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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.

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

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.

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.

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.

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.

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.

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.

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.

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.