Editorial and perspective
Drug Testing and Analysis Published online in Wiley Online Library
(www.drugtestinganalysis.com) DOI 10.1002/dta.1597
The current situation with cannabinoids This special issue focuses on recent developments with endocannabinoids, phytocannabinoids, and synthetic cannabinoids. This has now become a large area of research, and examples are presented of clinical use, analytical aspects, recreational use, legal problems, and driving while intoxicated. Part I of this special issue was published in 2013 as Vol. 5(1–2). Cannabis continues to be the most widely used illicit drug in most developed countries. For example, in England and Wales, 6.4% of adults used cannabis in the last year,[1] which is close to the average (6.8%) in the European Union.[2] Cannabis in its various forms has been used recreationally and as a ‘traditional’ medicine for thousands of years, but the modern era of research started with the elucidation of the structure of its main psychoactive ingredient, Δ9-tetrahydrocannabinol (THC), by Gaoni and Mechoulam[3] in 1964. By 1992, the naturally-occurring ligand (endocannabinoid) for the cannabinoid receptor in the brain had been isolated and its structure determined.[4] The endocannabinoid system is now recognized as playing an important role in the regulation of a wide range of important physiological functions including the immune response, food intake, cognition, emotion, perception, behavioural reinforcement, motor co-ordination, body temperature, and wake/sleep cycle. The various analytical approaches for the qualitative and quantitative analysis of endocannabinoids and phytocannabinoids in conventional and various alternative biological matrices, including the benefits and limitations of these procedures, is comprehensively reviewed by Battista et al.[5] in this issue. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), results from a study by Thieme et al.[6] presented in this issue, indicate that large doses of THC affect circulating endocannabinoids. This apparent response needs to be confirmed with a follow-up investigation incorporating a control group, preferably as a doubleblind cross-over study.
Medicinal uses
Drug Test. Analysis 2014, 6, 1–6
Copyright © 2013 John Wiley & Sons, Ltd.
1
Cannabis-based medicines are not new; tinctures of cannabis were available in the UK and elsewhere in the nineteenth century, and use continued until well into the twentieth century. There is growing evidence that cannabis-based medicines have potential for the treatment of a wide range of complex conditions, including symptomatic relief in multiple sclerosis, chronic neuropathic pain, intractable nausea and vomiting, loss of appetite and weight in the context of cancer or AIDS, as discussed by Robson in this issue.[7] Herbal cannabis (known, for example, as Bedrocan®) is a licensed medicine in the Netherlands for treating these conditions.[8] A new era of purified extracts of cannabis has been pioneered by GW Pharmaceuticals, and its product, Sativex®, has been licensed in a number of countries. The cultivation and processing of Cannabis sativa L. for production of prescription medicines is discussed by Potter[9] in this issue. To produce Sativex®, the first cannabis-based product recognized in the UK to have medicinal properties, GW
Pharmaceuticals had to overcome several problems. These included the inhomogeneity of the plant material and the fact that the ratios of active ingredients are affected by a range of factors including genetics, growing and storage conditions, the state of maturity at harvest, and the methods used to process and formulate the material. The pharmacologically active ingredients of Sativex® are THC and cannabidiol (CBD). This licensed medicinal product is used as a buccal spray preparation for the symptomatic relief of spasticity in adults suffering from multiple sclerosis when they have not responded adequately to other therapy.[10] Sativex® was granted regulatory approval for such relief in the UK and Spain in 2010 and subsequently in 19 other countries. This followed on from dronabinol, marketed as Marinol®, a pure isomer of THC that had previously been approved by the Food and Drugs Administration (FDA) for the treatment of nausea and vomiting induced by cytostatic therapy, and for the loss of appetite for HIV/AIDS–related cachexia.[11] Nabilone, a synthetic analogue of THC, had also been approved by the FDA as an anti-emetic to control nausea and vomiting associated with cancer chemotherapy,[12] being marketed as Cesamet® in Canada, the UK, the USA, and Mexico. Nabilone may be also used to relieve neuropathic pain in multiple sclerosis patients and also as adjunctive analgesic treatment in adult patients with advanced cancer. The wider use of cannabis-based medicines, not least to alleviate the pain of arthritis and also neuropathies of mixed aetiology, offers potential hope for millions of patients worldwide. But the development of licensed cannabis-based medicines is widely perceived to be hampered by drug prohibition laws in some countries. This stems back to Schedule 1 of the United Nations convention of 1961,[13] on the basis of cannabis having no medicinal use, that is clearly out of kilter with current knowledge regarding its potential therapeutic value. It is beyond this editorial to review the legal status of cannabis worldwide, but a few incongruities are highlighted below. In the UK, THC is classified in Schedule 1, but dronabinol is in Schedule 2 and Sativex® is in Schedule 4 (part 1). There may be legal nuances to support the differences in classification, but it may be argued that there is a lack of consistency in an area of law that should be clear to authorities and also researchers. In the USA, 17 states have permitted the medicinal use of cannabis, and two states (Colorado, Washington) have recently legalized its regulated recreational use. By contrast, also within the USA, obtaining cannabis or controlled cannabinoids such as THC for a clinical trial requires FDA-approved protocols to be submitted to a special ad hoc Public Health Service interdisciplinary review process.[14] Presumably this draconian measure remains because, under Federal law, cannabis is still in US Schedule 1 of the Controlled Substances Act. THC, as the principal psychoactive constituent of plants of the genus Cannabis L., has received much attention, but there are also anomalies concerning other cannabinoids. Research into the therapeutic potential of other phytocannabinoids has concentrated on Δ8- and Δ9-tetrahydrocannabivarin (THCV)[15] and cannabidiol. Research with THCV is hindered, at least in the
Drug Testing and Analysis
A. T. Kicman and L. A. King
UK, because of its status as a Schedule 1 drug,[16] despite the fact that it is not psychoactive and poses no likelihood of misuse. By contrast, 11-hydroxy-THC (11-OH-THC), a known active metabolite of THC in the human, is not controlled in the UK and most other countries With licensed cannabis medicines available, their non-licensed use to help patients in pain is a possibility. Whereas licensed medicines meet acceptable standards of efficacy, safety, and quality, they may also be prescribed for non-licensed use (referred to as ‘off-label’ in the UK and the USA). Such prescribing occurs where a physician may judge it is in the best interests of the patient on the basis of available evidence. With gathering evidence of efficacy, the prescription of cannabis-based medicines for non-licensed use seems a sensible option for pain control, especially because the common adverse effects may be considered to be relatively benign compared to other licensed drugs. For example, it is worth considering cannabis-based medicines, such as Sativex®, as a possible alternative pharmacological therapy for patients who find it difficult to deal with the side effects from the use of particular antidepressants or anticonvulsants for the treatment of neuropathic pain. With physicians being hesitant to prescribe cannabis-based medicines for non-licensed medicinal use, for reasons of cost, a desire for more evidence of efficacy or simply not wishing to take personal responsibility (as is the case for all ‘off-label’ drugs, whether controlled or not), it is not surprising that some patients are resorting to using cannabis illegally, despite the penalties and stigma if they are caught and prosecuted. A report of a cross-sectional survey of self-selected ‘medicinal use of cannabis’ in the UK over the period 1998 to 2002,[17] described that out of the 2969 questionnaires returned, a total of 947 (31.9%) subjects reported ever having used cannabis for medical purposes, including relieving symptoms of chronic pain (25%), multiple sclerosis and depression (22% each), arthritis (21%) and neuropathy (19%).
Harmful effects
2
By contrast to cannabis offering great medicinal value, the recreational use of cannabis can damage health, as reviewed by Hall and Degenhardt[18] in this issue. The adverse effect most often referred to is that cannabis can cause mental health problems, cannabis use being associated with psychotic symptoms (disordered thinking, hallucinations and delusions). When these symptoms meet a level of severity and are sustained over a significant period of time, then they are a manifestation of a psychotic disorder (psychotic illness). There is accumulating evidence that cannabis use plays a significant role in aggravating a psychosis; those most at risk being heavy cannabis users who have a history of psychotic symptoms or a family history of these disorders.[18] Whether there is a causal link between cannabis use and, specifically, developing a chronic psychosis remains to be proven; there is conflicting evidence, despite the large increase in cannabis use over several decades. In Europe, most cannabis is smoked in a mixture with tobacco. In their study of cigarette smokers and cannabis users, van Gastel et al.[19] concluded that smoking is an equally strong independent predictor of the frequency of psychotic-like experiences (PLEs) as monthly cannabis use, and that the association between moderate cannabis use and such experiences is confounded by cigarette smoking. The authors conclude that ‘our findings fit the hypothesis that individuals, who are prone to PLEs, particularly if associated with high
wileyonlinelibrary.com/journal/dta
distress, are more inclined to use cannabis. If this is the case, moderate cannabis use, like cigarette smoking, could be viewed as a mere indicator of risk for PLEs, and thus for mental health problems in general instead of a causative factor.’ It is also worth mentioning that cannabis use is considered to present far less of a risk to the health of the general population compared to alcohol, for example, as discussed by Nutt et al.[20–22] Moreover, the continuing emphasis on the potential of cannabis to cause mental illnesses tends to overshadow a probably much more common effect, which is insidious and psychosocial in nature, in that adolescents who become regular users have impaired educational attainment, as Hall and Degenhardt point out.[18] This impairment is at a critical time of life where educational achievement usually fashions the career that an individual will enter. There is a commonly held argument that cannabis should remain illegal to dissuade its use, but there is a powerful counter-argument that it should be legalized, its sale being licensed, available to adults only, in a form that is controlled in dose and quality. The potential of revenue to governments by taxation on its sale has also not gone unnoticed.[23–25] Moreover, recent attention has focused on the USA, where Colorado voters have overwhelmingly approved a plan for taxing their state’s legal cannabis market, with the revenue to be dedicated to school construction and regulation of cannabis sales. As an alternative to legalization, decriminalization, or non-prosecution strategies for possession would still leave a cannabis embargo on its supply, thus continuing to enrich organised crime. The current illegality of cannabis and the difficulties faced by importers has led to the availability of home-grown super-strength cannabis often known as Skunk.[26] This is usually produced by intensive cultivation using additional lighting, propagation of female cuttings, and hydroponic methods. It contains a much higher THC content compared to imported cannabis originating from its natural habitat, for example, as reported by Hardwick and King.[27] There are concerns that the higher THC content will lead to increases in psychosis, but that supposition is countered by the argument that some experienced users will titrate the amount they administer to achieve the effect they are seeking. Even so, Skunk has typically a much smaller content of CBD relative to THC, where CBD is the natural biosynthetic precursor of THC. The much reduced CBD content is probably key, as it is thought to confer the anxiolytic (anxiety reducing) properties that can counter the psychotic effects of THC.[28] In their report in this issue on cannabis potency in the Venice area, Zamengo et al.[29] show that the CBD/THC ratio in all cannabis samples decreased from 0.52 in 2010 to 0.18 in 2012. This was largely due to the increase in the proportion of herbal cannabis, including samples which had been grown by intensive methods. In their 2008 study, Hardwick and King[27] found a CBD/THC ratio of 0.6 for cannabis resin, but less than 0.006 in ‘sinsemilla’.
Synthetic cannabinoid receptor agonists The illegality of cannabis for recreational use in many countries has also led to the relatively new phenomenon of the sale of cannabimimetics, these often circumventing existing laws concerning drug misuse. Cannabimimetics are not compounds produced by the cannabis plant, but synthetic products that were investigated by the pharmaceutical industry as alternative medicinal products to the use of phytocannabinoids in an attempt to mimic some of the beneficial effects of cannabis, but without causing adverse psychoactive effects. This research has
Copyright © 2013 John Wiley & Sons, Ltd.
Drug Test. Analysis 2014, 6, 1–6
Drug Testing and Analysis
Editorial and perspective
Drug Test. Analysis 2014, 6, 1–6
an attempt to address these issues (aptamers are folded singlestranded nucleic acids that can be synthesized in vitro, with those that bind with high affinity and specificity for their target molecules being chosen). There is currently a gap between the convenience of immunoglobulin-based assays that lack the specificity and adaptability for the screening of the many synthetic cannabinoids and the hyphenated mass spectrometry approaches that can screen for these compounds and confirm their presence, but lack the simplicity and portability of immunoassay.
Workplace testing Acute cannabis intoxication compromises safety in the workplace and leads to an increased risk of having an accident. Oral fluid is increasingly becoming the matrix of choice for workplace drug testing and is the obvious candidate also for roadside drug testing. Current knowledge on cannabinoids in oral fluid is extensively reviewed in this issue by Lee and Huestis.[37] Data from urine and oral fluid analysis showed that cannabis was the most frequently detected drug amongst workers in the USA, the UK, Italy, and Brazil. One major advantage of oral fluid collection is that it may be performed under observation, thus impeding substitution or adulteration that has been known to occur when urine is voided unobserved to preserve an individual’s privacy. Another advantage of oral fluid is that the concentration of a drug and/or its metabolite(s) often can be better correlated with blood or plasma/ serum concentrations than with urine, a very important factor when evaluating whether or not an individual was likely to have been impaired at the time of collection; this is particularly germane to workplace and also roadside drug testing. Of note, however, are that cannabinoids are slowly absorbed through the oral cavity and consequently following the smoking of cannabis, THC deposited in the mouth predominantly contributes to the oral fluid concentration, with a relatively small contribution from the transfer of THC from the systemic circulation. By contrast, THCCOOH (11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid), a major secondary, biologically inactive metabolite of THC is not present in cannabis smoke. But as Lee and Huestis[37] point out, these two compounds are not significantly correlated from 0.25 hr to 6 hr after smoking due to their differing mechanisms of entry into oral fluid, a time period overlapping with the impairment window. A possible fruitful avenue of investigation is to examine whether 11-OH-THC in oral fluid is a valuable confirmatory marker of recent cannabis intoxication that circumvents the problem with encapsulation of phytocannabinoids in the oral cavity. 11-OH-THC is the intermediate (and active) metabolite in the formation of THCCOOH from THC, primarily by the activity of CYP 2C9 in the human liver. Another alternative matrix for analysis is head hair, which can reveal a history of drug administration and whether use was acute or chronic. Thieme et al.,[38] in this issue, focus on detection of THCCOOH in hair, as its incorporation can only result from cannabis use, as opposed to the presence of THC which can also occur because of environmental contamination (smoke). A sensitive and specific procedure for the analysis of this metabolite in hair by LC-tandem/multistage MS is challenging, primarily because of the small target concentration. THCCOOH has a low rate of incorporation in hair due it being an acidic compound, its presence being dwarfed by that of lipophilic organic acids that interfere on LC-MS systems, resulting in insufficient sensitivity for this target
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/dta
3
continued to the present day, with many hundreds of cannabinoid receptor CB1 agonists having been described in a recent review.[30] CB1 inverse agonists and antagonists may be of medicinal value; investigations are underway to evaluate whether the second generation of CB1 inverse agonists could be useful in the treatment of obesity and/or related diseases, as the endocannabinoid system appears to play an important role in energy homeostasis and eating behaviour, as reviewed in this issue by Bermudez-Silva et al.[31] Some of the early synthetic substances were based on the dibenzopyran nucleus and showed a structural affinity with THC. But, cannabimimetic activity arises in a wide range of compounds, few of which show any structural resemblance to the phytocannabinoids. Despite that extensive effort, nabilone, is the only synthetic compound to have been licensed. By contrast, a plethora of illicit cannabimimetics have been sold for recreational use, some being based on structures described in patents, but others appear to be entirely new, never having been described in any publication or having a Chemical Abstract Number. In this issue, the identification and quantification of synthetic cannabinoids in ‘Spice-like’ herbal mixtures is presented by Langer et al.[32] with particular reference to the situation in Germany, the authors recommending gas chromatography–mass spectrometry (GC-MS) for this purpose. To monitor recreational drugs being used at any one time within a community, sewage analysis can be of help, but of the cannabimimetics targeted by Reid et al.,[33] as described in this issue, some are prone to degradation. The authors did, however, discover that a urinary metabolite of JWH 018 was present in samples collected from three Norwegian cities, suggesting that its use is widespread in that country. Even with increased analytical capability, nonetheless, there are formidable legislative issues surrounding the cannabimimetics. King[34] in this issue comments that there are now so many cannabimimetics that is questionable whether they can be legally controlled, and concludes in his perspective: ‘Insofar as there is little evidence that current controls on new substances reduce harmful behaviours, then in response to the question ‘What should be done about controlling synthetic cannabinoids?’– the answer might be to do nothing. While this idea may be unpalatable to politicians, the reality is that few would notice, administrative costs to legislatures would be reduced and little additional harm would be caused.’ It is unclear if synthetic cannabinoid agonists pose any greater harm than the cannabis plant and its products. Although a few case reports of severe toxicity have appeared,[35] a review of 1898 cases involving cannabimimetics reported to the US National Poison Data System between 1 January 2010 and 1 October 2010 found that most exposures resulted in non-life-threatening effects not requiring treatment, although a minority of exposures resulted in more severe effects, including seizures.[36] In contrast to immunoassay techniques, hyphenated mass spectrometry is demonstrably well suited for the effective screening of synthetic cannabinoids in the laboratory. The cannabimimetic market is dynamic and it is inevitable that there will be a time-lag in the raising of immunoglobulins to the newer compounds. Furthermore, even with favourable cross-reactivity, it is reasonable to conclude that there will never be antibody based commercial kits and point-of-care devices available that have the blanket capability of detecting the ever increasing number of synthetic cannabinoids (and their metabolites) and thus this technology is limited. It is possible that, in the future, commercial suppliers will switch to the use of aptamers, a young science, in
Drug Testing and Analysis
A. T. Kicman and L. A. King
analyte. Thieme et al.[38] investigate various derivatives to solve these problematic issues and elegantly arrive at a solution using selective methylation as the final step in sample preparation. The authors’ success opens the avenue of toxicology laboratories being able to employ a method that achieves the sensitivity and specificity required using their existing LC-MS/MS systems rather than relying on the need for GC-MS/MS or GC-GC-MS instruments (using negative chemical ionization), the latter instruments still largely being considered only necessary for bespoke analytical toxicology purposes. Methylation of the 11-carboxyl group, but with the (acidic) phenolic hydroxyl group of THCCOOH remaining underivatized, makes methylated THCCOOH conducive to sensitive electrospray ionization in negative mode. In comparison, the fatty acids in hair extracts, not being phenolic, but having their carboxylic acid groups methylated, have consequently been converted to neutral compounds and thus are no longer susceptible to ionization in negative ion mode, thereby considerably reducing matrix interference. A further significant improvement in sensitivity and specificity for THCCOOH was achieved by MS3 using a QTRAP instrument, as may be expected. Moosmann et al.[39] in this issue investigate the THC precursor, Δ9-tetrahydrocannabinolic acid A (THCA-A) as a control marker of in-vivo exposure to cannabis smoke, as THCA-A is not incorporated into hair by metabolic processes in evidentially significant amounts. These investigators found that THCA-A was present in relatively low concentrations compared to forensic samples (from a previous investigation of theirs), indicating that the touching of hair with contaminated fingers is likely to be the major source of THC contamination. The authors conclude with a cautionary note: ‘These findings underline that hair-findings in general have to be interpreted with utmost care, particularly in case of drugs which are smoked or when cross-contamination via hand contact is likely to occur’. LC-MS/MS was also adopted by Salomone et al.[40] in this issue to evaluate the prevalence of cannabimimetics in hair. Their developed procedure, which incorporated an ultra-high performance liquid chromatography system (Waters Acquity UPLC ®), selected reaction monitoring and use of 5 internal standards, achieved sufficient sensitivity for the specific analysis of 23 synthetic cannabinoids, ranging from a limit of detection of 0.2 pg/mg to 1.3 pg/mg hair (the only exception being HU-210 at 24 pg/mg) and a lower limit of quantification of 0.7 pg/mg to 4.3 pg/mg (80 pg/mg for HU-210). The procedure was applied to the analysis to hair samples previously tested in their laboratory, with a number of cannabimimetics being detected and several positive samples notably having concentrations less than 50 pg/mg, this being the internationally accepted cut-off value for THC in hair. A challenge for those performing toxicological analysis of hair is how to interpret such concentrations and what cut-off value should be chosen for a particular cannabimimetic to distinguish between chronic consumption and occasional use or possible external contamination. The seemingly long terminal half-lives of commonly used synthetic cannabinoids in serum also present a major challenge to the clinical and forensic toxicologist. Kneisel et al.[41] in this issue report that JWH-081, JWH-122 or JWH-210 were detectable by LC-MS/MS for up to 102 days in serum samples collected from patients that had self-reported cessation of drug use.
Cannabis and driving
4
With respect to driving under the influence of cannabis, analysis is vital to obtaining accurate information on prevalence. It is a
wileyonlinelibrary.com/journal/dta
moot point whether the continuation of roadside surveys based solely on results of self-reporting are worthwhile, as pointed out by Van der Linden et al.[42] in this issue. There are many factors, however, to take into account regarding the relationship between blood/serum THC concentrations and driving behaviour, as reviewed by Wolff and Johnston[43] in this issue. This paper is based on a report to the UK Government published in early 2013[44] that was concerned with the effects of driving under the influence of a number of drugs other than alcohol. Just as in the population at large, cannabis remains the most commonly used drug in the UK amongst those who reported driving under the influence of illegal drugs in the previous 12 months. A significant dose-related decrease in driving performance exists following cannabis use; raised blood THC concentrations are significantly associated with an increased number of traffic crashes and death.[44,45] There is a clear need for objective measurement of THC if any drug-driving legislation is to be effective. It is generally accepted that the inactive metabolite THCCOOH is an unsuitable indicator of recent cannabis use or impairment. When attempting to frame legislation on the basis of THC concentrations in blood, a number of issues arise. First, in a roadside survey carried out in Belgium, Van der Linden et al.[42] showed that of 81 drivers who self-reported cannabis use, 34 had THC levels in oral fluid greater than the normal analytical limit (1 μg/L). However, only 8 had THC blood concentrations above this cut-off. Even in a sub-set of those who claimed to have used cannabis less than 4 hr before driving, THC was only detected in the blood of 3 of the 8 individuals. Secondly, when a driver has consumed alcohol and cannabis there is a greater risk to road safety than when either drug is used alone; this complicates the interpretation of blood levels. Thirdly, peak plasma concentrations of THC in habitual cannabis users may range up to 11μg/L, but, paradoxically, they are much lower than peak values in occasional smokers, which can reach over 260 μg/L. Finally, any legislation which sets maximum limits for THC in blood would require roadside screening devices and further equipment in police stations. Interpretation of the evidence of the risks of driving under the influence of cannabis has also been muddled by the conflation of two issues, as Wolff and Johnston point out in their article; that of the fitness to drive and that of criminality due to the illicit nature of the drug. This is most apparent in countries that apply thresholds (concentration limits) for alcohol and driving but apply or advocate a zero-tolerance course of action for THC. Fig. 1 in the Wolff and Johnston review[43] shows the risk of a traffic accident, using data reported by Laumon et al.,[46] based on a positive detection of cannabis at a blood THC concentration of >1 μg/L and for blood alcohol (EtOH) of ≥50 mg alcohol/dL. The risk approximately doubles for use of cannabis alone, whereas that for alcohol alone increases approximately 8-fold, being approximately 16-fold when alcohol and cannabis use are detected concurrently. Furthermore, the risk associated with cannabis seems to be similar to the risk when driving with a low-alcohol concentration (between 10 and 50 mg/dL),[47] notably under the current UK drink-driving threshold of 80 mg/dL); a serum concentration of THC of 3.8 μg/L (~2 μg/L whole blood) causes the same amount of impairment as 50 mg/dL of alcohol.[45] As an aside, almost nothing is known about the effect of cannabimimetics on driving performance, but it would be expected that those CB1 agonists that readily cross the blood–brain barrier would cause similar impairment to that caused by cannabis. After a driver has been found positive, the question then arises as to when a driving licence may be returned, as heavy
Copyright © 2013 John Wiley & Sons, Ltd.
Drug Test. Analysis 2014, 6, 1–6
Drug Testing and Analysis
Editorial and perspective consumption may suggest a long-term unfitness to drive. This is not a trivial consideration and can have cost implications for governments that consider it important to have follow-up investigations of offending drivers to answer this question. Fabritius et al.[48] in this issue recommend a way forward, based on the combination of findings from analysis of whole blood THC and a medical interview of the driver, in the context of the zero tolerance policy in their country, Switzerland. Their recommended guidance criteria were underpinned by THCCOOH data from a carefully controlled administration study, these being: (1) the driver can reclaim their licence if the whole blood THCCOOH concentration is below 3 μg/L and a medical interview suggests that cannabis smoking is occasional, i.e. no long and costly monitoring is necessary; (2) for THCCOOH concentrations greater than 40 μg/L (these being strongly correlated with heavy consumption) and the medical assessment indicates heavy consumption, a follow-up for a cannabis disorder must be undertaken until abstinence is proven; and (3) with a concentration between 3 and 40 μg/L and the medical assessment does not exclude regular use, it is advisable to seek the opinion of a forensic expert and have a ‘therapeutic follow-up’ until abstinence has been demonstrated, at which point the driving licence can be reclaimed.
Future developments
[7] [8] [9] [10] [11] [12] [13]
[14]
[15]
[16]
Although not discussed elsewhere in this special issue, the recent introduction of E-cigarettes in many countries could have major implications for drug policies. Even ‘standard’ E-cigarettes, which contain nicotine, have proved controversial from a regulatory aspect. However, these devices could easily be adapted to vaporise THC with an equal proportion of CBD as a controlled dose, thus circumventing the adverse respiratory effects of inhaling the high amounts of hydrocarbons and toxic by-products associated with smoking cannabis with or without tobacco.
[17] [18] [19]
[20] [21]
Andrew T. Kicman Drug Control Centre, Department of Forensic Science and Drug Monitoring, Division of Analytical and Environmental Sciences, King’s College London, UK E-mail: [email protected]
[22] [23] [24]
Leslie A. King 27 Ivar Gardens, Basingstoke, RG24 8YD, UK E-mail: [email protected]
[25]
References
Drug Test. Analysis 2014, 6, 1–6
[26] [27] [28]
[29]
[30] [31] [32]
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/dta
5
[1] Home Office. Drug misuse: Findings from the 2012/13 crime survey for England and Wales. London. Available at: http://socialwelfare.bl. uk/subject-areas/services-activity/substance-misuse/homeoffice/ 153597Drugs_Misuse201213.pdf [29 November 2013]. [2] EMCDDA. Annual report 2012: The state of the drugs problem in Europe. Available at: http://www.emcdda.europa.eu/attachements. cfm/att_190854_EN_TDAC12001ENC_.pdf [29 November 2013]. [3] Y. Gaoni, R. Mechoulam. Isolation, structure and partial synthesis of an active constituent of hashish. J. Am. Chem. Soc. 1964, 86, 1646. [4] W.A. Devane, L. Hanus, A. Breuer, R.G. Pertwee, L.A. Stevenson, G. Griffin, D. Gibson, A. Mandelbaum, A. Etinger, R. Mechoulam. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 1992, 258, 1946. [5] N. Battista, M. Sergi, C. Montesano, S. Napoletano, D. Compagnone, M. Maccarrone. Analytical approaches for the determination of phytocannabinoids and endocannabinoids in human matrices. Drug Test. Anal. 2014, 6, 7. [6] U. Thieme, G. Schelling, D. Hauer, R. Greif, T. Dame, R.P. Laubender, W. Bernhard, D. Thieme, P. Campololongo, L. Theiler. Quantification
of anadamide and 2-arichidonoylglycerol plasma levels to examine potential influences of tetrahydrocannabinol application on the endocannabinoid system in humans. Drug Test. Anal. 2014, 6, 17. P.J. Robson. Therapeutic potential of cannabinoid medicines. Drug Test. Anal. 2014, 6, 24. A. Hazekamp. An evaluation of the quality of medicinal grade cannabis in the Netherlands. Cannabinoids 2006, 1, 1. D. Potter. A review of the cultivation and processing of cannabis (Cannabis sativa L.) for production of prescription medicines in the UK. Drug Test. Anal. 2014, 6, 31. Martindale: The Complete Drug Reference (online). Drug monograph:Nabiximols. [29 November 2013] Martindale: The Complete Drug Reference (online). Drug monograph:Dronabinol. [29 November 2013] Martindale: The Complete Drug Reference (online). Drug monograph:Nabilone. [29 November 2013] United Nations. Single Convention on Narcotic Drugs, 1961. Available at: http://www.unodc.org/pdf/convention_1961_en.pdf [29 November 2013]. Department of Health and Human Services’ Guidance. Procedures for the Provision of Marijuana for Medical Research. Available at: http://grants.nih.gov/grants/guide/notice-files/not99-091. html [29 November 2013]. R.G. Pertwee, A. Thomas, L.A. Stevenson, R.A. Ross, S.A. Varvel, A.H. Lichtman, B.R. Martin, R.K. Razdan. The psychoactive plant cannabinoid, Delta(9)-tetrahydrocannabinol, is antagonized by Delta(8)and Delta(9)-tetrahydrocannabivarin in mice in vivo. Brit. J. Pharmacol. 2007, 150, 586. D.J. Nutt, L.A. King, D.E. Nichols. Effects of schedule I drug laws on neuroscience research and treatment innovation. Nat. Rev. Neurosci. 2013, 14, 577. M.A. Ware, H. Adams, G.W. Guy. The medicinal use of cannabis in the UK: Results of a nationwide survey. Int. J. Clin. Pract. 2005, 59, 291. W. Hall, L. Degenhardt. The adverse health effects of chronic cannabis use. Drug Test. Anal. 2014, 6, 39. W.A. van Gastel, J.H. MacCabe, C.D. Schubart, A. Vreeker, W. Tempelaar, R.S. Kahn, M.P.M. Boks. Cigarette smoking and cannabis use are equally strongly associated with psychotic-like experiences: A cross-sectional study in 1929 young adults. Psychol. Med. 2013, 43, 2393. D. Nutt. Government vs science over drug and alcohol policy. Lancet 2009, 374, 1731. D. Nutt, L.A. King, W. Saulsbury, C. Blakemore. Development of a rational scale to assess the harm of drugs of potential misuse. Lancet 2007, 369, 1047. D.J. Nutt, L.A. King, L.D. Phillips. Drug harms in the UK: A multicriteria decision analysis. Lancet, 2010, 376, 1558. S. Pudney. Drugs policy: What should we do about cannabis? Econ. Policy 2010, 25, 165. D. Werb, B. Nosyk, T. Kerr, B. Fischer, J. Montaner, E. Wood. Estimating the economic value of British Columbia’s British cannabis market: Implications for provincial cannabis policy. Int. J. Drug Policy 2012, 23, 436. M. Bryan, E. Del Bono, S. Pudney. Licensing and regulation of the cannabis market in England and Wales: Towards a cost-benefit analysis. Institute for Social and Economic Research. Available at: https:// www.iser.essex.ac.uk/d/153 [28 November 2013]. L.A. King, C. Carpentier, P. Griffiths. An overview of cannabis potency in Europe. EMCDDA Insights. Available at: http://www.emcdda.europa.eu/ attachements.cfm/att_33985_EN_Insight6.pdf [29 November 2013]. S. Hardwick, L.A. King. Home Office Cannabis Potency Study 2008. Home Office Scientific Development Branch, St Albans, UK, 2008. A. Englund, P.D. Morrison, J. Nottage, D. Hague, F. Kane, S. Bonaccorso, J.M. Stone, A. Reichenberg, R. Brenneisen, D. Holt, A. Feilding, L. Walker, R.M. Murray, S. Kapur. Cannabidiol inhibits THCelicited paranoid symptoms and hippocampal-dependent memory impairment. J. Psychopharmacol. 2013, 27, 19. L. Zamengo, G. Frison, C. Bettin, R. Sciarrone. Variability of cannabis potency in the Venice area (Italy): A survey over the period 2010– 12. Drug Test. Anal. 2014, 6, 46. J.W. Huffman. The Cannabinoid Receptors (Ed: P.H. Reggio). Humana Press, New York, NY, USA, 2009. S.Y. Romerzo-Zerbo, F.J. Bermudez-Silva. Cannabinoids, eating behaviour, and energy homeostasis. Drug Test. Anal. 2014, 6, 52. N. Langer, R. Lindigkeit, H.-M. Schiebel, L. Ernst, T. Beurle. Identification and quantification of synthetic cannabinoids in ’spice-like’
Drug Testing and Analysis
[33]
[34] [35]
[36]
[37] [38]
[39] [40]
[41]
A. T. Kicman and L. A. King
herbal mixtures: A snapshot of the German situation in the autumn of 2012. Drug Test. Anal. 2014, 6, 59. M.J. Reid, L. Derry, K.V. Thomas. Analysis of new classes of recreational drugs in sewage: Synthetic cannabinoids and amphetaminelike substances. Drug Test. Anal. 2014, 6, 72. L.A. King. Legal controls on cannabimimetics: an international dilemma? Drug Test. Anal. 2014, 6, 80. J. Lapoint, L.P. James, C.L. Moran, L.S. Nelson, R.S. Hoffman, J.H. Moran. Severe toxicity following synthetic cannabinoid ingestion. Clin. Toxicol. 2011, 49, 760. C.O. Hoyte, J. Jacob, A.A. Monte, M. Al-Jumaan, A.C. Bronstein, K.J. Heard. A characterization of synthetic cannabinoid exposures reported to the national poison data system in 2010. Ann. Emerg. Med. 2012, 60, 435. D. Lee, M.A. Huestis. Current knowledge on cannabinoids in oral fluid. Drug Test. Anal. 2014, 6, 88. D. Thieme, H. Sachs, M. Uhl. Proof of cannabis administration by sensitive detection of 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid in hair using selective methylation and application of liquid chromatography- tandem and multistage mass spectrometry. Drug Test. Anal. 2014, 6, 112. B. Moosmann, N. Roth, V. Auwärter. Hair analysis for THCA-A, THC and CBN after passive in vivo exposure to marijuana smoke. Drug Test. Anal. 2014, 6, 119. A. Salomone, C. Luciano, D. Di Corcia, E. Gerace, M. Vincenti. Hair analysis as a tool to evaluate the prevalence of synthetic cannabinoids in different populations of drug consumers. Drug Test. Anal. 2014, 6, 126. S. Kneisel, J. Teske, V. Auwärter. Analysis of synthetic cannabinoids in abstinence control: Long drug detection windows in serum and implications for practitioners. Drug Test. Anal. 2014, 6, 135.
[42] T. Van der Linden, P. Silverans, A.G. Verstraete. Comparison between self-report of cannabis use and toxicological detection of THC/ THCCOOH in blood and THC in oral fluid in drivers in a roadside survey. Drug Test. Anal. 2014, 6, 137. [43] K. Wolff, A. Johnston. Cannabis use: A perspective in relation to the proposed UK drug-driving legislation. Drug Test. Anal. 2014, 6, 143. [44] K. Wolff, R. Brimblecombe, J.C. Forfar, A.R. Forrest, E. Gilvarry, A. Johnston, J. Morgan, M.D. Osselton, L. Read, D. Taylor. Driving under the influence of drugs. Available at: http://www.apccs.police.uk/ fileUploads/APCC_Group_Emails/drug-driving-expert-panelreport-1.pdf [29 November 2013]. [45] H. Schulze, M. Schumacher, R. Urmeew, K. Auerbach. Final Report: Work performed, main results and recommendations. Available at: http://www.druid-project.eu/cln_031/nn_107548/Druid/EN/Dissemination/downloads__and__links/Final__Report,templateId=raw, property=publicationFile.pdf/Final_Report.pdf [29 November 2013]. [46] B. Laumon, B. Gadegbeku, J.L. Martin, M.B. Biecheler, S.A.M. Grp. Cannabis intoxication and fatal road crashes in France: Population based case–control study. Brit. Med. J. 2005, 331, 1371. [47] T. Hels, I.M. Bernhoft, A. Lyckegaard, S. Houwing, M. Hagenzieker, S.A. Legrand, C. Isalberti, T. Van der Linden, A.G. Verstraete. Risk of injury by driving with alcohol and other drugs. Available at: http://www. druid-project.eu/cln_031/nn_107548/Druid/EN/deliverales-list/downloads/Deliverable__2__3__5,templateId=raw,property=publicationFile. pdf/Deliverable_2_3_5.pdf [29 November 2013]. [48] M. Fabritius, B. Favrat, H. Chtioui, G. Battistella, J.-M. Annoni, M. Appenzeller, K. Dao, E. Fornari, E. Lauer, J.-F. Mall, P. Maeder, P. Mangin, C. Staub, C. Giroud. THCCOOH concentrations in whole blood: Are they useful in discriminating occasional from heavy smokers? Drug Test. Anal. 2014, 6, 155.
6 wileyonlinelibrary.com/journal/dta
Copyright © 2013 John Wiley & Sons, Ltd.
Drug Test. Analysis 2014, 6, 1–6
Drug Testing and Analysis Published online in Wiley Online Library
(www.drugtestinganalysis.com) DOI 10.1002/dta.1597
The current situation with cannabinoids This special issue focuses on recent developments with endocannabinoids, phytocannabinoids, and synthetic cannabinoids. This has now become a large area of research, and examples are presented of clinical use, analytical aspects, recreational use, legal problems, and driving while intoxicated. Part I of this special issue was published in 2013 as Vol. 5(1–2). Cannabis continues to be the most widely used illicit drug in most developed countries. For example, in England and Wales, 6.4% of adults used cannabis in the last year,[1] which is close to the average (6.8%) in the European Union.[2] Cannabis in its various forms has been used recreationally and as a ‘traditional’ medicine for thousands of years, but the modern era of research started with the elucidation of the structure of its main psychoactive ingredient, Δ9-tetrahydrocannabinol (THC), by Gaoni and Mechoulam[3] in 1964. By 1992, the naturally-occurring ligand (endocannabinoid) for the cannabinoid receptor in the brain had been isolated and its structure determined.[4] The endocannabinoid system is now recognized as playing an important role in the regulation of a wide range of important physiological functions including the immune response, food intake, cognition, emotion, perception, behavioural reinforcement, motor co-ordination, body temperature, and wake/sleep cycle. The various analytical approaches for the qualitative and quantitative analysis of endocannabinoids and phytocannabinoids in conventional and various alternative biological matrices, including the benefits and limitations of these procedures, is comprehensively reviewed by Battista et al.[5] in this issue. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), results from a study by Thieme et al.[6] presented in this issue, indicate that large doses of THC affect circulating endocannabinoids. This apparent response needs to be confirmed with a follow-up investigation incorporating a control group, preferably as a doubleblind cross-over study.
Medicinal uses
Drug Test. Analysis 2014, 6, 1–6
Copyright © 2013 John Wiley & Sons, Ltd.
1
Cannabis-based medicines are not new; tinctures of cannabis were available in the UK and elsewhere in the nineteenth century, and use continued until well into the twentieth century. There is growing evidence that cannabis-based medicines have potential for the treatment of a wide range of complex conditions, including symptomatic relief in multiple sclerosis, chronic neuropathic pain, intractable nausea and vomiting, loss of appetite and weight in the context of cancer or AIDS, as discussed by Robson in this issue.[7] Herbal cannabis (known, for example, as Bedrocan®) is a licensed medicine in the Netherlands for treating these conditions.[8] A new era of purified extracts of cannabis has been pioneered by GW Pharmaceuticals, and its product, Sativex®, has been licensed in a number of countries. The cultivation and processing of Cannabis sativa L. for production of prescription medicines is discussed by Potter[9] in this issue. To produce Sativex®, the first cannabis-based product recognized in the UK to have medicinal properties, GW
Pharmaceuticals had to overcome several problems. These included the inhomogeneity of the plant material and the fact that the ratios of active ingredients are affected by a range of factors including genetics, growing and storage conditions, the state of maturity at harvest, and the methods used to process and formulate the material. The pharmacologically active ingredients of Sativex® are THC and cannabidiol (CBD). This licensed medicinal product is used as a buccal spray preparation for the symptomatic relief of spasticity in adults suffering from multiple sclerosis when they have not responded adequately to other therapy.[10] Sativex® was granted regulatory approval for such relief in the UK and Spain in 2010 and subsequently in 19 other countries. This followed on from dronabinol, marketed as Marinol®, a pure isomer of THC that had previously been approved by the Food and Drugs Administration (FDA) for the treatment of nausea and vomiting induced by cytostatic therapy, and for the loss of appetite for HIV/AIDS–related cachexia.[11] Nabilone, a synthetic analogue of THC, had also been approved by the FDA as an anti-emetic to control nausea and vomiting associated with cancer chemotherapy,[12] being marketed as Cesamet® in Canada, the UK, the USA, and Mexico. Nabilone may be also used to relieve neuropathic pain in multiple sclerosis patients and also as adjunctive analgesic treatment in adult patients with advanced cancer. The wider use of cannabis-based medicines, not least to alleviate the pain of arthritis and also neuropathies of mixed aetiology, offers potential hope for millions of patients worldwide. But the development of licensed cannabis-based medicines is widely perceived to be hampered by drug prohibition laws in some countries. This stems back to Schedule 1 of the United Nations convention of 1961,[13] on the basis of cannabis having no medicinal use, that is clearly out of kilter with current knowledge regarding its potential therapeutic value. It is beyond this editorial to review the legal status of cannabis worldwide, but a few incongruities are highlighted below. In the UK, THC is classified in Schedule 1, but dronabinol is in Schedule 2 and Sativex® is in Schedule 4 (part 1). There may be legal nuances to support the differences in classification, but it may be argued that there is a lack of consistency in an area of law that should be clear to authorities and also researchers. In the USA, 17 states have permitted the medicinal use of cannabis, and two states (Colorado, Washington) have recently legalized its regulated recreational use. By contrast, also within the USA, obtaining cannabis or controlled cannabinoids such as THC for a clinical trial requires FDA-approved protocols to be submitted to a special ad hoc Public Health Service interdisciplinary review process.[14] Presumably this draconian measure remains because, under Federal law, cannabis is still in US Schedule 1 of the Controlled Substances Act. THC, as the principal psychoactive constituent of plants of the genus Cannabis L., has received much attention, but there are also anomalies concerning other cannabinoids. Research into the therapeutic potential of other phytocannabinoids has concentrated on Δ8- and Δ9-tetrahydrocannabivarin (THCV)[15] and cannabidiol. Research with THCV is hindered, at least in the
Drug Testing and Analysis
A. T. Kicman and L. A. King
UK, because of its status as a Schedule 1 drug,[16] despite the fact that it is not psychoactive and poses no likelihood of misuse. By contrast, 11-hydroxy-THC (11-OH-THC), a known active metabolite of THC in the human, is not controlled in the UK and most other countries With licensed cannabis medicines available, their non-licensed use to help patients in pain is a possibility. Whereas licensed medicines meet acceptable standards of efficacy, safety, and quality, they may also be prescribed for non-licensed use (referred to as ‘off-label’ in the UK and the USA). Such prescribing occurs where a physician may judge it is in the best interests of the patient on the basis of available evidence. With gathering evidence of efficacy, the prescription of cannabis-based medicines for non-licensed use seems a sensible option for pain control, especially because the common adverse effects may be considered to be relatively benign compared to other licensed drugs. For example, it is worth considering cannabis-based medicines, such as Sativex®, as a possible alternative pharmacological therapy for patients who find it difficult to deal with the side effects from the use of particular antidepressants or anticonvulsants for the treatment of neuropathic pain. With physicians being hesitant to prescribe cannabis-based medicines for non-licensed medicinal use, for reasons of cost, a desire for more evidence of efficacy or simply not wishing to take personal responsibility (as is the case for all ‘off-label’ drugs, whether controlled or not), it is not surprising that some patients are resorting to using cannabis illegally, despite the penalties and stigma if they are caught and prosecuted. A report of a cross-sectional survey of self-selected ‘medicinal use of cannabis’ in the UK over the period 1998 to 2002,[17] described that out of the 2969 questionnaires returned, a total of 947 (31.9%) subjects reported ever having used cannabis for medical purposes, including relieving symptoms of chronic pain (25%), multiple sclerosis and depression (22% each), arthritis (21%) and neuropathy (19%).
Harmful effects
2
By contrast to cannabis offering great medicinal value, the recreational use of cannabis can damage health, as reviewed by Hall and Degenhardt[18] in this issue. The adverse effect most often referred to is that cannabis can cause mental health problems, cannabis use being associated with psychotic symptoms (disordered thinking, hallucinations and delusions). When these symptoms meet a level of severity and are sustained over a significant period of time, then they are a manifestation of a psychotic disorder (psychotic illness). There is accumulating evidence that cannabis use plays a significant role in aggravating a psychosis; those most at risk being heavy cannabis users who have a history of psychotic symptoms or a family history of these disorders.[18] Whether there is a causal link between cannabis use and, specifically, developing a chronic psychosis remains to be proven; there is conflicting evidence, despite the large increase in cannabis use over several decades. In Europe, most cannabis is smoked in a mixture with tobacco. In their study of cigarette smokers and cannabis users, van Gastel et al.[19] concluded that smoking is an equally strong independent predictor of the frequency of psychotic-like experiences (PLEs) as monthly cannabis use, and that the association between moderate cannabis use and such experiences is confounded by cigarette smoking. The authors conclude that ‘our findings fit the hypothesis that individuals, who are prone to PLEs, particularly if associated with high
wileyonlinelibrary.com/journal/dta
distress, are more inclined to use cannabis. If this is the case, moderate cannabis use, like cigarette smoking, could be viewed as a mere indicator of risk for PLEs, and thus for mental health problems in general instead of a causative factor.’ It is also worth mentioning that cannabis use is considered to present far less of a risk to the health of the general population compared to alcohol, for example, as discussed by Nutt et al.[20–22] Moreover, the continuing emphasis on the potential of cannabis to cause mental illnesses tends to overshadow a probably much more common effect, which is insidious and psychosocial in nature, in that adolescents who become regular users have impaired educational attainment, as Hall and Degenhardt point out.[18] This impairment is at a critical time of life where educational achievement usually fashions the career that an individual will enter. There is a commonly held argument that cannabis should remain illegal to dissuade its use, but there is a powerful counter-argument that it should be legalized, its sale being licensed, available to adults only, in a form that is controlled in dose and quality. The potential of revenue to governments by taxation on its sale has also not gone unnoticed.[23–25] Moreover, recent attention has focused on the USA, where Colorado voters have overwhelmingly approved a plan for taxing their state’s legal cannabis market, with the revenue to be dedicated to school construction and regulation of cannabis sales. As an alternative to legalization, decriminalization, or non-prosecution strategies for possession would still leave a cannabis embargo on its supply, thus continuing to enrich organised crime. The current illegality of cannabis and the difficulties faced by importers has led to the availability of home-grown super-strength cannabis often known as Skunk.[26] This is usually produced by intensive cultivation using additional lighting, propagation of female cuttings, and hydroponic methods. It contains a much higher THC content compared to imported cannabis originating from its natural habitat, for example, as reported by Hardwick and King.[27] There are concerns that the higher THC content will lead to increases in psychosis, but that supposition is countered by the argument that some experienced users will titrate the amount they administer to achieve the effect they are seeking. Even so, Skunk has typically a much smaller content of CBD relative to THC, where CBD is the natural biosynthetic precursor of THC. The much reduced CBD content is probably key, as it is thought to confer the anxiolytic (anxiety reducing) properties that can counter the psychotic effects of THC.[28] In their report in this issue on cannabis potency in the Venice area, Zamengo et al.[29] show that the CBD/THC ratio in all cannabis samples decreased from 0.52 in 2010 to 0.18 in 2012. This was largely due to the increase in the proportion of herbal cannabis, including samples which had been grown by intensive methods. In their 2008 study, Hardwick and King[27] found a CBD/THC ratio of 0.6 for cannabis resin, but less than 0.006 in ‘sinsemilla’.
Synthetic cannabinoid receptor agonists The illegality of cannabis for recreational use in many countries has also led to the relatively new phenomenon of the sale of cannabimimetics, these often circumventing existing laws concerning drug misuse. Cannabimimetics are not compounds produced by the cannabis plant, but synthetic products that were investigated by the pharmaceutical industry as alternative medicinal products to the use of phytocannabinoids in an attempt to mimic some of the beneficial effects of cannabis, but without causing adverse psychoactive effects. This research has
Copyright © 2013 John Wiley & Sons, Ltd.
Drug Test. Analysis 2014, 6, 1–6
Drug Testing and Analysis
Editorial and perspective
Drug Test. Analysis 2014, 6, 1–6
an attempt to address these issues (aptamers are folded singlestranded nucleic acids that can be synthesized in vitro, with those that bind with high affinity and specificity for their target molecules being chosen). There is currently a gap between the convenience of immunoglobulin-based assays that lack the specificity and adaptability for the screening of the many synthetic cannabinoids and the hyphenated mass spectrometry approaches that can screen for these compounds and confirm their presence, but lack the simplicity and portability of immunoassay.
Workplace testing Acute cannabis intoxication compromises safety in the workplace and leads to an increased risk of having an accident. Oral fluid is increasingly becoming the matrix of choice for workplace drug testing and is the obvious candidate also for roadside drug testing. Current knowledge on cannabinoids in oral fluid is extensively reviewed in this issue by Lee and Huestis.[37] Data from urine and oral fluid analysis showed that cannabis was the most frequently detected drug amongst workers in the USA, the UK, Italy, and Brazil. One major advantage of oral fluid collection is that it may be performed under observation, thus impeding substitution or adulteration that has been known to occur when urine is voided unobserved to preserve an individual’s privacy. Another advantage of oral fluid is that the concentration of a drug and/or its metabolite(s) often can be better correlated with blood or plasma/ serum concentrations than with urine, a very important factor when evaluating whether or not an individual was likely to have been impaired at the time of collection; this is particularly germane to workplace and also roadside drug testing. Of note, however, are that cannabinoids are slowly absorbed through the oral cavity and consequently following the smoking of cannabis, THC deposited in the mouth predominantly contributes to the oral fluid concentration, with a relatively small contribution from the transfer of THC from the systemic circulation. By contrast, THCCOOH (11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid), a major secondary, biologically inactive metabolite of THC is not present in cannabis smoke. But as Lee and Huestis[37] point out, these two compounds are not significantly correlated from 0.25 hr to 6 hr after smoking due to their differing mechanisms of entry into oral fluid, a time period overlapping with the impairment window. A possible fruitful avenue of investigation is to examine whether 11-OH-THC in oral fluid is a valuable confirmatory marker of recent cannabis intoxication that circumvents the problem with encapsulation of phytocannabinoids in the oral cavity. 11-OH-THC is the intermediate (and active) metabolite in the formation of THCCOOH from THC, primarily by the activity of CYP 2C9 in the human liver. Another alternative matrix for analysis is head hair, which can reveal a history of drug administration and whether use was acute or chronic. Thieme et al.,[38] in this issue, focus on detection of THCCOOH in hair, as its incorporation can only result from cannabis use, as opposed to the presence of THC which can also occur because of environmental contamination (smoke). A sensitive and specific procedure for the analysis of this metabolite in hair by LC-tandem/multistage MS is challenging, primarily because of the small target concentration. THCCOOH has a low rate of incorporation in hair due it being an acidic compound, its presence being dwarfed by that of lipophilic organic acids that interfere on LC-MS systems, resulting in insufficient sensitivity for this target
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/dta
3
continued to the present day, with many hundreds of cannabinoid receptor CB1 agonists having been described in a recent review.[30] CB1 inverse agonists and antagonists may be of medicinal value; investigations are underway to evaluate whether the second generation of CB1 inverse agonists could be useful in the treatment of obesity and/or related diseases, as the endocannabinoid system appears to play an important role in energy homeostasis and eating behaviour, as reviewed in this issue by Bermudez-Silva et al.[31] Some of the early synthetic substances were based on the dibenzopyran nucleus and showed a structural affinity with THC. But, cannabimimetic activity arises in a wide range of compounds, few of which show any structural resemblance to the phytocannabinoids. Despite that extensive effort, nabilone, is the only synthetic compound to have been licensed. By contrast, a plethora of illicit cannabimimetics have been sold for recreational use, some being based on structures described in patents, but others appear to be entirely new, never having been described in any publication or having a Chemical Abstract Number. In this issue, the identification and quantification of synthetic cannabinoids in ‘Spice-like’ herbal mixtures is presented by Langer et al.[32] with particular reference to the situation in Germany, the authors recommending gas chromatography–mass spectrometry (GC-MS) for this purpose. To monitor recreational drugs being used at any one time within a community, sewage analysis can be of help, but of the cannabimimetics targeted by Reid et al.,[33] as described in this issue, some are prone to degradation. The authors did, however, discover that a urinary metabolite of JWH 018 was present in samples collected from three Norwegian cities, suggesting that its use is widespread in that country. Even with increased analytical capability, nonetheless, there are formidable legislative issues surrounding the cannabimimetics. King[34] in this issue comments that there are now so many cannabimimetics that is questionable whether they can be legally controlled, and concludes in his perspective: ‘Insofar as there is little evidence that current controls on new substances reduce harmful behaviours, then in response to the question ‘What should be done about controlling synthetic cannabinoids?’– the answer might be to do nothing. While this idea may be unpalatable to politicians, the reality is that few would notice, administrative costs to legislatures would be reduced and little additional harm would be caused.’ It is unclear if synthetic cannabinoid agonists pose any greater harm than the cannabis plant and its products. Although a few case reports of severe toxicity have appeared,[35] a review of 1898 cases involving cannabimimetics reported to the US National Poison Data System between 1 January 2010 and 1 October 2010 found that most exposures resulted in non-life-threatening effects not requiring treatment, although a minority of exposures resulted in more severe effects, including seizures.[36] In contrast to immunoassay techniques, hyphenated mass spectrometry is demonstrably well suited for the effective screening of synthetic cannabinoids in the laboratory. The cannabimimetic market is dynamic and it is inevitable that there will be a time-lag in the raising of immunoglobulins to the newer compounds. Furthermore, even with favourable cross-reactivity, it is reasonable to conclude that there will never be antibody based commercial kits and point-of-care devices available that have the blanket capability of detecting the ever increasing number of synthetic cannabinoids (and their metabolites) and thus this technology is limited. It is possible that, in the future, commercial suppliers will switch to the use of aptamers, a young science, in
Drug Testing and Analysis
A. T. Kicman and L. A. King
analyte. Thieme et al.[38] investigate various derivatives to solve these problematic issues and elegantly arrive at a solution using selective methylation as the final step in sample preparation. The authors’ success opens the avenue of toxicology laboratories being able to employ a method that achieves the sensitivity and specificity required using their existing LC-MS/MS systems rather than relying on the need for GC-MS/MS or GC-GC-MS instruments (using negative chemical ionization), the latter instruments still largely being considered only necessary for bespoke analytical toxicology purposes. Methylation of the 11-carboxyl group, but with the (acidic) phenolic hydroxyl group of THCCOOH remaining underivatized, makes methylated THCCOOH conducive to sensitive electrospray ionization in negative mode. In comparison, the fatty acids in hair extracts, not being phenolic, but having their carboxylic acid groups methylated, have consequently been converted to neutral compounds and thus are no longer susceptible to ionization in negative ion mode, thereby considerably reducing matrix interference. A further significant improvement in sensitivity and specificity for THCCOOH was achieved by MS3 using a QTRAP instrument, as may be expected. Moosmann et al.[39] in this issue investigate the THC precursor, Δ9-tetrahydrocannabinolic acid A (THCA-A) as a control marker of in-vivo exposure to cannabis smoke, as THCA-A is not incorporated into hair by metabolic processes in evidentially significant amounts. These investigators found that THCA-A was present in relatively low concentrations compared to forensic samples (from a previous investigation of theirs), indicating that the touching of hair with contaminated fingers is likely to be the major source of THC contamination. The authors conclude with a cautionary note: ‘These findings underline that hair-findings in general have to be interpreted with utmost care, particularly in case of drugs which are smoked or when cross-contamination via hand contact is likely to occur’. LC-MS/MS was also adopted by Salomone et al.[40] in this issue to evaluate the prevalence of cannabimimetics in hair. Their developed procedure, which incorporated an ultra-high performance liquid chromatography system (Waters Acquity UPLC ®), selected reaction monitoring and use of 5 internal standards, achieved sufficient sensitivity for the specific analysis of 23 synthetic cannabinoids, ranging from a limit of detection of 0.2 pg/mg to 1.3 pg/mg hair (the only exception being HU-210 at 24 pg/mg) and a lower limit of quantification of 0.7 pg/mg to 4.3 pg/mg (80 pg/mg for HU-210). The procedure was applied to the analysis to hair samples previously tested in their laboratory, with a number of cannabimimetics being detected and several positive samples notably having concentrations less than 50 pg/mg, this being the internationally accepted cut-off value for THC in hair. A challenge for those performing toxicological analysis of hair is how to interpret such concentrations and what cut-off value should be chosen for a particular cannabimimetic to distinguish between chronic consumption and occasional use or possible external contamination. The seemingly long terminal half-lives of commonly used synthetic cannabinoids in serum also present a major challenge to the clinical and forensic toxicologist. Kneisel et al.[41] in this issue report that JWH-081, JWH-122 or JWH-210 were detectable by LC-MS/MS for up to 102 days in serum samples collected from patients that had self-reported cessation of drug use.
Cannabis and driving
4
With respect to driving under the influence of cannabis, analysis is vital to obtaining accurate information on prevalence. It is a
wileyonlinelibrary.com/journal/dta
moot point whether the continuation of roadside surveys based solely on results of self-reporting are worthwhile, as pointed out by Van der Linden et al.[42] in this issue. There are many factors, however, to take into account regarding the relationship between blood/serum THC concentrations and driving behaviour, as reviewed by Wolff and Johnston[43] in this issue. This paper is based on a report to the UK Government published in early 2013[44] that was concerned with the effects of driving under the influence of a number of drugs other than alcohol. Just as in the population at large, cannabis remains the most commonly used drug in the UK amongst those who reported driving under the influence of illegal drugs in the previous 12 months. A significant dose-related decrease in driving performance exists following cannabis use; raised blood THC concentrations are significantly associated with an increased number of traffic crashes and death.[44,45] There is a clear need for objective measurement of THC if any drug-driving legislation is to be effective. It is generally accepted that the inactive metabolite THCCOOH is an unsuitable indicator of recent cannabis use or impairment. When attempting to frame legislation on the basis of THC concentrations in blood, a number of issues arise. First, in a roadside survey carried out in Belgium, Van der Linden et al.[42] showed that of 81 drivers who self-reported cannabis use, 34 had THC levels in oral fluid greater than the normal analytical limit (1 μg/L). However, only 8 had THC blood concentrations above this cut-off. Even in a sub-set of those who claimed to have used cannabis less than 4 hr before driving, THC was only detected in the blood of 3 of the 8 individuals. Secondly, when a driver has consumed alcohol and cannabis there is a greater risk to road safety than when either drug is used alone; this complicates the interpretation of blood levels. Thirdly, peak plasma concentrations of THC in habitual cannabis users may range up to 11μg/L, but, paradoxically, they are much lower than peak values in occasional smokers, which can reach over 260 μg/L. Finally, any legislation which sets maximum limits for THC in blood would require roadside screening devices and further equipment in police stations. Interpretation of the evidence of the risks of driving under the influence of cannabis has also been muddled by the conflation of two issues, as Wolff and Johnston point out in their article; that of the fitness to drive and that of criminality due to the illicit nature of the drug. This is most apparent in countries that apply thresholds (concentration limits) for alcohol and driving but apply or advocate a zero-tolerance course of action for THC. Fig. 1 in the Wolff and Johnston review[43] shows the risk of a traffic accident, using data reported by Laumon et al.,[46] based on a positive detection of cannabis at a blood THC concentration of >1 μg/L and for blood alcohol (EtOH) of ≥50 mg alcohol/dL. The risk approximately doubles for use of cannabis alone, whereas that for alcohol alone increases approximately 8-fold, being approximately 16-fold when alcohol and cannabis use are detected concurrently. Furthermore, the risk associated with cannabis seems to be similar to the risk when driving with a low-alcohol concentration (between 10 and 50 mg/dL),[47] notably under the current UK drink-driving threshold of 80 mg/dL); a serum concentration of THC of 3.8 μg/L (~2 μg/L whole blood) causes the same amount of impairment as 50 mg/dL of alcohol.[45] As an aside, almost nothing is known about the effect of cannabimimetics on driving performance, but it would be expected that those CB1 agonists that readily cross the blood–brain barrier would cause similar impairment to that caused by cannabis. After a driver has been found positive, the question then arises as to when a driving licence may be returned, as heavy
Copyright © 2013 John Wiley & Sons, Ltd.
Drug Test. Analysis 2014, 6, 1–6
Drug Testing and Analysis
Editorial and perspective consumption may suggest a long-term unfitness to drive. This is not a trivial consideration and can have cost implications for governments that consider it important to have follow-up investigations of offending drivers to answer this question. Fabritius et al.[48] in this issue recommend a way forward, based on the combination of findings from analysis of whole blood THC and a medical interview of the driver, in the context of the zero tolerance policy in their country, Switzerland. Their recommended guidance criteria were underpinned by THCCOOH data from a carefully controlled administration study, these being: (1) the driver can reclaim their licence if the whole blood THCCOOH concentration is below 3 μg/L and a medical interview suggests that cannabis smoking is occasional, i.e. no long and costly monitoring is necessary; (2) for THCCOOH concentrations greater than 40 μg/L (these being strongly correlated with heavy consumption) and the medical assessment indicates heavy consumption, a follow-up for a cannabis disorder must be undertaken until abstinence is proven; and (3) with a concentration between 3 and 40 μg/L and the medical assessment does not exclude regular use, it is advisable to seek the opinion of a forensic expert and have a ‘therapeutic follow-up’ until abstinence has been demonstrated, at which point the driving licence can be reclaimed.
Future developments
[7] [8] [9] [10] [11] [12] [13]
[14]
[15]
[16]
Although not discussed elsewhere in this special issue, the recent introduction of E-cigarettes in many countries could have major implications for drug policies. Even ‘standard’ E-cigarettes, which contain nicotine, have proved controversial from a regulatory aspect. However, these devices could easily be adapted to vaporise THC with an equal proportion of CBD as a controlled dose, thus circumventing the adverse respiratory effects of inhaling the high amounts of hydrocarbons and toxic by-products associated with smoking cannabis with or without tobacco.
[17] [18] [19]
[20] [21]
Andrew T. Kicman Drug Control Centre, Department of Forensic Science and Drug Monitoring, Division of Analytical and Environmental Sciences, King’s College London, UK E-mail: [email protected]
[22] [23] [24]
Leslie A. King 27 Ivar Gardens, Basingstoke, RG24 8YD, UK E-mail: [email protected]
[25]
References
Drug Test. Analysis 2014, 6, 1–6
[26] [27] [28]
[29]
[30] [31] [32]
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/dta
5
[1] Home Office. Drug misuse: Findings from the 2012/13 crime survey for England and Wales. London. Available at: http://socialwelfare.bl. uk/subject-areas/services-activity/substance-misuse/homeoffice/ 153597Drugs_Misuse201213.pdf [29 November 2013]. [2] EMCDDA. Annual report 2012: The state of the drugs problem in Europe. Available at: http://www.emcdda.europa.eu/attachements. cfm/att_190854_EN_TDAC12001ENC_.pdf [29 November 2013]. [3] Y. Gaoni, R. Mechoulam. Isolation, structure and partial synthesis of an active constituent of hashish. J. Am. Chem. Soc. 1964, 86, 1646. [4] W.A. Devane, L. Hanus, A. Breuer, R.G. Pertwee, L.A. Stevenson, G. Griffin, D. Gibson, A. Mandelbaum, A. Etinger, R. Mechoulam. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 1992, 258, 1946. [5] N. Battista, M. Sergi, C. Montesano, S. Napoletano, D. Compagnone, M. Maccarrone. Analytical approaches for the determination of phytocannabinoids and endocannabinoids in human matrices. Drug Test. Anal. 2014, 6, 7. [6] U. Thieme, G. Schelling, D. Hauer, R. Greif, T. Dame, R.P. Laubender, W. Bernhard, D. Thieme, P. Campololongo, L. Theiler. Quantification
of anadamide and 2-arichidonoylglycerol plasma levels to examine potential influences of tetrahydrocannabinol application on the endocannabinoid system in humans. Drug Test. Anal. 2014, 6, 17. P.J. Robson. Therapeutic potential of cannabinoid medicines. Drug Test. Anal. 2014, 6, 24. A. Hazekamp. An evaluation of the quality of medicinal grade cannabis in the Netherlands. Cannabinoids 2006, 1, 1. D. Potter. A review of the cultivation and processing of cannabis (Cannabis sativa L.) for production of prescription medicines in the UK. Drug Test. Anal. 2014, 6, 31. Martindale: The Complete Drug Reference (online). Drug monograph:Nabiximols. [29 November 2013] Martindale: The Complete Drug Reference (online). Drug monograph:Dronabinol. [29 November 2013] Martindale: The Complete Drug Reference (online). Drug monograph:Nabilone. [29 November 2013] United Nations. Single Convention on Narcotic Drugs, 1961. Available at: http://www.unodc.org/pdf/convention_1961_en.pdf [29 November 2013]. Department of Health and Human Services’ Guidance. Procedures for the Provision of Marijuana for Medical Research. Available at: http://grants.nih.gov/grants/guide/notice-files/not99-091. html [29 November 2013]. R.G. Pertwee, A. Thomas, L.A. Stevenson, R.A. Ross, S.A. Varvel, A.H. Lichtman, B.R. Martin, R.K. Razdan. The psychoactive plant cannabinoid, Delta(9)-tetrahydrocannabinol, is antagonized by Delta(8)and Delta(9)-tetrahydrocannabivarin in mice in vivo. Brit. J. Pharmacol. 2007, 150, 586. D.J. Nutt, L.A. King, D.E. Nichols. Effects of schedule I drug laws on neuroscience research and treatment innovation. Nat. Rev. Neurosci. 2013, 14, 577. M.A. Ware, H. Adams, G.W. Guy. The medicinal use of cannabis in the UK: Results of a nationwide survey. Int. J. Clin. Pract. 2005, 59, 291. W. Hall, L. Degenhardt. The adverse health effects of chronic cannabis use. Drug Test. Anal. 2014, 6, 39. W.A. van Gastel, J.H. MacCabe, C.D. Schubart, A. Vreeker, W. Tempelaar, R.S. Kahn, M.P.M. Boks. Cigarette smoking and cannabis use are equally strongly associated with psychotic-like experiences: A cross-sectional study in 1929 young adults. Psychol. Med. 2013, 43, 2393. D. Nutt. Government vs science over drug and alcohol policy. Lancet 2009, 374, 1731. D. Nutt, L.A. King, W. Saulsbury, C. Blakemore. Development of a rational scale to assess the harm of drugs of potential misuse. Lancet 2007, 369, 1047. D.J. Nutt, L.A. King, L.D. Phillips. Drug harms in the UK: A multicriteria decision analysis. Lancet, 2010, 376, 1558. S. Pudney. Drugs policy: What should we do about cannabis? Econ. Policy 2010, 25, 165. D. Werb, B. Nosyk, T. Kerr, B. Fischer, J. Montaner, E. Wood. Estimating the economic value of British Columbia’s British cannabis market: Implications for provincial cannabis policy. Int. J. Drug Policy 2012, 23, 436. M. Bryan, E. Del Bono, S. Pudney. Licensing and regulation of the cannabis market in England and Wales: Towards a cost-benefit analysis. Institute for Social and Economic Research. Available at: https:// www.iser.essex.ac.uk/d/153 [28 November 2013]. L.A. King, C. Carpentier, P. Griffiths. An overview of cannabis potency in Europe. EMCDDA Insights. Available at: http://www.emcdda.europa.eu/ attachements.cfm/att_33985_EN_Insight6.pdf [29 November 2013]. S. Hardwick, L.A. King. Home Office Cannabis Potency Study 2008. Home Office Scientific Development Branch, St Albans, UK, 2008. A. Englund, P.D. Morrison, J. Nottage, D. Hague, F. Kane, S. Bonaccorso, J.M. Stone, A. Reichenberg, R. Brenneisen, D. Holt, A. Feilding, L. Walker, R.M. Murray, S. Kapur. Cannabidiol inhibits THCelicited paranoid symptoms and hippocampal-dependent memory impairment. J. Psychopharmacol. 2013, 27, 19. L. Zamengo, G. Frison, C. Bettin, R. Sciarrone. Variability of cannabis potency in the Venice area (Italy): A survey over the period 2010– 12. Drug Test. Anal. 2014, 6, 46. J.W. Huffman. The Cannabinoid Receptors (Ed: P.H. Reggio). Humana Press, New York, NY, USA, 2009. S.Y. Romerzo-Zerbo, F.J. Bermudez-Silva. Cannabinoids, eating behaviour, and energy homeostasis. Drug Test. Anal. 2014, 6, 52. N. Langer, R. Lindigkeit, H.-M. Schiebel, L. Ernst, T. Beurle. Identification and quantification of synthetic cannabinoids in ’spice-like’
Drug Testing and Analysis
[33]
[34] [35]
[36]
[37] [38]
[39] [40]
[41]
A. T. Kicman and L. A. King
herbal mixtures: A snapshot of the German situation in the autumn of 2012. Drug Test. Anal. 2014, 6, 59. M.J. Reid, L. Derry, K.V. Thomas. Analysis of new classes of recreational drugs in sewage: Synthetic cannabinoids and amphetaminelike substances. Drug Test. Anal. 2014, 6, 72. L.A. King. Legal controls on cannabimimetics: an international dilemma? Drug Test. Anal. 2014, 6, 80. J. Lapoint, L.P. James, C.L. Moran, L.S. Nelson, R.S. Hoffman, J.H. Moran. Severe toxicity following synthetic cannabinoid ingestion. Clin. Toxicol. 2011, 49, 760. C.O. Hoyte, J. Jacob, A.A. Monte, M. Al-Jumaan, A.C. Bronstein, K.J. Heard. A characterization of synthetic cannabinoid exposures reported to the national poison data system in 2010. Ann. Emerg. Med. 2012, 60, 435. D. Lee, M.A. Huestis. Current knowledge on cannabinoids in oral fluid. Drug Test. Anal. 2014, 6, 88. D. Thieme, H. Sachs, M. Uhl. Proof of cannabis administration by sensitive detection of 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid in hair using selective methylation and application of liquid chromatography- tandem and multistage mass spectrometry. Drug Test. Anal. 2014, 6, 112. B. Moosmann, N. Roth, V. Auwärter. Hair analysis for THCA-A, THC and CBN after passive in vivo exposure to marijuana smoke. Drug Test. Anal. 2014, 6, 119. A. Salomone, C. Luciano, D. Di Corcia, E. Gerace, M. Vincenti. Hair analysis as a tool to evaluate the prevalence of synthetic cannabinoids in different populations of drug consumers. Drug Test. Anal. 2014, 6, 126. S. Kneisel, J. Teske, V. Auwärter. Analysis of synthetic cannabinoids in abstinence control: Long drug detection windows in serum and implications for practitioners. Drug Test. Anal. 2014, 6, 135.
[42] T. Van der Linden, P. Silverans, A.G. Verstraete. Comparison between self-report of cannabis use and toxicological detection of THC/ THCCOOH in blood and THC in oral fluid in drivers in a roadside survey. Drug Test. Anal. 2014, 6, 137. [43] K. Wolff, A. Johnston. Cannabis use: A perspective in relation to the proposed UK drug-driving legislation. Drug Test. Anal. 2014, 6, 143. [44] K. Wolff, R. Brimblecombe, J.C. Forfar, A.R. Forrest, E. Gilvarry, A. Johnston, J. Morgan, M.D. Osselton, L. Read, D. Taylor. Driving under the influence of drugs. Available at: http://www.apccs.police.uk/ fileUploads/APCC_Group_Emails/drug-driving-expert-panelreport-1.pdf [29 November 2013]. [45] H. Schulze, M. Schumacher, R. Urmeew, K. Auerbach. Final Report: Work performed, main results and recommendations. Available at: http://www.druid-project.eu/cln_031/nn_107548/Druid/EN/Dissemination/downloads__and__links/Final__Report,templateId=raw, property=publicationFile.pdf/Final_Report.pdf [29 November 2013]. [46] B. Laumon, B. Gadegbeku, J.L. Martin, M.B. Biecheler, S.A.M. Grp. Cannabis intoxication and fatal road crashes in France: Population based case–control study. Brit. Med. J. 2005, 331, 1371. [47] T. Hels, I.M. Bernhoft, A. Lyckegaard, S. Houwing, M. Hagenzieker, S.A. Legrand, C. Isalberti, T. Van der Linden, A.G. Verstraete. Risk of injury by driving with alcohol and other drugs. Available at: http://www. druid-project.eu/cln_031/nn_107548/Druid/EN/deliverales-list/downloads/Deliverable__2__3__5,templateId=raw,property=publicationFile. pdf/Deliverable_2_3_5.pdf [29 November 2013]. [48] M. Fabritius, B. Favrat, H. Chtioui, G. Battistella, J.-M. Annoni, M. Appenzeller, K. Dao, E. Fornari, E. Lauer, J.-F. Mall, P. Maeder, P. Mangin, C. Staub, C. Giroud. THCCOOH concentrations in whole blood: Are they useful in discriminating occasional from heavy smokers? Drug Test. Anal. 2014, 6, 155.
6 wileyonlinelibrary.com/journal/dta
Copyright © 2013 John Wiley & Sons, Ltd.
Drug Test. Analysis 2014, 6, 1–6
