Extensive phytocannabinoid profiles of seized cannabis and cannabis-based medicines - Identification of potential distinguishing markers.

As the frequency of cannabis-based therapy increases, the ability to distinguish intake of cannabis-based medicines from recreational cannabis use becomes desirable. Minor cannabinoids have been suggested to indicate recreational cannabis use in biological matrices but are unreliable when presumably also present in directly plantderived medicines. Thus, for therapeutics such as medical cannabis, Sativex® and Dronabinol, a more thorough investigation of cannabinoid profiles is required to identify possible distinguishing markers. In this study, 16 phytocannabinoids were quantified in samples of seized and medical cannabis, Sativex® and Dronabinol from two different manufacturers, using a validated LC-MS/MS method. Analytes included delta-9- tetrahydrocannabinol, tetrahydocannabinolic acid A, cannabidiol, cannabidiolic acid, cannabigerol, cannabigerolic acid, cannabinol, cannabinolic acid, cannabichromene, cannabichromenic acid, cannabicyclol, cannabicyclolic acid, tetrahydrocannabivarin, tetrahydrocannabivarinic acid, cannabidivarin and cannabidivarinic acid. Resultant cannabinoid profiles were compared, and markers were suggested. Characteristics of Sativex® included a specific cannabidiol/tetrahydrocannabinol ratio and presence of cannabichromene, while acidic cannabinoids, cannabigerol and cannabinol occurred in only low amounts. As expected, the predominant ingredient in Dronabinol was tetrahydrocannabinol, but minor cannabinoids were quantified as well. Medical marihuana and seized cannabis were compared separately in a principal component analysis. Several medical marihuana varieties were found to significantly differ from seized cannabis, mostly regarding contents of tetrahydocannabinolic acid A and tetrahydrocannabivarinic acid and cannabidiolic and cannabidivarinic acid respectively.

Detection of the synthetic drug 4-fluoroamphetamine (4-FA) in serum and urine.

4-Fluoroamphetamine (4-FA) was detected in the blood and urine of two individuals suspected for driving under the influence (DUI). The test for amphetamines in urine subjected to immunoassay screening using the CEDIA DAU assay proved positive. Further investigations revealed a 4-FA cross-reactivity of about 6% in the CEDIA amphetamine assay. 4-FA was qualitatively detected in a general unknown screening for drugs using GC/MS in full scan mode. No other drugs or fluorinated phenethylamines were detected. A validated GC/MS method was established in SIM mode for serum analysis of 4-FA with a limit of detection (LOD) of 1 ng/mL and a lower limit of quantification (LLOQ) of 5 ng/mL. Intra-assay precision was approx. 4% and inter-assay precision approx. 8%. Applying this method, the 4-FA serum concentrations of the two subjects were determined to be 350 ng/mL and 475 ng/mL, respectively. Given the pharmacological data of amphetamine, 4-FA psychoactive effects are to be expected at these serum levels. Both subjects exhibited sympathomimetic effects and psychostimulant-like impairment accordingly.

In vitro production of GHB in blood and serum samples under various storage conditions.

UNLABELLED: The in vitro production of GHB was observed in freshly collected, untreated whole blood samples using glass BD-Vacutainers and polypropylene S-monovettes. GHB concentrations were determined daily over a period of one week and after 3, 6 and 9 weeks again. Furthermore, the GHB concentration in 40 untreated random whole blood samples stored at 4°C for a longer period of time (10 samples 12 month, 10 samples 24 month and 20 samples 36 month) was also determined. For comparison, the in vitro production of GHB in freshly collected and prepared serum samples was observed. GHB serum concentrations were determined three times over a period of one week and once again after six weeks. Sample preparation was performed by means of methanolic extraction following the precipitation of whole blood and serum samples. A methanolic standard calibration was done in a low range of 0.005-0.1 µg/mL (LOD: 0.004, LLOQ: 0.013). For quantification a spiked blood bank serum with a determined GHB concentration of 0.09 µg/mL was used. Corrected calibrations in the range of 0.09-5.09 µg/mL were used (LOD: 0.08 µg/mL, LLOQ: 0.30 µg/mL), recovery: 91.3% (high level: 4.09 µg/mL) 50.5% (low level: 0.19 µg/mL). RESULTS: Relevant elevation of GHB was observed in all whole blood samples stored in liquid form (4°C or room temperature). In two of the 40 whole blood samples stored over a longer period of time at 4°C, GHB concentrations in the range of 13 µg/mL were even determined. These findings constitute grounds for caution. Even a GHB cut-off level of 5 µg/mL cannot be considered as "absolutely positive" proof of a case of exogenous administration, at least in untreated liquid blood samples in long time storage. However, no significant elevations of GHB were otherwise observed in any of the serum samples independently of storage temperature nor in the whole blood samples that were frozen for storage. CONCLUSIONS: The results suggest that the cut-off for exogenous GHB of 5 µg/mL could be lowered significantly, with the consequence of winning valuable time for the potential victim, but only if serum is collected for GHB determination or if the whole blood sample is frozen immediately after collection and the procedure well documented.

Concentrations of delta9-tetrahydrocannabinol and 11-nor-9-carboxytetrahydrocannabinol in blood and urine after passive exposure to Cannabis smoke in a coffee shop.

Cannabinoid concentrations in blood and urine after passive exposure to cannabis smoke under real-life conditions were investigated in this study. Eight healthy volunteers were exposed to cannabis smoke for 3 h in a well-attended coffee shop in Maastricht, Netherlands. An initial blood and urine sample was taken from each volunteer before exposure. Blood samples were taken 1.5, 3.5, 6, and 14 h after start of initial exposure, and urine samples were taken after 3.5, 6, 14, 36, 60, and 84 h. The samples were subjected to immunoassay screening for cannabinoids and analyzed using gas chromatography-mass spectrometry (GC-MS) for Delta(9)-tetrahydrocannabinol (THC), 11-nor-hydroxy-Delta(9)-tetrahydrocannabinol (THC-OH), and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH). It could be demonstrated that all volunteers absorbed THC. However, the detected concentrations were rather small. None of the urine samples produced immunoassay results above the cutoff concentration of 25 ng/mL. THC-COOH concentrations up to 5.0 and 7.8 ng/mL before and after hydrolysis, respectively, were found in the quantitative GC-MS analysis of urine. THC could be detected in trace amounts close to the detection limit of the used method in the first two blood samples after initial exposure (1.5 and 3.5 h). In the 6 h blood samples, THC was not detectable anymore. THC-COOH could be detected after 1.5 h and was still found in 3 out of 8 blood samples after 14 h in concentrations between 0.5 and 1.0 ng/mL.

Detection of Delta9-tetrahydrocannabinol and amphetamine-type stimulants in oral fluid using the Rapid Stat point-of-collection drug-testing device.

The Rapid Stat assay, a point-of-collection drug-testing device for detection of amphetamines, cannabinoids, cocaine, opiates, methadone, and benzodiazepines in oral fluid, was evaluated for cannabis and amphetamine-type stimulants. The Rapid Stat tests (n = 134) were applied by police officers in routine traffic checks. Oral fluid and blood samples were analyzed using gas chromatography-mass spectrometry (GC-MS) for Delta(9)-tetrahydrocannabinol, amphetamine, methamphetamine, methylenedioxymethamphetamine, methylenedioxyethylamphetamine, and methylenedioxyamphetamine. The comparison of GC-MS analysis of oral fluid with the Rapid Stat results for cannabis showed a sensitivity of 85%, a specificity of 87%, and a total confirmation rate of 87%. When compared with serum, the sensitivity of the cannabis assay decreased to 71%, the specificity to 60%, and the total confirmation rate to 66%. These findings were possibly caused by an incorrect reading of the THC test results. Comparison of the Rapid Stat amphetamine assay with GC-MS in oral fluid showed a sensitivity of 94%, a specificity of 97%, and a total confirmation rate of 97%. Compared with serum, a sensitivity of 100%, a specificity of 90%, and a total confirmation rate of 92% was found. The amphetamine assay must, therefore, be regarded as satisfactory.

Effect of the shampoo Ultra Clean on drug concentrations in human hair.

The influence of the special shampoo Ultra Clean (Zydot Unlimited, Tulsa, Oklahoma) on the results of hair analyses was investigated. Hair samples from persons (n = 14) with a known history of drug abuse were collected at autopsy. The hair samples were divided into separate strands which were analyzed both after washing with Ultra Clean and without treatment. Hair analyses were performed by methanol extraction under sonication, purification by solid phase extraction and GC/MS in SIM mode according to routine procedures for tetrahydrocannabinol (THC), cocaine, amphetamine, methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), methylenedioxyethylamphetamine (MDE), heroin, 6-monoacetylmorphine (6-MAM), morphine, codeine, dihydrocodeine and methadone. All drugs originally present in the hair fibers were still detected after a single application of Ultra Clean. However, a slight decrease in drug concentrations could mostly be observed e.g. cocaine (n = 10) -5%, 6-MAM (n = 12) -9%, morphine (n = 12) -26%, THC (n = 4) -36%. The findings clearly demonstrated that drug substances had not been sufficiently removed from human hair by a single Ultra Clean treatment to drop their concentrations below the limit of detection of the analytical method applied.

Analysis of LSD in human body fluids and hair samples applying ImmunElute columns.

Immunoaffinity extraction units (LSD ImmunElute) are commercially available for the analysis of lysergic acid diethylamide (LSD) in urine. The ImmunElute resin contains immobilized monoclonal antibodies to LSD. We applied the ImmunElute procedure to serum and also to human hair samples. For hair analysis the samples were first extracted with methanol under sonication. The extracts were then purified using the ImmunElute resin. LSD analysis was carried out with HPLC and fluorescence detection. The immunoaffinity extraction provides highly purified extracts for chromatographic analysis. The limit of detection (signal-to-noise ratio = 3) has been determined to be < 50 pg regardless of which sample material was used. The procedure was applied to authentic hair samples from drug abusers (n = 11). One of these samples tested positive with an amount of 110 pg LSD in 112 mg extracted hair corresponding to a concentration of 1 pg/mg.

False-positive LSD testing in urine samples from intensive care patients.

Unexpected positive results for lysergic acid diethylamide (LSD) were found in urine samples from 12 patients in an intensive care unit in a routine screening using the CEDIA DAU assay. None of these test results could be confirmed by high-performance liquid chromatography analysis, but all samples contained the mucolytic drug ambroxol. Further studies demonstrated that ambroxol exhibits a significant cross-reactivity in the CEDIA DAU LSD assay. Therefore, positive LSD results obtained with the CEDIA DAU assay have to be critically evaluated, particularly during the cold season, when infections of the respiratory tract often result in more frequent use of mucolytic medications.