On-site testing of cannabis. A controlled study after smoking cannabis. Per Mansson, Biosensor Applications Sweden

Transcription

1 Per Mansson, Biosensor Applications Sweden Magnus Nygren Biosensor Applications Sweden Carl Lundberg, APAC Security Pty Ltd, Australia Jan Ramaekers, Faculty of Psychology and Neuroscience, Maastricht Abstract Background Collection and analysis of drugs in oral fluid using conventional on-site test devices is time consuming, often taking more than 10 minutes. The BIOSENS system, utilizes an alternative approach which implies collection of mucus from the mucosal surface from the tongue with a special designed Oral Mucus Collector (OMC), subsequently analyzed in a BIOSENS system. The collection procedure takes a few seconds and the analysis procedure approximately 60 seconds. Aims To determine the window of detection and cut off values for THC (Δ-9-tetrahydrocannabinol) using the BIOSENS system after smoking one single cannabis cigarette. Methods Eight subjects smoked one cannabis cigarette (Δ-9 THC content 0.3mg/kg body weight). Conventinal oral fluid samples and oral mucus samples were collected prior smoking and 1, 2, 3, 4, 5, 6h after smoking. The mucus samples were collected using a BIOSENS Oral Mucus Collector and conventional oral fluid samples were collected with Quantisal Oral Fluid Devices (Immunalysis) subsequently analyzed for THC at laboratory. The clinical part of the study was conducted at the Faculty of Psychology and Neuroscience, Maastricht in the Netherlands. Results The window of detection using the BIOSENS system was found to be 4 hours for four of the eight subjects, 3h for one subject, 2h for one subject, 1h for one subject and less than 1h for one subject. The cut off values were calculated from the conventional oral fluid samples (Quantisal), at 20ng/ml 71% sensitivity was achieved and at 80ng/ml 94% sensitivity was achieved with the BIOSENS system The specificity was 100% (No false positives) Discussion and conclusions The window of detection after smoking a single cannabis cigarette were 3-4hours and the very short sample collection/analysis time (<2min) opens up possibilities to detect DUI (Driving Under Influence) in practical way. The window of detection is similar to the acute impairment window reported elsewhere. Apart from cannabis, we have observed that other drugs are accumulated in the mucosal surface of the tongue as well. The overall results 1 (5)

2 showed that the BIOSENS system is applicable as an efficient screening method for detecting recent drug use. Introduction Detection of cannabis is known to be a problem for most on-site oral testing devices. The collection and analysis of a saliva sample using most on-site oral devices is reported to take more than 10 minutes for non-drug takers and considerable longer when taking samples from persons under influence of certain drugs 1,2. The BIOSENS system is based on the principle that drug molecules are accumulated in mucus on the mucosal layer on the tongue, which can be collected by a simple wiping procedure. It takes approximately five to ten seconds to acquire sufficient amount of the mucus for a BIOSENS analysis. The high efficacy in the collection of drugs is explained by the principle that drug molecules are accumulated on the surfaces giving a fortified concentration compared to the surrounding oral fluid. Apart from cannabis (THC) the accumulation effect has been observed for other drugs such as opiates, cocaine and amphetamines, which can be explained by hydrophobic interaction as well as ionic trapping to the negatively charged mucosal layer of the tongue. Experimental Study Objectives To determine the window of detection and cut off values for Δ-9 THC measured by the BIOSENS system after smoking one single cannabis cigarette. Study design The study was conducted at the Faculty of Psychology and Neuroscience, Maastricht in the Netherlands in accordance with ethical principles as enunciated in the Declaration of Helsinki in compliance with The Netherlands law. (Principal Investigator Jan Ramaekers). Mucus samples for BIOSENS analysis and conventional oral fluid samples for laboratory analysis (Quantisal collector devices) were taken before and at different intervals after smoking cannabis or tobacco cigarette. The Cannabis cigarettes were prepared beforehand for each individual by using Medicinal Cannabis from the Cannabis Bureau in the Netherlands. The Δ-9 THC-content was adjusted individually to the body weight (0.3mg/kg) and mixed with tobacco to a standard sized cigarette. The Δ-9 THC content in the cannabis was 11%. Eight volunteers smoked one cannabis cigarette (volunteer 4, 5, 6, 7, 8, 10, 11 and 12) and four volunteers smoked one tobacco cigarette without cannabis (subject 1, 2, 3 and 9). Oral mucus collection and analysis using the BIOSENS system. The BIOSENS system consists of a desorption unit, an extraction unit and a sensor unit. The mucus samples were collected by wiping the tongue a few seconds using a BIOSENS Oral Mucus Collector (OMC), which were heated in the desorption unit in the system to desorb and transfer the drugs in the sample to a cold finger, subsequently extracted in the extraction unit with a small volume of buffer containing a known amount of antibodies against the various drugs that were to be detected in the system. The mixture of inhibited and 2 (5)

3 active antibodies was introduced into the sensor unit in the system after a few seconds inhibition time. The sensing principle to detect presence of antibodies is based on Surface Acoustic Wave technology (SAW) 3. The SAW sensor chip in a BIOSENS system contains 12 different SAW-sensors that are individually surface coated with an antigen analogue selective against a specific antibody. When a drug is present in the sample, the drug will inhibit all or parts of its specific antibody and the SAW-sensor will respond by a decrease in the response providing information of the presence (or absence) of the drug in the mucus sample. Thus a response of 100% means a complete inhibition of the antibodies. The method is very rapid (about 1-2 minutes for sampling and analysis). Laboratory analysis of THC in oral fluid by liquid chromatography-mass spectrometry (LC/MS) Conventional oral fluid samples were collected with Quantisal collection devices, subsequently analyzed for THC at Immunalysis Corporation, 829 Towne Center Drive, Pomona, CA) using LC/MS/MS-technology. (Liquid Chromatography/Mass-Spectrometry,). The limit of quantification for THC was 1ng/ml. Results and discussion Window of detection Figure 1 and 2 shows the response data obtained by using the BIOSENS system from samples collected from the eight cannabis smokers at different times after smoking. Samples showing an inhibition value close to 100% indicate a high concentration of THC. No false positives were observed from mucus samples taken before smoking cannabis or in samples taken from persons smoking only tobacco cigarettes in the study. The figures show substantial inter-individual variations as well as intra-individual variations. The low BIOSENS responses in the Subjects # 5, 7 and 8 correlate very well with the low THCconcentrations obtained from laboratory analysis of conventional oral fluid samples. (Figure 3). The cut off values for the BIOSENS were found to be 20ng/ml at a sensitivity of 71% and 80ng/ml at a sensitivity of 94%. The elimination half-lives of the THC in the samples vary between the subjects but did not differ from published data (half-life around 1-1.5h) 4. 3 (5)

4 Subject No THC response (Degree of antibody inhibition) BIOSENS response from cannabis smokers 100% 80% 60% 40% 20% 0% -20% Subject 4 Subject 5 Subject 6 Subject 7 Subject 8 Subject 10 Subject 11 Subject 12 0h (before smoking) 5% 3% -4% 2% 8% -3% 7% -4% 1h after smoking 80% 25% 98% 30% 14% 65% 87% 70% 2h after smoking 68% 23% 78% 26% 10% 25% 63% 50% 3h after smoking 43% 17% 58% 11% 3% 42% 65% 30% 4h after smoking 66% 21% 51% 12% 12% 17% 57% 30% After meal (at 4h) 35% 6% 14% 9% 9% 2% 13% 5h after smoking 16% 0% 15% 9% -5% 5% 12% 11% 6h after smoking 28% 7% 8% 5% 10% 11% 12% 6% Fig.1: THC responses obtained of the BIOSENS system from oral mucus samples collected from cannabis smokers at different times after smoking. The subjects had a meal after 4 hours BIOSENS window of detection Δ-9 THC #12 #11 #10 #8 #7 #6 #5 # Window of detection (hours) Fig.2: Window of detection of THC in oral mucus samples after smoking one cannabis cigarette 4 (5)

5 THC ng/ml Quantisal results THC ng/ml Subject 4 Subject 5 Subject 6 Subject 7 Subject 8 Subject 10 Subject 11 Subject 12 0h (before smoking) h after smoking h after smoking h after smoking , h after smoking , After a meal , h after smoking , h after smoking ,8 1, Fig.3: THC concentration in oral fluid samples taken by Quantisal devices and analyzed by Liquid Chromatography/Mass-Spectrometry, LC/MS/MS Conclusion The results showed that the BIOSENS system is an applicable and accurate tool for detecting recent cannabis smoking and relating impairment. The whole procedure including collection and analysis of an oral mucus sample took between one and two minutes and provides high sensitivity and low false positive rate. The results from the study confirmed that BIOSENS "wiping collection technique" utilizes the fact that the concentration of Δ-9 THC is higher in the mucus sample from the surface of the tongue compared to samples of oral fluid collected by conventional methods References 1 Bosker, W.M, Huestis, M.A. (2009). Oral fluid testing for drugs of abuse. Clin Chem. 55(11): Drummer, O.H. (2006) Drug testing in oral fluid. Clin Biochem Rev. 27(3): Gronewolds, TM. (2007). Surface acoustic wave sensors in the bioanalytical field: recent trends and challenges. Anal Chim Acta. 603(2): Toennes SW, Ramaekers JG, Theunissen EL, Moeller MR, Kauert GF. (2010). Pharmacokinetic properties of delta9-tetrahydrocannabinol in oral fluid of occasional and chronic users. J Anal Toxicol. 34(4): (5)