation and rest, to finishsmoking a cannabis cigarette completely. These compara-tive data on THC dosing regimens certainly do not supportthe claim by Drs Nordstrom and Hart that the totalamount of THC delivered to subjects were comparable inboth studies. Of course, the final confirmation of theirclaim can only come from comparative plasma THCdata. These however are not available, as Hart et al (2001)did not assess plasma THC. In addition, their claim is atodds with a previous conclusion by Hart et al (2002)that the behavioral effects of smoked cannabis containing3.1% THC is similar to the effects of 20mg oral THC.Again, no plasma THC data were provided but it is wellestablished that bioavailability of oral THC is only 10% andgenerally three times lower as compared to smoked THC(McGilveray, 2005; Ohlsson et al, 1980). This seems toindicate that THC concentrations during the smokedcondition in this and perhaps other experiments may havebeen very low as well. Of course, this is very speculative butit goes to show how data on plasma THC concentrationswould have come in very handy.