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The incidence of drugs of impairment in oral fluid from random roadside testing
Forensic Science International 215 (2012) 28–31
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
The incidence of drugs of impairment in oral fluid from random roadside testing Mark Chu a,*, Dimitri Gerostamoulos a,b, Jochen Beyer a,b, Luke Rodda a,b, Martin Boorman c, Olaf H. Drummer a,b a
Victorian Institute of Forensic Medicine, Monash University, Australia Department of Forensic Medicine, Monash University, Australia c Road Policing Traffic Drug and Alcohol Section, Victoria Police, Australia b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 October 2010 Received in revised form 11 May 2011 Accepted 11 May 2011 Available online 12 June 2011
Oral fluid (OF) has become a popular specimen to test for presence of drugs, particularly in regards to road safety. In Victoria, OF specimens from drivers have been used to test for the presence of methylamphetamine (MA) and D9-tetrahydrocannabinol (THC) since 2003 and 3,4-methylenedioxy-Nmethylamphetamine (MDMA) since 2006. LC–MS/MS has been used to test the most recent 853 submitted OF specimens from Victoria Police for 31 drugs of abuse including those listed in the Australian Standard AS4760-2006. At least one proscribed drug was detected in 96% of drivers, of which MA was the most common (77%), followed by THC (42%), MDMA (17%) and the combination of all three (3.9%). Opioids were detected in 14% of drivers of which 4.8% were positive for 6-acetylmorphine and 3.3% for methadone. The incidence of the opioids tramadol (1.2%) and oxycodone (1.1%) were relatively low. Cocaine (8.0%) was as commonly detected as benzodiazepines (8.0%), and was almost always found in combination with MA (7.9%). Samples positive to benzodiazepines were largely due to diazepam (3.5%) and alprazolam (3.4%), with only 0.2% of drivers combining the two. Ketamine was also detected in 1.5% of cases. While the incidences of the proscribed drugs itself are concerning, it is clear that many drivers are also using other drugs capable of causing impairment. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Oral fluid LC–MS/MS Drug prevalence Drugs of abuse Road safety Impairment
1. Introduction The use of oral fluid (OF) as a biological specimen to test for the presence of drugs has become more commonplace, largely attributed to it being a relatively fast and non-invasive specimen to collect compared to blood and urine [1–9]. OF has become of particular interest in road safety, where the presence of a drug is not only more likely to suggest a driver is adversely affected at the time of sampling [10,11] compared to urine, but also offers the simplicity and non-invasiveness of sample collection compared to blood [3,12,13]. In what was then a world-first, legislation was passed by the State Government of Victoria enabling random roadside OF testing of drivers with no prior suspicion of impairment to determine the presence of methylamphetamine (MA) and D9tetrahydrocannabinol (THC) since December 2004 [3,13]. This law was further amended to include 3,4-methylenedioxy-Nmethylamphetamine (MDMA) in September 2006 [13]. This formed part of the police procedure where drivers are initially
* Corresponding author at: Victorian Institute of Forensic Medicine, 57-83 Kavanagh St, Southbank 3006, Australia. Tel.: +61 3 9684 4334; fax: +61 3 9682 7353. E-mail address: [email protected] (M. Chu). 0379-0738/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2011.05.012
tested at the roadside through a tongue swipe (the Securetec DrugWipe1 TWIN), and if positive to one or both drug groups, OF is collected and tested using a second immunoassay device (Cozart Rapiscan1) in a custom-built vehicle on-site. If this device also reveals a positive result for either or both drug groups, then the drivers are not permitted to continue driving their vehicles for a minimum period of 24 h and an OF specimen is sent for laboratory confirmation. The presence of one or more of the proscribed drugs at any concentration is deemed to be an offence [2,3,13]. Since its inception, over 100,000 drivers have been screened for drugs on the roadside in Victoria. The Australian states of New South Wales, Queensland, South Australia, Western Australia and Tasmania now conduct similar testing to Victoria. Previously, OF specimens received for laboratory confirmation were analyzed using two separate GC/MS-EI methods for the methylamphetamines and THC using methods developed in our laboratory [3]. However, with the establishment of an Australian Standard for OF (AS4760-2006), a multi-analyte LC–MS/MS method was developed in our laboratory that enabled the simultaneous detection of the 11 drugs contained in AS47602006 in addition to a further 20 common drugs; many of which are capable of causing impairment. When implemented in mid2009, the monitoring of potential drugs of impairment beyond the three proscribed in the legislation has since been possible,
M. Chu et al. / Forensic Science International 215 (2012) 28–31
29
Table 1 List of 31 analyzed drugs using the LC–MS/MS method. Target concentration (ng/mL) as per Australian Standard AS4760-2006
Drug AS4760-2006
Additional drugs detected
Morphine 25 Codeine 25 6-Acetyl morphine (6-AM) 10 Amphetamine 25 Methylamphetamine (MA) 25 MDMA 25 25 Methylenedioxyamphetamine (MDA) Cocaine 25 Benzoylecgonine (BZE) 25 Ecgonine methyl ester (EME) 25 D9-Tetrahydrocannabinol (THC) 10 oxycodone, tramadol, methadone, EDDP, pethidine, cocaethylene, alprazolam, clonazepam, diazepam, flunitrazepam, nitrazepam, nordiazepam, oxazepam, temazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, 7-aminonitrazepam, ketamine, zolpidem, THC-COOH
LOQ = 2.5 ng/mL for all analytes; EDDP, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; THC-COOH, 11-nor-D9-tetrahydrocannabinol-9-carboxylic acid; MDMA, 3,4methylenedioxy-N-methylamphetamine.
with the more significant findings from the analysis of the data discussed in this paper. 2. Methods All OF specimens sent for analysis from June 2009 to August 2010 were received in sealed collection tubes included with the Cozart1 Rapiscan testing kit, and were mixed in a 1:3 dilution with Cozart1 Rapiscan buffer. Specimens were stored in the laboratory at 20 8C until analysis. A rapid liquid–liquid extraction was performed as described previously using 200 mL of diluted OF specimen [14]. Extracts were analyzed on an ABSCIEX API 5000 LC–MS/MS system in ESI, MRM mode. The method was fully validated according to international standards in both neat and dilute OF [15,16]. The method was selective for all 31 drugs listed in Table 1. As the method paper with full validation data is currently under review for publication at the time of submission, the authors have therefore chosen not to include the full range of validation data within this paper and will not present concentrations of the drugs detected until the paper is accepted for publication. The calibration range for all 31 analytes included in the method (Table 1) was 2.5– 100 ng/mL using calibrators prepared in OF diluted with Cozart Rapiscan1 buffer. Quality control samples were used to assess assay performance; controls containing all drugs in AS4760-2006 at target cut-offs (Table 1) were prepared and run interspersed with OF specimens in every assay. Accuracy was calculated for each analyte as bias determined by calculating the percent deviation of the mean of all calculated concentration values at a specific level from the respective nominal concentration. Precision data (given as relative standard deviations (RSDs)) for within-day (repeatability) and time-different intermediate precision (combination of within- and between-day effects) of the method were calculated using one-way analysis of variance (ANOVA) with the grouping-variable ‘day’. The acceptance intervals of within-day (repeatability) and intermediate precision were 15% RSD and 15% for bias of the nominal values. The results from all OF specimens analyzed using LC–MS/MS were collated in a Microsoft1 Access 2002 SP3 database.
(Table 2). Of these, 815 (96%) were confirmed to have at least one proscribed drug present, with MA being the most common drug in 661 (77%) drivers. THC was the next most frequently detected drug with 355 (42%) of drivers positive, with MDMA present in 141 (17%) of drivers. The most frequent combination of drugs was MA and THC, observed in 174 (20%) drivers, followed by MA and MDMA in 129 (15%) and MDMA and THC in 6 (0.7%) drivers. A total of 33 (3.9%) drivers were positive to all three drugs. Opioids were detected in 120 (14%) drivers, with codeine, morphine and 6-acetylmorphine (6-AM) detected in 78 (9.1%), 66 (7.7%) and 41 (4.8%) of drivers, respectively (Table 3). The incidences of morphine and 6-AM combined with MA (6.8% and 4.1%) was approximately double that of their respective combinations with THC (3.5% and 2.0%) but did not suggest any trend difference given the relative incidences of the proscribed drugs. Cocaine was the next most highly represented drug with 68 (8.0%) of drivers being positive (all cases also contained the primary metabolite of cocaine, benzoylecgonine and 22 (2.6%) positive to EME). Cocaine was more commonly associated with MA (67 cases, 7.9%) with only one case not found in combination, compared to THC in 19 (2.2%) cases, however, this data did suggest that all three drugs are frequently used together. Of the drugs not included in AS4760-2006, other opioids and benzodiazepines were the most common drugs detected (Table 4). Methadone was present in 28 (3.3%) of drivers, with its main metabolite EDDP observed in 20 (2.3%) of cases. Interestingly, almost half of methadone-positive drivers were also positive for 6AM and thus had used both heroin and methadone. Tramadol and oxycodone were observed less frequently, in 10 (1.2%) and 9 (1.1%)
3. Results The method provided an LOQ of 2.5 ng/mL in dilute OF which satisfied the requirements of AS4760-2006 for those specifically targeted drugs as defined in neat OF. The LOQ fulfilled the minimum requirement of LOQ signal-to-noise ratio (S/N) of 10:1. Accuracy data were within the required interval of 15% for all drugs listed in Table 1, including MA (range 10.0 to 0.9%), MDMA (range 8.2 to 11.3%) and THC (range 8.2 to 1.8%). Intermediate precision was within the required limits of 15% RSD for all drugs, including MA (range 7.9–11.8%), MDMA (range 7.0–12.0) and THC (range 4.6–10.2%). The OF specimens analyzed were consecutive presumptive positive cases from June 2009 to August 2010 as deemed by the two roadside results. As expected the three targeted proscribed drugs had the highest representation of all drugs in the OF specimens that were received by the laboratory for confirmation
Table 2 Incidence of the 3 proscribed drugs in 853 oral fluid specimens. Cases
Incidence (%)
Total
853
Positive for at least 1 proscribed drug MA positive (all) MDMA positive (all) THC (all)
815 661 141 355
(96%) (77%) (17%) (42%)
Drug combinations MA + THC MA + MDMA THC + MDMA MA + MDMA + THC
174 129 6 33
(20%) (15%) (0.7%) (3.9%)
MA, methylamphetamine; MDMA, 3,4-methylenedioxy-N-methylamphetamine; THC, D9-tetrahydrocannabinol. Note: Given multiple use of drugs, percentages do not add to 100%.
30
M. Chu et al. / Forensic Science International 215 (2012) 28–31
Table 3 Incidence of other AS4760-2006 drugs detected in 853 oral fluid specimens. Cases
Incidence (%)
Total
853
Opioids (all) Codeine Morphine 6-AM Cocaine and metabolites (all) Cocaine BZE EME
120 78 66 41 68 68 68 22
(14%) (9.1%) (7.7%) (4.8%) (8.0%) (8.0%) (8.0%) (2.6%)
65 58 35 31 30 17 67 29 19
(7.6%) (6.8%) (4.1%) (3.6%) (3.5%) (2.0%) (7.9%) (3.5%) (2.2%)
Drug combinations MA + codeine MA + morphine MA + 6-AM THC + codeine THC + morphine THC + 6-AM MA + cocaine MDMA + cocaine THC + cocaine
6-AM, 6-acetyl morphine; BZE, benzoylecgonine; EME, ecgonine methyl ester; MA, methylamphetamine; MDMA, 3,4-methylenedioxy-N-methylamphetamine; THC, D9-tetrahydrocannabinol. Note: Given multiple use of drugs, percentages do not add to 100%.
cases, respectively. Not surprisingly due to their high prescription rate, diazepam and nordiazepam were the most common benzodiazepines detected, with 4% of drivers confirmed positive for each drug. Alprazolam was the next most detected with 29 (3.4%) cases, followed by oxazepam in 8 (0.9%) and temazepam in 4 (0.5%), respectively. Diazepam and alprazolam were only detected in combination in 2 (0.2%) of drivers. Only single cases were observed for nitrazepam, flunitrazepam and the 7-amino metabolites of nitrobenzodiazepines. Cocaethylene and ketamine were detected in 15 (1.8%) and 13 drivers (1.5%), respectively. 4. Discussion In the 853 OF specimens from drivers analyzed using LC–MS/ MS since mid-2009, the false-positive rate for proscribed drugs
Table 4 Incidence of drugs not included in AS4760-2006 detected in 853 oral fluid specimens. Drug
Incidence (%)
Opioids (all) Methadone EDDP Tramadol Oxycodone Benzodiazepines (all) Diazepam Nordiazepam Alprazolam Oxazepam Temazepam
120 28 20 10 9 68 30 33 29 8 4
(14%) (3.3%) (2.3%) (1.2%) (1.1%) (8.0%) (3.5%) (3.9%) (3.4%) (0.9%) (0.5%)
Other Cocaethylene Ketamine
15 (1.8%) 13 (1.5%)
Drug combinations Methadone + 6-AM Diazepam + oxazepam Diazepam + alprazolam
13 (1.5%) 4 (0.5%) 2 (0.2%)
EDDP, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; 6-AM, 6-acetyl morphine. Note: Given multiple use of drugs, percentages do not add to 100%.
was 4%. This low rate has remained relatively constant over the testing program, and reflects the benefit of having two sequential immunoassay devices to test for the presence of target drugs before sending a specimen for laboratory confirmation [3]. Only 3 (0.4%) of 38 (4.5%) drivers who had no proscribed drugs present were positive for drugs of any other type; one detected with alprazolam, one detected with codeine, and one detected with 6AM, amphetamine, codeine, methadone and morphine. This suggests that the cause of the false-positive results is unlikely to be due to cross-reactivity with MA, MDMA or THC. Official figures from Victoria Police show that up to the end of December 2009, 100,092 drivers were screened for drugs with 1,555 confirmed positive to one or more of the target drugs; a detection rate of 1:64 drivers. This detection rate has decreased from a rate of 1:42 from the 13,176 roadside drug tests performed in the first year of the random drug-testing program [3], but has remained relatively constant since then. In contrast to many European countries where amphetamine abuse is much more common [17–20], MA was the most commonly detected drug. The incidence of drivers with THC is also substantial at 40%; further confirming the concerning number of people who risk driving while impaired, as THC has been shown to be detectable in OF for periods that reflect the use of the drug within timeframes where the drug can still exert a negative effect on the skills required to drive a motor vehicle [21– 23]. However, it is important to note that this incidence may be under-representing due to possible false-negative cases going undetected. THC has traditionally posed problems in immunoassay devices, resulting in manufacturers continually seeking to decrease cut-off concentrations [24]. As THC can exist in very low concentrations in OF and could be below the detection threshold for the devices used in Victoria, it is possible that a number of drivers who have used cannabis relatively recently are not being detected due to their low concentrations [10]. Recent data suggests that the OF to blood ratio is not at 1:1 but closer to 10:1 (or higher), suggesting that low detection thresholds may not be as important as previously thought [25–27]. Typically, concentrations of both MA and MDMA are significantly higher in OF compared to blood are thus less likely to be under the cutoff concentrations of the immunoassay devices [3,18,28]. The manufacturers of the Securetec DrugWipe1 TWIN list the cut-off concentrations of MA/MDMA and THC to be 100 ng/mL/100 ng/mL and 30 ng/mL, respectively. The manufacturers for the Cozart Rapiscan1 list the cut-off concentrations of MA/MDMA and THC to be 60 ng/mL/60 ng/ mL and 150 ng/mL, respectively. A total of 20% of drivers had both MA and THC detected together in this study, showing that the two drugs are still being commonly used together [3]. In the high number of cases containing both MA and MDMA, it is likely that MDMA is present as a contaminant in the processing and handling of MA which is commonly observed in Australia [29] and further supported by the typically low concentrations of MDMA observed in these cases compared to those of MA (data not shown). The incidences of drugs other than the 3 proscribed in Victoria are also a cause for alarm. While the frequency of drugs such as heroin and cocaine are higher than envisioned when this data collection was initiated, it is important to recognize that these drivers were positive to a targeted drug and would not have been identified had they used these drugs without also using MA, MDMA or THC. While cocaine in particular has traditionally not been a common drug of abuse in Victoria, these figures suggest that its use now may be on the rise. The incidence of cocaine in injured drivers presenting to a Victorian major trauma centre in 2007 was reported as 1.4% in the blood of drivers [30].
M. Chu et al. / Forensic Science International 215 (2012) 28–31
Ketamine, at an incidence of 1.5%, has not been reported before in Australian drivers although it is a commonly abused drug in Asia. A recent publication from Hong Kong revealed an incidence of ketamine (in urine) from drugged drivers in non-fatal accidents at a trauma centre of 45% in 395 drivers [31]. With the data from Victorian drivers, it will be possible to monitor changing drug trends on Victorian roads and enable decisions to be made regarding further additions to the proscribed drugs in the legislation. The effectiveness and benefits of removing impaired drivers from the road will also be of great interest as the database continues to grow. 5. Conclusion The change to measuring drugs of abuse in oral fluid using LC– MS/MS in the place of older GC/MS-EI methods has provided useful information on the prevalence of other drugs of impairment not currently targeted in the driving population. This knowledge will increase substantially as Victoria Police and the Victorian Government are committed to further reduce preventable road trauma. References [1] D.J. Crouch, J.M. Walsh, L. Cangianelli, O. Quintela, Laboratory evaluation and field application of roadside oral fluid collectors and drug testing devices, Ther. Drug Monit. 30 (2008) 188–195. [2] O.H. Drummer, Introduction and review of collection techniques and applications of drug testing of oral fluid, Ther. Drug Monit. 30 (2008) 203–206. [3] O.H. Drummer, D. Gerostamoulos, M. Chu, P. Swann, M. Boorman, I. Cairns, Drugs in oral fluid in randomly selected drivers, Forensic Sci. Int. 170 (2007) 105–110. [4] C. Engblom, T. Gunnar, A. Rantanen, P. Lillsunde, Driving under the influence of drugs—amphetamine concentrations in oral fluid and whole blood samples, J. Anal. Toxicol. 31 (2007) 276–280. [5] D. Furr-Holden, R.B. Voas, T. Kelley-Baker, B. Miller, Drug and alcohol-impaired driving among electronic music dance event attendees, Drug Alcohol Depend. 85 (2006) 83–86. [6] H. Gjerde, J. Mordal, A.S. Christophersen, J.G. Bramness, J. Morland, Comparison of drug concentrations in blood and oral fluid collected with the intercept sampling device, J. Anal. Toxicol. 34 (2010) 204–209. [7] A. Pehrsson, T. Gunnar, C. Engblom, H. Seppa, A. Jama, P. Lillsunde, Roadside oral fluid testing: comparison of the results of drugwipe 5 and drugwipe benzodiazepines on-site tests with laboratory confirmation results of oral fluid and whole blood, Forensic Sci. Int. 175 (2008) 140–148. [8] A.G. Verstraete, Oral fluid testing for driving under the influence of drugs: history, recent progress and remaining challenges, Forensic Sci. Int. 150 (2005) 143–150. [9] J.M. Walsh, J.J. de Gier, A.S. Christopherson, A.G. Verstraete, Drugs and driving, Traffic Inj. Prev. 5 (2004) 241–253. [10] O.H. Drummer, Drug testing in oral fluid, Clin. Biochem. Rev. 27 (2006) 147–159.
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[11] H. Gjerde, A. Verstraete, Can the prevalence of high blood drug concentrations in a population be estimated by analysing oral fluid? A study of tetrahydrocannabinol and amphetamine, Forensic Sci. Int. 195 (2010) 153–159. [12] W.M. Bosker, M.A. Huestis, Oral fluid testing for drugs of abuse, Clin. Chem. 55 (2009) 1910–1931. [13] M. Boorman, K. Owens, The Victorian legislative framework for the random testing drivers at the roadside for the presence of illicit drugs: an evaluation of the characteristics of drivers detected from 2004 to 2006, Traffic Inj. Prev. 10 (2009) 16–22. [14] J. Beyer, M. Chu, D. Gerostamoulos, O.H. Drummer, Detection and Validated Quantification of 31 Drugs of Abuse in Oral Fluid, The International Association of Forensic Toxicologists, Geneva, Switzerland, 2009. [15] F.T. Peters, O.H. Drummer, F. Musshoff, Validation of new methods, Forensic Sci. Int. 165 (2007) 216–224. [16] F.T. Peters, T. Kraemer, H.H. Maurer, Drug testing in blood: validated negative-ion chemical ionization gas chromatographic–mass spectrometric assay for determination of amphetamine and methamphetamine enantiomers and its application to toxicology cases, Clin. Chem. 48 (2002) 1472–1485. [17] Problem amphetamine and methamphetamine use in Europe, European Monitoring Centre for Drugs and Drug Addiction, Lisbon, 2010. [18] J. Davey, N. Leal, J. Freeman, Screening for drugs in oral fluid: illicit drug use and drug driving in a sample of Queensland motorists, Drug Alcohol Rev. 26 (2007) 301–307. [19] B. Milne, J. Bell, B. Lampropoulos, S. Towns, Alcohol, drugs and Australian young people, Int. J. Adolesc. Med. Health 19 (2007) 245–253. [20] B. Murphy, Motor accidents and blood samples in New South Wales, J. Law Med. 15 (2008) 644–653. [21] F. Grotenhermen, G. Leson, G. Berghaus, O.H. Drummer, H.P. Kruger, M. Longo, H. Moskowitz, B. Perrine, J.G. Ramaekers, A. Smiley, R. Tunbridge, Developing limits for driving under cannabis, Addiction 102 (2007) 1910–1917. [22] M.A. Huestis, E.J. Cone, Relationship of Delta9-tetrahydrocannabinol concentrations in oral fluid and plasma after controlled administration of smoked cannabis, J. Anal. Toxicol. 28 (2004) 394–399. [23] J.G. Ramaekers, M.R. Moeller, P. van Ruitenbeek, E.L. Theunissen, E. Schneider, G. Kauert, Cognition and motor control as a function of Delta9-THC concentration in serum and oral fluid: limits of impairment, Drug Alcohol Depend. 85 (2006) 114– 122. [24] K. Pil, A. Verstraete, Current developments in drug testing in oral fluid, Ther. Drug Monit. 30 (2008) 196–202. [25] G.F. Kauert, J.G. Ramaekers, E. Schneider, M.R. Moeller, S.W. Toennes, Pharmacokinetic properties of delta9-tetrahydrocannabinol in serum and oral fluid, J. Anal. Toxicol. 31 (2007) 288–293. [26] S.W. Toennes, J.G. Ramaekers, E.L. Theunissen, M.R. Moeller, G.F. Kauert, Pharmacokinetic properties of delta9-tetrahydrocannabinol in oral fluid of occasional and chronic users, J. Anal. Toxicol. 34 (2010) 216–221. [27] S.M. Wille, E. Raes, P. Lillsunde, T. Gunnar, M. Laloup, N. Samyn, A.S. Christophersen, M.R. Moeller, K.P. Hammer, A.G. Verstraete, Relationship between oral fluid and blood concentrations of drugs of abuse in drivers suspected of driving under the influence of drugs, Ther. Drug Monit. 31 (2009) 511–519. [28] J. Davey, J. Freeman, Screening for drugs in oral fluid: drug driving and illicit drug use in a sample of Queensland motorists, Traffic Inj. Prev. 10 (2009) 231–236. [29] Illicit Drug Data Report 2008–2009, Australian Crime Commission, 2010. [30] C.W. Ch’ng, M. Fitzgerald, J. Gerostamoulos, P. Cameron, D. Bui, O.H. Drummer, J. Potter, M. Odell, Drug use in motor vehicle drivers presenting to an Australian, adult major trauma centre, Emerg. Med. Aust. 19 (2007) 359–365. [31] O.F. Wong, K.L. Tsui, T.S. Lam, N.N. Sze, S.C. Wong, F.L. Lau, S.H. Liu, Prevalence of drugged drivers among non-fatal driver casualties presenting to a trauma centre in Hong Kong, Hong Kong Med. J. 16 (2010) 246–251.
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
The incidence of drugs of impairment in oral fluid from random roadside testing Mark Chu a,*, Dimitri Gerostamoulos a,b, Jochen Beyer a,b, Luke Rodda a,b, Martin Boorman c, Olaf H. Drummer a,b a
Victorian Institute of Forensic Medicine, Monash University, Australia Department of Forensic Medicine, Monash University, Australia c Road Policing Traffic Drug and Alcohol Section, Victoria Police, Australia b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 October 2010 Received in revised form 11 May 2011 Accepted 11 May 2011 Available online 12 June 2011
Oral fluid (OF) has become a popular specimen to test for presence of drugs, particularly in regards to road safety. In Victoria, OF specimens from drivers have been used to test for the presence of methylamphetamine (MA) and D9-tetrahydrocannabinol (THC) since 2003 and 3,4-methylenedioxy-Nmethylamphetamine (MDMA) since 2006. LC–MS/MS has been used to test the most recent 853 submitted OF specimens from Victoria Police for 31 drugs of abuse including those listed in the Australian Standard AS4760-2006. At least one proscribed drug was detected in 96% of drivers, of which MA was the most common (77%), followed by THC (42%), MDMA (17%) and the combination of all three (3.9%). Opioids were detected in 14% of drivers of which 4.8% were positive for 6-acetylmorphine and 3.3% for methadone. The incidence of the opioids tramadol (1.2%) and oxycodone (1.1%) were relatively low. Cocaine (8.0%) was as commonly detected as benzodiazepines (8.0%), and was almost always found in combination with MA (7.9%). Samples positive to benzodiazepines were largely due to diazepam (3.5%) and alprazolam (3.4%), with only 0.2% of drivers combining the two. Ketamine was also detected in 1.5% of cases. While the incidences of the proscribed drugs itself are concerning, it is clear that many drivers are also using other drugs capable of causing impairment. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Oral fluid LC–MS/MS Drug prevalence Drugs of abuse Road safety Impairment
1. Introduction The use of oral fluid (OF) as a biological specimen to test for the presence of drugs has become more commonplace, largely attributed to it being a relatively fast and non-invasive specimen to collect compared to blood and urine [1–9]. OF has become of particular interest in road safety, where the presence of a drug is not only more likely to suggest a driver is adversely affected at the time of sampling [10,11] compared to urine, but also offers the simplicity and non-invasiveness of sample collection compared to blood [3,12,13]. In what was then a world-first, legislation was passed by the State Government of Victoria enabling random roadside OF testing of drivers with no prior suspicion of impairment to determine the presence of methylamphetamine (MA) and D9tetrahydrocannabinol (THC) since December 2004 [3,13]. This law was further amended to include 3,4-methylenedioxy-Nmethylamphetamine (MDMA) in September 2006 [13]. This formed part of the police procedure where drivers are initially
* Corresponding author at: Victorian Institute of Forensic Medicine, 57-83 Kavanagh St, Southbank 3006, Australia. Tel.: +61 3 9684 4334; fax: +61 3 9682 7353. E-mail address: [email protected] (M. Chu). 0379-0738/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2011.05.012
tested at the roadside through a tongue swipe (the Securetec DrugWipe1 TWIN), and if positive to one or both drug groups, OF is collected and tested using a second immunoassay device (Cozart Rapiscan1) in a custom-built vehicle on-site. If this device also reveals a positive result for either or both drug groups, then the drivers are not permitted to continue driving their vehicles for a minimum period of 24 h and an OF specimen is sent for laboratory confirmation. The presence of one or more of the proscribed drugs at any concentration is deemed to be an offence [2,3,13]. Since its inception, over 100,000 drivers have been screened for drugs on the roadside in Victoria. The Australian states of New South Wales, Queensland, South Australia, Western Australia and Tasmania now conduct similar testing to Victoria. Previously, OF specimens received for laboratory confirmation were analyzed using two separate GC/MS-EI methods for the methylamphetamines and THC using methods developed in our laboratory [3]. However, with the establishment of an Australian Standard for OF (AS4760-2006), a multi-analyte LC–MS/MS method was developed in our laboratory that enabled the simultaneous detection of the 11 drugs contained in AS47602006 in addition to a further 20 common drugs; many of which are capable of causing impairment. When implemented in mid2009, the monitoring of potential drugs of impairment beyond the three proscribed in the legislation has since been possible,
M. Chu et al. / Forensic Science International 215 (2012) 28–31
29
Table 1 List of 31 analyzed drugs using the LC–MS/MS method. Target concentration (ng/mL) as per Australian Standard AS4760-2006
Drug AS4760-2006
Additional drugs detected
Morphine 25 Codeine 25 6-Acetyl morphine (6-AM) 10 Amphetamine 25 Methylamphetamine (MA) 25 MDMA 25 25 Methylenedioxyamphetamine (MDA) Cocaine 25 Benzoylecgonine (BZE) 25 Ecgonine methyl ester (EME) 25 D9-Tetrahydrocannabinol (THC) 10 oxycodone, tramadol, methadone, EDDP, pethidine, cocaethylene, alprazolam, clonazepam, diazepam, flunitrazepam, nitrazepam, nordiazepam, oxazepam, temazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, 7-aminonitrazepam, ketamine, zolpidem, THC-COOH
LOQ = 2.5 ng/mL for all analytes; EDDP, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; THC-COOH, 11-nor-D9-tetrahydrocannabinol-9-carboxylic acid; MDMA, 3,4methylenedioxy-N-methylamphetamine.
with the more significant findings from the analysis of the data discussed in this paper. 2. Methods All OF specimens sent for analysis from June 2009 to August 2010 were received in sealed collection tubes included with the Cozart1 Rapiscan testing kit, and were mixed in a 1:3 dilution with Cozart1 Rapiscan buffer. Specimens were stored in the laboratory at 20 8C until analysis. A rapid liquid–liquid extraction was performed as described previously using 200 mL of diluted OF specimen [14]. Extracts were analyzed on an ABSCIEX API 5000 LC–MS/MS system in ESI, MRM mode. The method was fully validated according to international standards in both neat and dilute OF [15,16]. The method was selective for all 31 drugs listed in Table 1. As the method paper with full validation data is currently under review for publication at the time of submission, the authors have therefore chosen not to include the full range of validation data within this paper and will not present concentrations of the drugs detected until the paper is accepted for publication. The calibration range for all 31 analytes included in the method (Table 1) was 2.5– 100 ng/mL using calibrators prepared in OF diluted with Cozart Rapiscan1 buffer. Quality control samples were used to assess assay performance; controls containing all drugs in AS4760-2006 at target cut-offs (Table 1) were prepared and run interspersed with OF specimens in every assay. Accuracy was calculated for each analyte as bias determined by calculating the percent deviation of the mean of all calculated concentration values at a specific level from the respective nominal concentration. Precision data (given as relative standard deviations (RSDs)) for within-day (repeatability) and time-different intermediate precision (combination of within- and between-day effects) of the method were calculated using one-way analysis of variance (ANOVA) with the grouping-variable ‘day’. The acceptance intervals of within-day (repeatability) and intermediate precision were 15% RSD and 15% for bias of the nominal values. The results from all OF specimens analyzed using LC–MS/MS were collated in a Microsoft1 Access 2002 SP3 database.
(Table 2). Of these, 815 (96%) were confirmed to have at least one proscribed drug present, with MA being the most common drug in 661 (77%) drivers. THC was the next most frequently detected drug with 355 (42%) of drivers positive, with MDMA present in 141 (17%) of drivers. The most frequent combination of drugs was MA and THC, observed in 174 (20%) drivers, followed by MA and MDMA in 129 (15%) and MDMA and THC in 6 (0.7%) drivers. A total of 33 (3.9%) drivers were positive to all three drugs. Opioids were detected in 120 (14%) drivers, with codeine, morphine and 6-acetylmorphine (6-AM) detected in 78 (9.1%), 66 (7.7%) and 41 (4.8%) of drivers, respectively (Table 3). The incidences of morphine and 6-AM combined with MA (6.8% and 4.1%) was approximately double that of their respective combinations with THC (3.5% and 2.0%) but did not suggest any trend difference given the relative incidences of the proscribed drugs. Cocaine was the next most highly represented drug with 68 (8.0%) of drivers being positive (all cases also contained the primary metabolite of cocaine, benzoylecgonine and 22 (2.6%) positive to EME). Cocaine was more commonly associated with MA (67 cases, 7.9%) with only one case not found in combination, compared to THC in 19 (2.2%) cases, however, this data did suggest that all three drugs are frequently used together. Of the drugs not included in AS4760-2006, other opioids and benzodiazepines were the most common drugs detected (Table 4). Methadone was present in 28 (3.3%) of drivers, with its main metabolite EDDP observed in 20 (2.3%) of cases. Interestingly, almost half of methadone-positive drivers were also positive for 6AM and thus had used both heroin and methadone. Tramadol and oxycodone were observed less frequently, in 10 (1.2%) and 9 (1.1%)
3. Results The method provided an LOQ of 2.5 ng/mL in dilute OF which satisfied the requirements of AS4760-2006 for those specifically targeted drugs as defined in neat OF. The LOQ fulfilled the minimum requirement of LOQ signal-to-noise ratio (S/N) of 10:1. Accuracy data were within the required interval of 15% for all drugs listed in Table 1, including MA (range 10.0 to 0.9%), MDMA (range 8.2 to 11.3%) and THC (range 8.2 to 1.8%). Intermediate precision was within the required limits of 15% RSD for all drugs, including MA (range 7.9–11.8%), MDMA (range 7.0–12.0) and THC (range 4.6–10.2%). The OF specimens analyzed were consecutive presumptive positive cases from June 2009 to August 2010 as deemed by the two roadside results. As expected the three targeted proscribed drugs had the highest representation of all drugs in the OF specimens that were received by the laboratory for confirmation
Table 2 Incidence of the 3 proscribed drugs in 853 oral fluid specimens. Cases
Incidence (%)
Total
853
Positive for at least 1 proscribed drug MA positive (all) MDMA positive (all) THC (all)
815 661 141 355
(96%) (77%) (17%) (42%)
Drug combinations MA + THC MA + MDMA THC + MDMA MA + MDMA + THC
174 129 6 33
(20%) (15%) (0.7%) (3.9%)
MA, methylamphetamine; MDMA, 3,4-methylenedioxy-N-methylamphetamine; THC, D9-tetrahydrocannabinol. Note: Given multiple use of drugs, percentages do not add to 100%.
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M. Chu et al. / Forensic Science International 215 (2012) 28–31
Table 3 Incidence of other AS4760-2006 drugs detected in 853 oral fluid specimens. Cases
Incidence (%)
Total
853
Opioids (all) Codeine Morphine 6-AM Cocaine and metabolites (all) Cocaine BZE EME
120 78 66 41 68 68 68 22
(14%) (9.1%) (7.7%) (4.8%) (8.0%) (8.0%) (8.0%) (2.6%)
65 58 35 31 30 17 67 29 19
(7.6%) (6.8%) (4.1%) (3.6%) (3.5%) (2.0%) (7.9%) (3.5%) (2.2%)
Drug combinations MA + codeine MA + morphine MA + 6-AM THC + codeine THC + morphine THC + 6-AM MA + cocaine MDMA + cocaine THC + cocaine
6-AM, 6-acetyl morphine; BZE, benzoylecgonine; EME, ecgonine methyl ester; MA, methylamphetamine; MDMA, 3,4-methylenedioxy-N-methylamphetamine; THC, D9-tetrahydrocannabinol. Note: Given multiple use of drugs, percentages do not add to 100%.
cases, respectively. Not surprisingly due to their high prescription rate, diazepam and nordiazepam were the most common benzodiazepines detected, with 4% of drivers confirmed positive for each drug. Alprazolam was the next most detected with 29 (3.4%) cases, followed by oxazepam in 8 (0.9%) and temazepam in 4 (0.5%), respectively. Diazepam and alprazolam were only detected in combination in 2 (0.2%) of drivers. Only single cases were observed for nitrazepam, flunitrazepam and the 7-amino metabolites of nitrobenzodiazepines. Cocaethylene and ketamine were detected in 15 (1.8%) and 13 drivers (1.5%), respectively. 4. Discussion In the 853 OF specimens from drivers analyzed using LC–MS/ MS since mid-2009, the false-positive rate for proscribed drugs
Table 4 Incidence of drugs not included in AS4760-2006 detected in 853 oral fluid specimens. Drug
Incidence (%)
Opioids (all) Methadone EDDP Tramadol Oxycodone Benzodiazepines (all) Diazepam Nordiazepam Alprazolam Oxazepam Temazepam
120 28 20 10 9 68 30 33 29 8 4
(14%) (3.3%) (2.3%) (1.2%) (1.1%) (8.0%) (3.5%) (3.9%) (3.4%) (0.9%) (0.5%)
Other Cocaethylene Ketamine
15 (1.8%) 13 (1.5%)
Drug combinations Methadone + 6-AM Diazepam + oxazepam Diazepam + alprazolam
13 (1.5%) 4 (0.5%) 2 (0.2%)
EDDP, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; 6-AM, 6-acetyl morphine. Note: Given multiple use of drugs, percentages do not add to 100%.
was 4%. This low rate has remained relatively constant over the testing program, and reflects the benefit of having two sequential immunoassay devices to test for the presence of target drugs before sending a specimen for laboratory confirmation [3]. Only 3 (0.4%) of 38 (4.5%) drivers who had no proscribed drugs present were positive for drugs of any other type; one detected with alprazolam, one detected with codeine, and one detected with 6AM, amphetamine, codeine, methadone and morphine. This suggests that the cause of the false-positive results is unlikely to be due to cross-reactivity with MA, MDMA or THC. Official figures from Victoria Police show that up to the end of December 2009, 100,092 drivers were screened for drugs with 1,555 confirmed positive to one or more of the target drugs; a detection rate of 1:64 drivers. This detection rate has decreased from a rate of 1:42 from the 13,176 roadside drug tests performed in the first year of the random drug-testing program [3], but has remained relatively constant since then. In contrast to many European countries where amphetamine abuse is much more common [17–20], MA was the most commonly detected drug. The incidence of drivers with THC is also substantial at 40%; further confirming the concerning number of people who risk driving while impaired, as THC has been shown to be detectable in OF for periods that reflect the use of the drug within timeframes where the drug can still exert a negative effect on the skills required to drive a motor vehicle [21– 23]. However, it is important to note that this incidence may be under-representing due to possible false-negative cases going undetected. THC has traditionally posed problems in immunoassay devices, resulting in manufacturers continually seeking to decrease cut-off concentrations [24]. As THC can exist in very low concentrations in OF and could be below the detection threshold for the devices used in Victoria, it is possible that a number of drivers who have used cannabis relatively recently are not being detected due to their low concentrations [10]. Recent data suggests that the OF to blood ratio is not at 1:1 but closer to 10:1 (or higher), suggesting that low detection thresholds may not be as important as previously thought [25–27]. Typically, concentrations of both MA and MDMA are significantly higher in OF compared to blood are thus less likely to be under the cutoff concentrations of the immunoassay devices [3,18,28]. The manufacturers of the Securetec DrugWipe1 TWIN list the cut-off concentrations of MA/MDMA and THC to be 100 ng/mL/100 ng/mL and 30 ng/mL, respectively. The manufacturers for the Cozart Rapiscan1 list the cut-off concentrations of MA/MDMA and THC to be 60 ng/mL/60 ng/ mL and 150 ng/mL, respectively. A total of 20% of drivers had both MA and THC detected together in this study, showing that the two drugs are still being commonly used together [3]. In the high number of cases containing both MA and MDMA, it is likely that MDMA is present as a contaminant in the processing and handling of MA which is commonly observed in Australia [29] and further supported by the typically low concentrations of MDMA observed in these cases compared to those of MA (data not shown). The incidences of drugs other than the 3 proscribed in Victoria are also a cause for alarm. While the frequency of drugs such as heroin and cocaine are higher than envisioned when this data collection was initiated, it is important to recognize that these drivers were positive to a targeted drug and would not have been identified had they used these drugs without also using MA, MDMA or THC. While cocaine in particular has traditionally not been a common drug of abuse in Victoria, these figures suggest that its use now may be on the rise. The incidence of cocaine in injured drivers presenting to a Victorian major trauma centre in 2007 was reported as 1.4% in the blood of drivers [30].
M. Chu et al. / Forensic Science International 215 (2012) 28–31
Ketamine, at an incidence of 1.5%, has not been reported before in Australian drivers although it is a commonly abused drug in Asia. A recent publication from Hong Kong revealed an incidence of ketamine (in urine) from drugged drivers in non-fatal accidents at a trauma centre of 45% in 395 drivers [31]. With the data from Victorian drivers, it will be possible to monitor changing drug trends on Victorian roads and enable decisions to be made regarding further additions to the proscribed drugs in the legislation. The effectiveness and benefits of removing impaired drivers from the road will also be of great interest as the database continues to grow. 5. Conclusion The change to measuring drugs of abuse in oral fluid using LC– MS/MS in the place of older GC/MS-EI methods has provided useful information on the prevalence of other drugs of impairment not currently targeted in the driving population. This knowledge will increase substantially as Victoria Police and the Victorian Government are committed to further reduce preventable road trauma. References [1] D.J. Crouch, J.M. Walsh, L. Cangianelli, O. Quintela, Laboratory evaluation and field application of roadside oral fluid collectors and drug testing devices, Ther. Drug Monit. 30 (2008) 188–195. [2] O.H. Drummer, Introduction and review of collection techniques and applications of drug testing of oral fluid, Ther. Drug Monit. 30 (2008) 203–206. [3] O.H. Drummer, D. Gerostamoulos, M. Chu, P. Swann, M. Boorman, I. Cairns, Drugs in oral fluid in randomly selected drivers, Forensic Sci. Int. 170 (2007) 105–110. [4] C. Engblom, T. Gunnar, A. Rantanen, P. Lillsunde, Driving under the influence of drugs—amphetamine concentrations in oral fluid and whole blood samples, J. Anal. Toxicol. 31 (2007) 276–280. [5] D. Furr-Holden, R.B. Voas, T. Kelley-Baker, B. Miller, Drug and alcohol-impaired driving among electronic music dance event attendees, Drug Alcohol Depend. 85 (2006) 83–86. [6] H. Gjerde, J. Mordal, A.S. Christophersen, J.G. Bramness, J. Morland, Comparison of drug concentrations in blood and oral fluid collected with the intercept sampling device, J. Anal. Toxicol. 34 (2010) 204–209. [7] A. Pehrsson, T. Gunnar, C. Engblom, H. Seppa, A. Jama, P. Lillsunde, Roadside oral fluid testing: comparison of the results of drugwipe 5 and drugwipe benzodiazepines on-site tests with laboratory confirmation results of oral fluid and whole blood, Forensic Sci. Int. 175 (2008) 140–148. [8] A.G. Verstraete, Oral fluid testing for driving under the influence of drugs: history, recent progress and remaining challenges, Forensic Sci. Int. 150 (2005) 143–150. [9] J.M. Walsh, J.J. de Gier, A.S. Christopherson, A.G. Verstraete, Drugs and driving, Traffic Inj. Prev. 5 (2004) 241–253. [10] O.H. Drummer, Drug testing in oral fluid, Clin. Biochem. Rev. 27 (2006) 147–159.
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