Accident Analysis and Prevention 62 (2014) 26–31

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Prevalence of synthetic cannabinoids in blood samples from Norwegian drivers suspected of impaired driving during a seven weeks period Silja Skogstad Tuv ∗ , Hege Krabseth, Ritva Karinen, Kirsten M. Olsen, Elisabeth L. Øiestad, Vigdis Vindenes Norwegian Institute of Public Health, Division of Forensic Medicine and Drug Abuse Research, P.O. 4404, Nydalen, N-0403 Oslo, Norway

a r t i c l e

i n f o

Article history: Received 7 June 2013 Received in revised form 5 September 2013 Accepted 15 September 2013 Keywords: Synthetic cannabinoids Prevalence DUID UPLC–MS/MS AM-2201 JWH-018

a b s t r a c t From early year 2000 different herbal products containing synthetic cannabinoids (SC) have appeared on the drug market all over the world, and new substances are frequently introduced. The prevalence of SC use in different populations is however still mainly unknown, also in Norway. This information is difficult to obtain, but studies of drivers suspected of driving under the influence of drugs (DUID), might provide important information. The aim of this study was to assess the prevalence of SC in drivers suspected of being under the influence of drugs in Norway, and investigate if SCs impair driving performance. For two periods of three and four weeks all blood samples from drivers suspected of DUID in Norway were analyzed for the presence of 12 and 18 different SCs, respectively. A new ultra performance liquid chromatography tandem mass spectrometry method was developed. A total of 726 cases were analyzed during our study period, and SCs were detected in 16 cases (2.2%) in total. The mean age of these drivers was 29.6 years. High concentrations of other psychoactive drugs were detected in all the blood samples where a SC was found. AM-2201 and JWH-018 were the most frequently detected SCs, each found in five cases. In addition RSC-4, JWH-122, JWH-081 and JWH-250 were detected. None of the drivers had reported using SCs prior to driving. Despite the limited number of SCs investigated in this 7 week study period, a considerable percent of the cases were positive. Other psychoactive drugs of abuse were always found concomitant with the SCs, and the age of these drivers indicates that experienced drug users also ingest SCs. Since other drugs were found in all the samples, the psychomotor impairment caused by the SCs is difficult to estimate. Our study shows the importance of screening analyses of biological samples from different populations to assess the prevalence of drug use, since self-reporting might be encumbered with significant under-reporting. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction In the course of the past few years several types of synthetic cannabinoids (SC) have appeared on the drug marked word wide, as well as in Norway (Vardakou et al., 2010; Fattore and Fratta, 2011; Tuv et al., 2012). These compounds, known as e.g. “K2” and “Spice” are available on the Internet, often referred to as “legal highs” (Fattore and Fratta, 2011). To be able to keep up with the frequent appearance of new substances on the drug market, Norway has regulated groups of SCs under schedule 1 control (Slv, 2013) from February 14th 2013. Prior to this legislation, only eight SCs were registered as illicit drugs. The prevalence of use of these

∗ Corresponding author. Tel.: +47 21 07 78 81. E-mail address: [email protected] (S.S. Tuv). 0001-4575/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.aap.2013.09.009

compounds in the drug abuser population is not known. It is in addition not known to what extent this population drives a car after intake of SCs and to what degree SCs impair driving skills and cause increased risk of traffic accidents. Some studies have investigated the prevalence of SC use, both by surveys and analysing biological material. Winstock et al. investigated the population associated with the dance music scene in England, and 13% reported having used “Spice” (Winstock et al., 2011). Surveys conducted each year among households in England and Wales, revealed that the prevalence of Spice use was 0.4% among the age group 15–24 and 0.1% among the group 25–59 years (Smith and Flatley, 2011). The prevalence among college students from USA is reported to be 9% for use of “Spice and other smokable blends” (Hu et al., 2011b). Among athletes one study found SC in 2 out of 7500 urine samples tested (Moller et al., 2011) and in another study the prevalence of JWH-018, JWH-073 or any of their

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metabolites, were found to be 4.5%, when urine samples from 5956 athletes were tested (Heltsley et al., 2012). SC receptor agonists are a large family of chemically distinct compounds functionally similar to delta-9-tetrahydrocannabinol (THC), the main psychoactive compound of cannabis, and bind to the same cannabinoid receptors in the brain and in peripheral organs (CB-1 and CB-2 receptors) and mimic the effects of cannabis (Showalter et al., 1996; Grotenhermen, 2003; Wintermeyer et al., 2010). THC is among the drugs most frequently detected in blood samples from suspected drugged drivers (Christophersen et al., 1990; Waller et al., 1997), and there is strong evidence from experimental studies that THC has significant effects on the cognitive and psychomotor tasks associated with driving (Ramaekers et al., 2000). Studies concerning injured and fatally injured drivers in Australia reported cannabis as present in 10.8% (Longo et al., 2000) and 13.5% (Drummer et al., 2003) of cases, respectively. The psychoactive effects of SC are perceived to be even stronger than cannabis (Griffifths et al., 2010). Symptoms of SC toxicity are similar to the euphoric and psychoactive effects of cannabis with additional sympathomimetic symptoms, including diaphoresis, agitation/panic attacks, anxiety and restlessness (Piggee, 2009; Banerji et al., 2010; Wells and Ott, 2011; Vearrier and Osterhoudt, 2010; Bebarta et al., 2012). Little is known about the frequency of SC use among car drivers, and to which degree impaired driving is seen. Recently Musshoff et al. (Emcdda, 2010) published a series of seven cases of DUI with analytically confirmed ingestions of SCs. Based on analytical results and signs of impairment, they concluded that consumption of SCs can lead to impairment similar to typical performance deficits caused by cannabis use which are not compatible with safe driving. Particularly the centrally sedating effects and the impairment of fine motor skills are of concern, but awareness to further dangerous adverse reactions related to the ingestion of SCs is also needed. The aim of this study was to assess the prevalence of SCs in DUID suspects in Norway, and compare findings of SC in blood samples with results from the clinical examination performed by a physician. 2. Materials and methods At the Division of Forensic Medicine and drug abuse research at the Norwegian Institute of Public Health (NIPH), all blood samples collected from drivers suspected for drugged driving from the whole country, are analyzed. The Norwegian population consists of about five million inhabitants, and about 8.000 DUID cases are analyzed each year (Fhi, 2010). About 3000 of these cases are analyzed only for ethanol, and about 5000 are screened for a broad selection of legal and illegal psychoactive drugs. SCs are, however, not included in this routine screening. Blood samples are usually drawn within 1–2 h after apprehension (Vindenes et al., 2013). 2.1. Samples Whole blood samples were collected from Norwegian drivers apprehended for suspicion of driving under the influence of drugs (DUID). The samples were collected for two periods that lasted for three and four weeks; November 14th 2011 to December 2nd 2011 (n = 297), and March 26th 2012 to April 23th 2012 (n = 429). Blood samples were collected in 5 mL glass BD Vacutainer evacuated tubes containing 20 mg sodium fluoride and 143 I.U. heparin (BD Diagnostics, Velliver Industrial Estate, Plymoth, UK).

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2.2. Analysis All samples were analyzed shortly after arrival at the institute for more than 40 different medicinal and psychoactive drugs in addition to the chosen SCs (Kristoffersen et al., 2006; Oiestad et al., 2011; Dahl et al., 2012). During the first screening period 11 SCs were analyzed in all the blood samples sent to our Institute; JWH-018, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, JWH-250, AM-2201, RCS-4, JWH019 and WIN 55,212-2. During the second period we included eight additional SCs; JWH-015, JWH-020, JWH-251, AM-694, RCS-4-C4, RCS-8, CP 47,497 and HU-210. These latter substances were not available in our laboratory during the first screening period. The SC analyses were performed using an ultra-performance liquid chromatography–tandem mass spectrometer (UPLC–MS/MS) (Presley et al., 2013). Sample preparation (0.5 mL blood) was a liquid–liquid extraction with ethylacetate/heptane following evaporation to dryness. The dry residue was then reconstituted with 80 ␮L of ethanol. Chromatographic separation was achieved using an Acquity UPLC HSS T3 column (2.1 × 100, 1.8 ␮m). The mobile phase consisted of ammonium formate buffer pH = 3.1 (A) and methanol (B), with a gradient from 10% B to 90% B. Mass detection was performed by positive ion mode electrospray tandem mass spectrometry for all compounds. Within-day relative standard deviations varied from 4% to 17%, and day-to-day variations were in the range of 11–22%. 2.3. Clinical test of impairment (CTI) In Norway drivers suspected of DUID are usually examined by a physician to assess drug impairment. The Norwegian Clinical Test of Impairment (CTI) consists of 25 tests and observations related to common signs of drug impairment, including 7 tests of alertness, cognitive function and vestibular function, four observations on eyes, two observations on signs on intravenous drug abuse, four tests of motor activity and coordination and eight observations concerning appearance. The CTI has been describes elsewhere (Bramness et al., 2003). Based on the results from the tests the physician makes a conclusion regarding the overall degree of impairment of the subject, using a four-step scale: not impaired, mildly impaired, moderately impaired or greatly impaired. In the cases where a CTI had been performed, we compared these results with the drug findings in the blood samples. In relation to the CTI the driver is questioned by the physician about history of drug abuse prior to the driving. Both medicinal and recreational drug use is reported if the driver admits to such use, and also the ingested doses and time point for last intake of drug before apprehension. 2.4. Legal status of the SCs during the study period During our study periods, only eight SCs were regulated under schedule 1 control; JWH-018, JWH-073, JWH-081, JWH-122, JWH203, JWH-210, JWH-250 and AM-2201. All the other SCs were only regulated by medicines laws. From February 14th 2013, groups of SCs are registered under schedule 1 control, to make it easier to keep up with the very fast changes in the drug market regarding the SCs. 3. Results A total of 726 cases were analyzed during the seven weeks investigated, and 16 were SC positive. Eleven of these cases were a result of suspicion of DUID by the police, while four of the cases were collected as a result of traffic accidents and one case was collected as suspicion of drug use.

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AM-2201 was detected in five cases; in three of these, the driver was judged as impaired, and no CTI was performed in the last two. JWH-018 was also detected in five cases; three of the drivers were judged as not impaired, one as mildly and one as greatly impaired. JWH-122 was detected in three of the cases, but no CTI was performed in these cases. Two of these were involved in a traffic accident. JWH-081, RCS-4 and JWH-250 were each detected in one case. The CTI concluded with mildly to moderately impairment in all of these cases.

4. Discussion

Fig. 1. Different synthetic cannabinoids found in DUID-cases in Norway.

SCs were found in 16 cases during this period, which represents 2.2% of all the DUID cases in this period. The cases where only ethanol has been analyzed have not been included in the total number of cases. We identified six different SCs, see Fig. 1. AM-2201 and JWH-018 were most frequently detected, each in five different cases. Other SCs detected were JWH-122 (three cases), JWH-250 (one case), JWH-081 (one case) and RCS-4 (one case). Of the 16 cases positive for a SC, 15 of the drivers were male. The mean age was 29.6 years, ranging from 17 to 49 years, and the median was 29.5 years. The samples were collected from different parts of the country. In 12 of the cases we had information regarding ingestion of illegal drugs prior to driving. In four of the cases no information regarding drug use/abuse was provided. None of the drivers had reported use of SC prior to driving. LSD was found in two of the cases. No information regarding ingestion of LSD was provided as clinical examinations were not carried out for these subjects. In all the cases where SCs were found, other psychoactive drugs of abuse were detected concomitant, see Table 1. THC was the most frequently detected substance in these samples (12 cases), followed by clonazepam (8 cases), amphetamine/methamphetamine (7 cases), diazepam/nordiazepam (5 cases), LSD (2 cases), nitrazepam (1 case), alprazolam (1 case), oxazepam (1 case), ethanol (1 case), ketamine (1 case) and methylphenidate (1 case). 3.1. Concentration AM-2201 was detected in five of the cases with concentrations ranging from 0.07 to 1.33 ␮g/L, with an average of 0.47 ␮g/L. In the five cases where JWH-018 was present, the concentration varied between 0.08 and 0.46 ␮g/L, and averaged 0.20 ␮g/L. See Table 1 for further results. 3.2. CTI – clinical test of impairment The CTIs were performed by physicians in 12 of the 16 cases positive for SCs. In eight of these cases the physician judged the drivers as impaired; five as mildly, one as moderately and two as greatly impaired. In four cases the drivers were judged as not impaired and in four cases CTIs were not performed. In two of the latter, there was information that the suspect had been involved in a serious traffic accident. In the last two cases no information on why a CTI was not performed exists, this could be due to low medical coverage in the district where the DUID happened or involvement of other crimes by the suspected.

All blood samples from drivers suspected of DUID in Norway for a period of seven weeks, were analyzed for a selection of SCs, in order to give an estimate of the prevalence of use of these drugs among apprehended drivers. In 2.2% of the cases SCs were detected, and six different SCs were found. All SCs were found in combination with other drugs of abuse, but only one SC was detected in each case. AM-2201 and JWH-018 were the most frequently detected SCs. Since other drugs of abuse were found in all the SC cases, the impairment of driving skills caused by the SCs was difficult to assess. The degree of psychomotor impairment and the increase in risk of a traffic accident after ingestion of SCs, are not known. Estimating the risk of traffic accidents is difficult, and epidemiologic studies are required, but such data are not available for the SCs. Controlled clinical studies investigating the psychomotor impairment of the SCs have not been performed. The CTI has been shown to provide information regarding such impairment (Bramness et al., 2003; Gustavsen et al., 2006), although such tests provide evidence of drug effects, they do not specifically demonstrate driving impairment (Jones, 2007). In all of our cases where a SC was detected, other psychoactive substances were found concomitant in high concentrations. This makes it difficult to conclude to which degree the SCs might be contributing to the impairment. In one of the cases, only THC was found in addition to a SC, but in all the other cases, at least two other substances were detected in the blood sample. In samples received at NIPH, psychoactive drugs are detected in about 95% of the cases. The samples are collected from drivers where the police has suspected drugged driving, or from accidents, and are not collected during e.g. general traffic control. The most frequently detected drugs are THC (31%), methamphetamine (29%), amphetamines (26%) and clonazepam (24%); numbers from NIPH statistics for 2011. Drugs like GHB, codeine and zopiclone are all found in a prevalence of around 3% in these cases, and have thus, almost the same prevalence as the SCs in our screening period. The mean number of different drugs found in each case is 2–3 (Fhi, 2010). The high presence of multidrug findings in the cases with SCs is consequently similar to what is normally seen in DUID cases in Norway and the persons using SCs do not seem to differ from this group. Aberrations registered in the CTI in this study varied, but the most frequent findings were positive Romberg’s test, drowsiness, positive memory test, positive finger–nose test, impaired orientation, watery eyes, abnormal facial expression and abnormal articulation. These findings cannot be directly related to SC, but one cannot exclude their contribution to impairment. Most SCs are extremely potent, and doses reported to give psychoactive effects are reported to be as low as 0.5–5 milligram for some of the SCs. This challenges the detection of these substances in biological material as concentrations are expected to be low. We detected six different SCs; JWH-018 and AM-2201 being the most abundant ones. Looking isolated on blood concentrations of SCs, the Norwegian material has lower concentrations (mean

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Table 1 Type and concentrations of synthetic cannabinoids and other psychoactive drugs detected in the whole blood samples. Subject

Gender

Age

Synthetic cannabinoid

␮g/L

Other findings

␮g/L

Clinical examination

1

Male

48

JWH-081

0.19

Male

35

JWH-250

0.47

3

Male

29

JWH-018

0.24

4

Male

17

AM-2201

0.07

5

Male

20

RCS-4

1.0

6 7

Male Male

25 31

JWH-122 AM-2201

1.2 0.25

8

Female

35

JWH-018

0.13

9

Male

30

JWH-018

0.10

10

Male

41

AM-2201

0.28

11

Male

33

JWH-018

0.46

12

Male

29

JWH-122

0.50

13

Male

27

JWH-122

1.67

14

Male

26

JWH-018

0.08

15

Male

30

AM-2201

0.40

16

Male

17

AM-2201

1.33

1.0 1.9 0.09 0.02 0.9 0.6 0.02 0.04 0.8 0.005 0.1 0.06 0.01 0.004 0.02 0.09 0.6 0.6 0.002 0.09 0.8 0.05 0.003 0.07 0.002 0.09 0.1 0.04 1.5 0.0009 0.7 0.2 1.7 2.4 0.11 ppm 0.003 0.0005 0.005 0.06 0.0005 0.03 0.002 0.005 0.04 0.0006 0.02 0.002

Impaired (mildly)

2

Amphetamine Methamphetamine Diazepam Clonazepam Amphetamine Methamphetamine Clonazepam Amphetamine Methamphetamine THC Methamphetamine Clonazepam Clonazepam THC THC Methamphetamine Diazepam Nordiazepam THC Amphetamine Methamphetamine Nordiazepam THC Clonazepam THC Diazepam Nitrazepam Alprazolam Oxazepam THC Methadone Methamphetamine Diazepam Nordiazepam Ethanol THC LSD THC Ketamine LSD Clonazepam THC Methylphenidate Clonazepam THC Clonazepam THC

and range) compared with earlier published cases (Musshoff et al., 2013; Yeakel and Logan, 2013). The highest concentration reported in Norway was a case with 1.67 ␮g/L of JWH-122. Cases from USA and Germany have reported concentrations of this drug as high as 7.6 and 2.5 ␮g/L (Musshoff et al., 2013; Yeakel and Logan, 2013). The concentrations of the detected SCs, do not correlate with the degree of impairment judged by the physician. All the concentrations are as expected very low, but again, since there are one or more other potent psychoactive drugs ingested at the same time, the concentrations of SCs alone will not indicate the degree of impairment. The detected SCs have different affinity to the cannabinoid receptors. As an example, JWH-018 binds the cannabinoid receptor 1 (CB1) four times stronger than tetrahydrocannabinol (Fattore and Fratta, 2011). This is assumed to give JWH-018 higher potency of providing psychoactive effects than tetrahydrocannabinol. Also, AM-2201 is reported to bind the CB1 receptor 40 times stronger than tetrahydrocannabinol (Thakur et al., 2005). AM-2201 was detected in five cases, three of the drivers were judged as impaired by the physician. In the last two cases, a CTI was not performed. For JWH-018 three of the drivers were judged as not impaired, and two as impaired. From these findings it might seem that AM-2201 is more likely to induce impairment compared to JWH-018, but the

Impaired (moderately)

Not impaired

Impaired (mildly) Impaired (mildly) Not impaired Not performed

Impaired (mildly)

Not impaired Not performed

Not impaired

Not performed (traffic accident) Not performed (traffic accident) Impaired (greatly) (traffic accident) Impaired (greatly)

Impaired (mildly)

numbers are however too small to conclude and the effects from the other drugs also interfere with the evaluation. Different studies have claimed that SCs are frequently sold in products containing more than one SC (Zuba et al., 2011; Choi et al., 2012). We did however only find one type of SC in each case. It can however not be excluded that other SCs might have been present in the ingested product at trace concentrations levels, or that other than the substances in our repertoire had been ingested. Musshoff et al. (2013) reported multiple SCs in all of the analyzed blood samples, except from one. In the material published by Yeakel and Logan, 50% of the cases had more than one SC in blood (Yeakel and Logan, 2013). In all the Norwegian cases other drugs of abuse were detected in addition to the SCs, implying maybe a different pattern of use or the use of various products in Norway than in Germany and USA. In two of the cases where JWH-122 was detected, LSD was also found. Since Norway introduced legal limits for 20 drugs other than alcohol February 1st 2012 (Vindenes et al., 2012), LSD was included in the routine screening program for DUID cases. These two cases are however the only cases where LSD was detected in 2012. Both cases are reported to be traffic accidents, and clinical examinations have not been performed. The increase in risk of traffic accidents

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after ingestion of LSD, has not been reported previously, but from the effects of this hallucinogenic drug (Passie et al., 2008), driving under the influence of LSD is likely to be a hazardous activity. To our knowledge, this is thus the first report of driving under the influence where LSD has been detected in a biological sample. The prevalence of use of SCs in the general population is not known in Norway nor in other countries. Some studies do exist, but these often explore small environments with subgroups of the population not representative to the population in general. A study from USA found a lifetime prevalence of 9% among college students for SCs (Hu et al., 2011a). Questionnaires are frequently used to estimate prevalence of drug use in certain populations (Winstock et al., 2011), but might be encumbered with recall bias, and also selective reporting. Despite the legal status of most of the SCs in Norway during our study period, none of the drivers had reported using SCs prior to driving. Six of the drivers had reported smoking cannabis prior to driving, but 12 of the cases were positive for THC. This study emphasizes the importance of using biological samples to determine the prevalence of drug use in a certain population. Despite the selected group of drivers apprehended by the police for DUID, the biological samples from these cases provide important information regarding drug use in a country, and can reveal new trends in the drug market. Such information is difficult to obtain from other sources where biological samples are not available. The gender distribution among our 16 SC cases reveals that only one of the samples was collected from a woman. From another Norwegian study analyzing samples from the same population of drivers suspected of DUID, were THC was the only substance detected in the blood, the prevalence of men was 96% (n = 437) and women 4% (n = 19) (Khiabani et al., 2006). The distribution of gender in the DUID cases differs however between the different drugs. For such cases where only zopiclone has been detected, 25% of the samples were from female (Gustavsen et al., 2009). The number of drivers included in our study is very small, and from these limited data, it can be anticipated that there is a higher prevalence of use among males, compared to females. We found a mean age among the drivers with SCs in their blood samples at 29.6 years, ranging from 17 to 49 years, and the median is 29.5 year. This reflects an adult population, and not teenagers getting such drugs at the Internet. The combination of other drugs of abuse indicates that also more experienced drug users ingest SCs. The selection of SCs screened in this study was chosen based on data from the Criminal Police Laboratory regarding seizures of SCs in Norway. Since the number of new designer drugs are numerous, and changes very quickly, it should always be questioned if other substances should be screened for. In the case where ketamine was detected, the suspect was involved in a traffic accident, and ketamine might thus have been given as medical treatment. We do however not have any information regarding this from the police. Ingestion of ketamine as a recreational drug is assumed to be rare in Norway, but in 2012 there have been seizures also in Norway. 5. Conclusions Our study has shown that the prevalence of SCs in DUID suspects in Norway was 2.2%. This frequency is surprising high, almost as high as other commonly abused drugs like GHB, zopiclone and codeine. AM-2201 and JWH-018 were the most frequently detected SCs. Due to concomitant findings of other potent psychoactive drugs of abuse it is not possible to indicate the degree of reduced driving skills seen after ingestion of the SCs. The age of the SCs users show that such drugs are not only ingested by teenagers, but also experienced drug users, and the majority were male. Analyses of biological samples are of great importance to provide information regarding prevalence of drug use in a country. None of the drivers in

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