Analysis of Δ-tetrahydrocannabinol in oral fluid samples using solid-phase extraction and high-performance liquid chromatography–electrospray ionization mass spectrometry

An analytical method using solid-phase extraction (SPE) and high-performance liquid chromatography–mass spectrometry (LC–MS) has been developed and validated for the confirmation of Δ⁹-tetrahydrocannabinol (THC) in oral fluid samples. Oral fluid was extracted using Bond Elut LRC-Certify solid-phase extraction columns (10 cm³, 300 mg) and elution performed with n-hexane/ethyl acetate. Quantitation made use of the selected ion-recording mode (SIR) using the most abundant characteristic ion [THC + H⁺], m/z 315.31 and the fragment ion, m/z 193.13 for confirmation, and m/z 318.00 for the protonated internal standard, [d₃-THC + H⁺]. The method proved to be precise for THC, in terms of both intra-day and inter-day analyses, with coefficients of variation less than 10%, and the calculated extraction efficiencies for THC ranged from 76 to 83%. Calibration standards spiked with THC between 2 and 100 ng/mL showed a linear relationship (r² = 0.999). The method presented was applied to the oral fluid samples taken from the volunteers during the largest music event in Portugal, named Rock in Rio-Lisboa. Oral fluid was collected from 40 persons by expectoration and with Salivette^®. In 55% of the samples obtained by expectorating, THC was detected with concentration ranges from 1033 to 6552 ng/mL and in 45% of cases THC was detected at concentrations between 51 and 937 ng/mL. However, using Salivette^® collection, 26 of the 40 cases had an undetectable THC.     

Plasma,oral fluid and sweat wipe ecstasy concentrations in controlled and real life conditions

In a double-blind placebo controlled study on psychomotor skills important for car driving (Study 1), a 75 mg dose of +/- 3,4-methylenedioxymethamphetamine (MDMA) was administered orally to 12 healthy volunteers who were known to be recreational MDMA-users. Toxicokinetic data were gathered by analysis of blood, urine, oral fluid and sweat wipes collected during the first 5h after administration. Resultant plasma concentrations varied from 21 to 295 ng/ml, with an average peak concentration of 178 ng/ml observed between 2 and 4h after administration. MDA concentrations never exceeded 20 ng/ml. Corresponding MDMA concentrations in oral fluid, as measured with a specific LC-MS/MS method (which required only 50 microl of oral fluid), generally exceeded those in plasma and peaked at an average concentration of 1215 ng/ml. A substantial intra- and inter-subject variability was observed with this matrix, and values ranged from 50 to 6982 ng/ml MDMA. Somewhat surprisingly, even 4-5h after ingestion, the MDMA levels in sweat only averaged 25 ng/wipe. In addition to this controlled study, data were collected from 19 MDMA-users who participated in a driving simulator study (Study 2), comparing sober non-drug conditions with MDMA-only and multiple drug use conditions. In this particular study, urine samples were used for general drug screening and oral fluid was collected as an alternative to blood sampling. Analysis of oral fluid samples by LC-MS/MS revealed an average MDMA/MDEA concentration of 1121 ng/ml in the MDMA-only condition, with large inter-subject variability. This was also the case in the multiple drug condition, where generally, significantly higher concentrations of MDMA, MDEA and/or amphetamine were detected in the oral fluid samples. Urine screening revealed the presence of combinations such as MDMA, MDEA, amph, cannabis, cocaine, LSD and psilocine in the multiple-drug condition.     

Evolution of the content of THC and other major cannabinoids in drug-type cannabis cuttings and seedlings during growth of plants

In Europe, authorities frequently ask forensic laboratories to analyze seized cannabis plants to prove that cultivation was illegal (drug type and not fiber type). This is generally done with mature and flowering plants. However, authorities are often confronted with very young specimens. The aim of our study was to evaluate when the chemotype of cannabis plantlets can be surely determined through analysis of eight major cannabinoids content during growth. Drug-type seedlings and cuttings were cultivated, sampled each week, and analyzed by high-performance liquid chromatography with diode array detection. The chemotype of clones was recognizable at any developmental stage because of high total Δ(9)-tetrahydrocannabinol (THC) concentrations even at the start of the cultivation. Conversely, right after germination seedlings contained a low total THC content, but it increased quickly with plant age up, allowing chemotype determination after 3 weeks. In conclusion, it is not necessary to wait for plants' flowering to identify drug-type cannabis generally cultivated in Europe.     

Validated method for the simultaneous determination of Delta9-THC and Delta9-THC-COOH in oral fluid, urine and whole blood using solid-phase extraction and liquid chromatography-mass spectrometry with electrospray ionization

A fully validated, sensitive and specific method for the extraction and quantification of Delta(9)-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-Delta(9)-THC (THC-COOH) and for the detection of 11-hydroxy-Delta(9)-THC (11-OH THC) in oral fluid, urine and whole blood is presented. Solid-phase extraction and liquid chromatography-mass spectrometry (LC-MS) technique were used, with electrospray ionization. Three ions were monitored for THC and THC-COOH and two for 11-OH THC. The compounds were quantified by selected ion recording of m/z 315.31, 329.18 and 343.16 for THC, 11-OH THC and THC-COOH, respectively, and m/z 318.27 and 346.26 for the deuterated internal standards, THC-d(3) and THC-COOH-d(3), respectively. The method proved to be precise for THC and THC-COOH both in terms of intra-day and inter-day analysis, with intra-day coefficients of variation (CV) less than 6.3, 6.6 and 6.5% for THC in saliva, urine and blood, respectively, and 6.8 and 7.7% for THC-COOH in urine and blood, respectively. Day-to-day CVs were less than 3.5, 4.9 and 11.3% for THC in saliva, urine and blood, respectively, and 6.2 and 6.4% for THC-COOH in urine and blood, respectively. Limits of detection (LOD) were 2 ng/mL for THC in oral fluid and 0.5 ng/mL for THC and THC-COOH and 20 ng/mL for 11-OH THC, in urine and blood. Calibration curves showed a linear relationship for THC and THC-COOH in all samples (r(2)>0.999) within the range investigated. The procedure presented here has high specificity, selectivity and sensitivity. It can be regarded as an alternative method to GC-MS for the confirmation of positive immunoassay test results, and can be used as a suitable analytical tool for the quantification of THC and THC-COOH in oral fluid, urine and/or blood samples.     

The modern trends in alcohol, drugs and driving research

Research on alcohol, drugs and driving can be broadly separated into experimental and epidemiological studies. Every approach has its inherent advantages and disadvantages. Experimental studies can result in an interpretation by single cause, but can only identify potential risks, and the results can sometimes be of limited value because of the use of non-realistic doses or because of the drug use history or inter-individual differences of the volunteers. Recent studies have used higher, more realistic doses and paid more attention to the combination of alcohol and drugs and have shown that the chronic use of illicit drugs can be associated with some cognitive and/or psychomotor impairment, and can lead to a decrease in driving performance even when the subject is no longer intoxicated.Epidemiological studies include roadside surveys, studies in a subset of drivers, accident risk studies, responsibility analyses, surveys and pharmaco-epidemiological studies. Between studies, results may be incomparable due to testing different populations, different kinds of samples, etc. More large-scale roadside studies are conducted now.Advances in analytical toxicology have also contributed to a better understanding of the risks associated with driving under the influence. While older studies measured the inactive metabolite THC-COOH and did not show an increased risk in cannabis-positive drivers, more recent studies measured the active THC in blood and did show a concentration dependent increase in crash risk. The use of LC–MS/MS has allowed more broad-range screening as this technique can measure many different drugs in a small sample volume. While some older studies used saliva but had many analytical problems (including an insufficient sample volume in up to a third of the cases), newer methods of saliva sampling and analysis give better results. The use of saliva for roadside surveys allows non-invasive sampling, but the lack of correlation with the concentrations in blood makes interpretation of results difficult.The results of both epidemiological and experimental studies should be combined to obtain a good estimate of the impact of certain drugs on driving performance and accident risk. In 2006–07 a committee of international experts drafted guidelines for future research into drugs and driving. These have been taken on board by the DRUID project, a large-scale EU funded project on driving under the influence of drugs, alcohol and medicines.     

An analytical evaluation of eight on-site oral fluid drug screening devices using laboratory confirmation results from oral fluid

A decaying cadaver emits volatile organic compounds that are used by necrophilous and necrophagous insects in order to find their brood substrate. Although volatile organic compounds (VOCs) that are released by carcasses have been identified, little is known about the specific compounds that are used by these insects while searching for a brood substrate. Therefore, we have investigated the chemical ecology involved in the attraction of the necrophagous hide beetle Dermestes maculatus, which feeds as an adult and larva upon decomposing carcasses. Our aims have been to identify the responsible compounds in the odours of the carcass that are important for the attraction of the beetles. Furthermore, we have studied sex- and age-related differences in beetle attraction and tested whether the hide beetle can distinguish between various stages of decomposition by means of the emitted odours. Headspace collection of volatiles released from piglet carcasses (bloated stage, post-bloating stage, advanced decay and dry remains), coupled gas chromatography-mass spectrometry (GC-MS), gas chromatography with electroantennographic detection (GC-EAD) and bioassays were conducted to identify the volatiles responsible for the attraction of the beetles. Freshly emerged male beetles were attracted by the odour of piglets in the post-bloating stage (9 days after death; T(mean) = 27 °C) and the EAD-active compound benzyl butyrate. Statistical analysis revealed a higher relative proportion of benzyl butyrate in the odour bouquet of the post-bloating stage in comparison with the other stages. We therefore conclude that this compound plays an important role in the attraction of hide beetles to carcass odour. This underlines the potential use of D. maculatus for the estimation of the post mortem interval. The decomposition stage at which the female beetles are attracted to the odour of a cadaver remains unknown, as does the nature of this attraction. Pheromones (sexual or aggregation pheromones) might play an essential role correlated with their attraction to carrion and consequently with their attraction to the substrate for mating and ovipositioning.     

The impact of cybercrime on businesses: a novel conceptual framework and its application to Belgium

Despite growing indications and fears about the impact of cybercrime, only few academic studies have so far been published on the topic to complement those published by consultancy firms, cybersecurity companies and private institutes. The review of all these studies shows that there is no consensus on how to define and measure cybercrime or its impact. Against this background, this article pursues two aims: 1) to develop a thorough conceptual framework to define and operationalize cybercrime affecting businesses as well as its impact, harms, and costs; and 2) to test this conceptual framework with a survey of businesses based in Belgium, which was administered in summer 2016 and elicited 310 valid responses. Consisting of five types, our conceptualization of cybercrime is, unlike others, technology-neutral and fully compatible with the legislation. Drawing on Greenfield and Paoli’s Harm Assessment Framework (The British Journal of Criminology, 53, 864–885, 2013), we understand impact as the overall harm of cybercrime, that is, the “sum” of the harms to material support, or costs, and the harms to other interest dimensions i.e., functional (or operational) integrity, reputation and privacy. Whereas we ask respondents to provide a monetary estimate of the costs, respondents are invited to rate the severity of the harms on the basis of an ordinal scale. We claim that this “double track” gives a fuller, more valid assessment of cybercrime impact. Whereas most affected businesses do not report major costs or harm, 15% to 20% of them rate the harms to their internal operational activities as serious or more, with cyber extortion regarded as most harmful.     

Oral fluid testing for driving under the influence of drugs: history, recent progress and remaining challenges

In recent years the demand for drug testing in oral fluid in cases of driving under the influence has been increasing. The main advantages of saliva/oral fluid are the possibility for non-medical personnel to collect it without embarrassment and a better correlation between presence of drugs in oral fluid and impairment. Several surveys have been performed since the 1980s using saliva, and researchers encountered problems related to insufficient sample volume and insufficient sensitivity of the analytical methods. Steady progress has been shown in sample collection, knowledge of toxicokinetics in oral fluid, reliability of on-site and laboratory-based immunoassays and confirmation methods. In a few countries, legislation was passed that allows the use of saliva as a matrix for screening or confirmation.Despite this progress, some more work needs to be done, principally in the areas of the sensitivity and reliability of on-site screening devices, particularly for cannabis and benzodiazepines, knowledge about passive contamination and more generalised proficiency testing before oral fluid testing for DUID will have the reliability needed to be used forensically.