Common Drug-Testing Methodologies Dr Charles Appleton Queensland Medical Laboratory Substance Abuse in the Workplace Toxicology

Common Drug-Testing Methodologies Scope Which methodologies – strengths & limitations Screening Confirmation The Standards & Accreditation Includes compliance & accreditation, quality control Quality practice Testing outside of Standards Toxicology

Common Drug-Testing Methodologies Approach The Standards The methodologies Where does synthetic cannabinoid testing sit? Toxicology

Common Drug-Testing Methodologies AS/NZS 4308:2008 for urine AS 4760-2006 for oral fluid define procedures for Collection Transportation Analysis Reporting (cover a relatively small number of substances but the principles are applicable outside of the standard) Toxicology

Common Drug-Testing Methodologies Methods of analysis: Screening – predominantly immunoassay - workplace/laboratory Confirmation – exclusively mass spectrography - generally gas or liquid chromatography MS is the detection and characterisation stage Toxicology

Common Drug-Testing Methodologies Immunoassay: Generally a “class” method Cannabinoids** Opiates** Sympathomimetic Amines** Cocaine** Benzodiazepines* Barbiturates Phencyclidine Methadone and others Toxicology

Common Drug-Testing Methodologies Immunoassay: Thresholds (mg/L) differ from Country to Country Recommended by: Standards Australia SAMHSA (AS/NZS 4038) Cannabinoids 50 50 Cocaine metabolites 300 300 Sympathomimetic Amines 300 1000 Opiates 300 2000 Benzodiazepines 200 Phencyclidine 25 Toxicology

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Courtesy of Bio-Rad Laboratories Ltd Toxicology

Toxicology

Common Drug-Testing Methodologies Confirmation procedures Sample cleanup/extraction Chromatographic separation of different drugs and metabolites of those drugs gas, high performance liquid chromatography Identification and quantitation of the individual parent and metabolite peaks mass spectrography Toxicology

Toxicology

Common Drug-Testing Methodologies Urine testing - Important considerations: Sensitivity and specificity Adulteration/creatinine Other considerations: Historical positives Legitimate “innocent” positives False positives Passive positives Toxicology

Common Drug-Testing Methodologies Adulteration - Process of compromising or attempting to compromise the integrity of the sample after passage but prior to testing Toxicology

Common Drug-Testing Methodologies Adulteration (urine) - water – dilute substances in the sample to a level below the threshold for detection oxidising agents – chemically “burn” the drug nitrites, acid, etc – chemically alter the drug or the drug-antibody interaction Note – adulterant checks do not detect sample substitution Toxicology

Common Drug-Testing Methodologies Creatinine - normal product of muscle metabolism concentration in urine is determined by the amount of muscle in the subject’s body and the amount of water that his kidneys are excreting at the time of sample collection there is a (usually) minor contribution from diet an indicator of the overall concentration of the sample Toxicology

Common Drug-Testing Methodologies Urine Creatinine - average concentration 10-12 mmol/L in males 8-10 mmol/L in females “usual range”- 5-15 mmol/L creatinine less than 0.5 mmol/L (50 mg/L) “not consistent with human urine” creatinine 0.5 – 1.7 mmol/L (50 – 200 mg/L) “may indicate dilution in some individuals” Toxicology

Cases – Cannabinoids Non-negative initial urine screen GCMS ASSAY - URINE THC CONFIRMATION Date Lab No d9-THCA U.Creat Ratio ug/L mmol/L 14/12/09 12 1.4 8.6 The urine was very dilute. This suggests a large water intake prior to passage of the urine, or perhaps adulteration of the sample with water after collection. This may be used to dilute out any drug metabolites to concentrations below detection limits. Toxicology

Common Drug-Testing Methodologies Urine testing - Important considerations: Sensitivity and specificity Adulteration/creatinine Other considerations: Historical positives Legitimate “innocent” positives False positives Passive positives Toxicology

Common Drug-Testing Methodologies Urine testing - Historical positives Characteristic of lipid-soluble (dissolve in body fat) substances Seen predominantly after prolonged high dose use Within the Standard, consideration applies to cannabis and benzodiazepine users only Less well-defined with respect to other drugs such as synthetic cannabinoids Toxicology

Cases – Cannabinoids Non-negative initial urine screen GCMS ASSAY - URINE THC CONFIRMATION Date Lab No d9-THCA U.Creat Ratio ug/L mmol/L 06/11/09 596 37.6 16 13/11/09 screen neg 8.2 20/11/09 18 45.1 0.4 Large fluctuations in the urinary creatinine will complicate interpretation of absolute values. Toxicology

Common Drug-Testing Methodologies Urine testing - Legitimate “innocent” positives Over-the-counter medications Prescribed medications Food constituents Products of metabolism of other drugs Toxicology

Cases – Opiates Non-negative initial urine screen MS ASSAY - URINE OPIATE CONFIRMATION Date Codeine Morphine U.Creat Cod/Crea Mor/Crea (ug/L) (ug/L) (mmol/L) Ratio Ratio 18/12/09 265 556 18.7 14 30 Assayed to AS/NZS 4308:2008 requirements. The cutoff level for both Codeine and Morphine is 300ug/L. MS confirms the initial opiate screen and reveals a pattern indicative of recent use of codeine. There is no suggestion of use of illicit opiates. Toxicology

The metabolic pathway of the opiates. XII-Biotech-C-Opiate Chemistry-3 Toxicology

Cases – Opiates Non-negative initial urine screen MS ASSAY - URINE OPIATE CONFIRMATION Date Codeine Morphine U.Creat Cod/Crea Mor/Crea (ug/L) (ug/L) (mmol/L) Ratio Ratio 13/01/10 <50 413 19.6 <3 21 (27) MS confirms the initial opiate screen and reveals the presence of a small amount of morphine as well as a trace of codeine. Although it is not possible to exclude the possibility that this may reflect use of morphine or of heroin recently with use of codeine some days prior to that, this is the pattern typically seen after ingestion of foodstuffs containing poppy seed. There is no absolute indication of use of illicit opiates. Toxicology

Cases – Sympathomimetic Amines Non-negative initial urine screen MS ASSAY - URINE SYMPATHOMIMETIC AMINE CONFIRMATION Cut-off Amphetamine >1500 ug/L 150 ug/L Methamphetamine >1500 ug/L 150 ug/L MDMA Not detected 150 ug/L MDA Not detected 150 ug/L Phentermine Not detected 500 ug/L Ephedrine Not detected 500 ug/L Pseudoephedrine Not detected 500 ug/L Creatinine 14.8 mmol/L Amphetamine 1580 ug/L Methamphetamine 6100 ug/L Toxicology

Sympathomimetic Amines – the faces Toxicology

Cases – Sympathomimetic Amines Non-negative initial urine screen MS ASSAY - URINE SYMPATHOMIMETIC AMINE CONFIRMATION Cut-off Amphetamine >1500 ug/L 150 ug/L Methamphetamine Not detected 150 ug/L MDMA Not detected 150 ug/L MDA Not detected 150 ug/L Phentermine Not detected 500 ug/L Ephedrine Not detected 500 ug/L Pseudoephedrine Not detected 500 ug/L Creatinine 15.9 mmol/L Assayed to AS/NZS 4308:2008. Subject prescribed Dexamphetamine Toxicology

Cases – Sympathomimetic Amines Non-negative initial urine screen MS ASSAY - URINE SYMPATHOMIMETIC AMINE CONFIRMATION Cut-off Amphetamine Not detected 150 ug/L Methamphetamine Not detected 150 ug/L MDMA Not detected 150 ug/L MDA Not detected 150 ug/L Phentermine >1500 ug/L 500 ug/L Ephedrine Not detected 500 ug/L Pseudoephedrine Not detected 500 ug/L Creatinine 17.9 mmol/L Phentermine 31300 ug/L Toxicology

Cases – Sympathomimetic Amines Non-negative initial urine screen MS ASSAY - URINE SYMPATHOMIMETIC AMINE CONFIRMATION Cut-off Amphetamine 917 ug/L 150 ug/L Methamphetamine 3040 ug/L 150 ug/L MDMA Not detected 150 ug/L MDA Not detected 150 ug/L Phentermine Not detected 500 ug/L Ephedrine Not detected 500 ug/L Pseudoephedrine Not detected 500 ug/L Creatinine 12.6 mmol/L Subject has Parkinson disease. Toxicology 30 30

Cases – Benzodiazepines Non-negative initial urine screen MS ASSAY - URINE BENZODIAZEPINE CONFIRMATION Cut-off Oxazepam 151 ug/L 200 ug/L Temazepam 215 ug/L 200 ug/L Diazepam Not detected 200 ug/L Nordiazepam <50 ug/L 200 ug/L 7-Aminoclonazepam Not detected 100 ug/L 7-Aminoflunitrazepam Not detected 100 ug/L 7-Aminonitrazepam Not detected 100 ug/L Alpha OH-alprazolam Not Detected 100 ug/L Creatinine 11.0 mmol/L Assayed to AS/NZS 4308:2008. Subject reports prescribed Valium. Toxicology

Metabolite patterns of benzodiazepines (Source – taken & amended from www.nature.com/clpt/jpurnal/v76/n6/fig_tab/clpt2005539ft.html) Most simply inactivated by demethylation/hydroxylation/amination/conjugation but: Toxicology

Common Drug-Testing Methodologies Urine testing - False positives Initial screen yields a non-negative finding but subsequent confirmation fails to reveal the presence of any recognised substance May be due to antibody cross-reactivity, presence of a related substance which is not included in the confirmation testing, analytical interference, etc Toxicology

Cases – Opiates Non-negative initial urine screen MS ASSAY - URINE OPIATE CONFIRMATION Date Codeine Morphine U.Creat Cod/Crea Mor/Crea (ug/L) (ug/L) (mmol/L) Ratio Ratio 13/01/10 <50 61 11.9 <4 5 No trace of codeine is detected. Subject reports taking Duro-Tuss. Toxicology

Sympathomimetic Amines – the faces Toxicology

Sympathetic Amines – the family Toxicology

Sympathetic Amines – the family Toxicology 37 37

Cases – Benzodiazepines Non-negative initial urine screen MS ASSAY - URINE BENZODIAZEPINE CONFIRMATION Cut-off Oxazepam Not detected 200 ug/L Temazepam Not detected 200 ug/L Diazepam Not detected 200 ug/L Nordiazepam Not detected 200 ug/L 7-Aminoclonazepam Not detected 100 ug/L 7-Aminoflunitrazepam Not detected 100 ug/L 7-Aminonitrazepam Not detected 100 ug/L Alpha OH-alprazolam Not Detected 100 ug/L Creatinine 3.7 mmol/L Assayed to AS/NZS 4308:2008. Subject reports prescribed Zoloft. Toxicology

Cases – Benzodiazepines Non-negative initial urine screen Toxicology 39 39

Common Drug-Testing Methodologies Urine testing - Passive positives This consideration applies to smoked substances Essentially only cannabis at presence although synthetic cannabinoids may become a concern Opium and cocaine smoking is no longer a common practice Toxicology

Cases – Cannabinoids Non-negative initial urine screen GCMS ASSAY - URINE THC CONFIRMATION Date Lab No d9-THCA U.Creat Ratio ug/L mmol/L 10/12/09 375 12.2 30 The subject is a flight attendant who claims that she attended a party in which cannabis may have been used 2 days prior to sample collection. Toxicology

Common Drug-Testing Methodologies Urine Conclusions Ensure that reports are adequate for the purpose Interpretation is often not intuitive – potentially demand additional laboratory resources Toxicology

Common Drug-Testing Methodologies Oral fluid testing (AS 4760:2005) Important considerations: History of the Standard Pros & Cons AS 4760-2006 43 43

Procedures for specimen collection and the detection and quantitation of drugs in oral fluid John Henry, Standards Australia, 2003: “Delegates agreed that a standard on saliva-based drug testing would be required eventually. We are not currently in possession of the information necessary for a standard to be commenced.” Forum On Saliva-based Drug Testing (Held at Standards Australia’s Head Office in Sydney on Wednesday 23rd May, 2003) AS 4760-2006 44 44

Procedures for specimen collection and the detection and quantitation of drugs in oral fluid 2005 – Draft Standard DR 05590 released for public comment 2005 – Victorian police commence trial of random roadside testing of oral fluid for 4 classes 2006 – 1st November, AS 4760-2006 published 2006 – random road-side testing of oral fluid for drugs became widespread – 2 classes only AS 4760-2006 45 45

Procedures for specimen collection and the detection and quantitation of drugs in oral fluid Prior to 2005 – oral fluid drug testing performed in few Australian industries Increasing pressure from unions to change from urine testing to oral fluid testing 25th Aug, 2008 – Saliva testing OK: AIRC “In an important decision for employers across Aust, AIRC SDP Jonathan Hamberger has given the tick to saliva testing for workplace drug tests” (OHN 753). AS 4760-2006 46 46

Shell Refining (Australia) Pty Ltd, Clyde Refinery v Construction, Forestry, Mining and Energy Union (DR2008/1238) [125] Once these two issues* are satisfactorily resolved, any random drug testing should be conducted using oral fluids. Until then it would not be unreasonable for the company to implement a urine based testing regime on an interim basis. *existence of accredited laboratories *wish to test for drugs other than those in the Standard AS 4760-2006 47 47

Procedures for specimen collection and the detection and quantitation of drugs in oral fluid Monday, 05 December 2011 2:13pm FWA backs "role" for urine testing, finds saliva tests flawed AS 4760-2006 48 48

Oral screening linked to false negatives HWE Mining Pty Ltd v Construction, Forestry, Mining and Energy Union (FWA 8288 (30 November 2011)) Oral screening linked to false negatives Vice President Lawler accepted expert evidence indicating oral screening was flawed: saliva testing devices were linked to a "significant incidence" of false negative results for some drugs, including cannabis; a large number of Victorian motorists who tested positive to cannabis in laboratory tests after accidents had returned a negative result in the roadside saliva test; on-site saliva testing devices had a low sensitivity for cannabis and their effectiveness could be reduced by the use of particular substances; and there was no Australian Standard to test saliva for the prescription sedative benzodiazepine. On-site saliva tests could only detect high concentrations and could not detect levels where users would be impaired. In the light of these matters, HWE was "eminently reasonable" in its bid to retain urine testing, Vice President Lawler said. AS 4760-2006 49 49

Procedures for specimen collection and the detection and quantitation of drugs in oral fluid Wednesday, 28 March 2012 12:34pm Saliva swabs better indicator of "likely impairment" from cannabis FWA Senior Deputy President Jonathan Hamberger sided with the union on a number of issues, most notably the drug-test method. His decision in favour of oral testing appeared in conflict with another recent ruling by FWA on a similar issue. AS 4760-2006 50 50

Communications, Electrical and Plumbing Union Endeavour Energy v Communications, Electrical and Plumbing Union (FWA s739 (28 March 2012)) Saliva swabs better indicator of "likely impairment" from cannabis Senior Deputy President Hamberger expressed concern about the fairness of urine testing, given it could identify the presence of cannabis consumed days or even weeks prior to the test. "This means a person may be found to have breached the policy even though their actions were taken in their own time and in no way affect their capacity to do their job safely," he said. "The employer has a legitimate right (and indeed obligation) to try and eliminate the risk that employees might come to work impaired by drugs or alcohol such that they could pose a risk to health or safety. Beyond that the employer has no right to dictate what drugs or alcohol its employees take in their own time. "It is precisely because it only detects for recent use that oral fluid testing is a better indicator of the likely impairment as a result of smoking cannabis." FWA found that oral fluid testing was the appropriate method for determining whether employees were under the influence of drugs at work. AS 4760-2006 51 51

Common Drug-Testing Methodologies Oral fluid testing (AS 4760:2005) Important considerations: History of the Standard Pros & Cons AS 4760-2006 52 52

Advantages with oral fluid testing Privacy less of a consideration Recency of use no problem with “historical positives” Interpretation of reports generally straightforward AS 4760-2006 53 53

Problems with oral fluid testing Workplace health and safety risk. Only detects drug users who may potentially present affected if they have used prior to testing on the day of the test. Contrary to common claim, test result does not directly relate to performance/impairment On-site testing devices yet to meet Standard More work needed on interferences, adulterants, etc Laboratory confirmation includes ALL drugs listed in the Standard. AS 4760-2006 54 54

Review of findings FALSE NEGATIVES Look at 24 months 2010 & 2011 only 4 codeine in 83 controls 1 codeine in a false positive cocaine 1 amphetamine in 83 controls (1) cocaine in 83 controls 6 THC in samples positive for ATS only AS 4760-2006 55 55

Onsite test performance THC – looking at 2010 & 2011 only 13.9% of all referred non-negative samples 1.1% insufficient to confirm 78.7% false positive 20.2% true positive AS 4760-2006 56 56

Onsite test performance Opiates – looking at 2010 & 2011 only 42.5% of all referred non-negative samples 1.4% insufficient to confirm 31.4% false positive 67.2% true positive all (3) codeine 0 codeine with morphine 0 codeine with heroin 0 morphine AS 4760-2006 57 57

Onsite test performance Amphetamine Type Stimulants – looking at 2010 & 2011 only 19.0% of all referred non-negative samples 2.3% insufficient to confirm 63.3% false positive 34.4% true positive 31.2% methamphetamine 2.3% amphetamine (1%) MDMA AS 4760-2006 58 58

Onsite test performance Cocaine <1.0% of all referred non-negative samples 1 insufficient to confirm 4 false positive but (one) false negative AS 4760-2006 59 59

Common Drug-Testing Methodologies Oral Fluid Conclusions A relatively new Standard – not “mature” yet The on-site testing devices have some way to go before becoming “reliable” False and innocent positives are costly False negatives are worrying Need better “feel” for what the levels actually mean Need more experience re interferences Basically, this is a young field AS 4760-2006 60 60

Common Drug-Testing Methodologies Synthetic Cannabinoids Definition: Substances which Are at least partially man-made Bind to CNS CB1 receptor with a medium to high affinity When binding to CB1, act as agonist Are commonly marketed so as to avoid detection of unlawful drug use Toxicology

Synthetic Cannabinoids Scope: History Chemical nature Manufacture considerations Legality What does the Standard bring to bear? Analytical aspects Toxicology

Synthetic Cannabinoids History: 1965 THC synthesised 1980 Cannabinoid receptors recognised 1984 John W Huffman’s group commenced synthesising substances for medical use 2004 “Spice” herbal mixtures sold in Germany 2008 CP-47,497 & JWH-018 identified in “Spice” Toxicology 63 63

Synthetic Cannabinoids History - Australia: 2010 use increasing in WA mining industry January 2011 my first Australian enquiry 7 Australian laboratories offering testing Antibody screening test available early 2012 Toxicology 64 64

Synthetic Cannabinoids Scope: History Chemical nature Manufacture considerations Legality What does the Standard bring to bear? Analytical aspects Toxicology

Synthetic Cannabinoids Chemical nature – The Classes Classical cannabinoids e.g. THC, HU-210 Nonclassical cannabinoids e.g. CP-47,497 Hybrid cannabinoids e.g. AM-4030 Aminoalkylindoles e.g. JWH-018, JWH-073 Eicosanoids e.g. methanandamide Others Toxicology

Synthetic Cannabinoids Chemical nature – The Classes Toxicology

Synthetic Cannabinoids Scope: History Chemical nature Manufacture considerations Legality What does the Standard bring to bear? Analytical aspects Toxicology

Synthetic Cannabinoids Manufacture and supply: Impure substances imported Solution sprayed onto herbs Dried down, packaged and sold No quality control of dosage or distribution No continuity of composition – manufacturer attempts to “keep ahead” of detection Concerns with manufacturing modifications Toxicology

Synthetic Cannabinoids Scope: History Chemical nature Manufacture considerations Legal considerations What does the Standard bring to bear? Analytical aspects Toxicology

Synthetic Cannabinoids Legal considerations: Worldwide variation in banning these In Australia: Toxicology

Synthetic Cannabinoids Synthetic 'cannabis' gets nationwide ban - Aja Styles – The Australian July 7, 2011 Banned - Kronic and other synthetic cannabis-like substances moved to the prohibited substances list. Eight synthetic cannabis-like substances will be classified as prohibited substances throughout Australia from July 8, with plans to rule out any attempts to circumvent state bans on substances like Kronic. Parliamentary Secretary for Health and Ageing, Catherine King said the changes to classification of specific chemical compounds would enable a nationwide, uniform prohibition on these drugs. The chemicals to be prohibited ( using the common name) are: AM-694, JWH – 250, JWH – 200, JWH – 073, JWH – 122, JWH- 018, Cannabicyclohexanol, CP 47,497 – most of these can be found in retail products known as Kronic, Spice, Karma, Voodoo, Kaos and K2. "In response to calls for uniform restrictions on these types of substances, the Commonwealth has considered the matter and made a decision to prohibit eight of the most widely-used and abused synthetic cannabinoids." Yet broader restrictions are still being considered with advice on such restrictions being sought from Advisory Committee on Medicines Scheduling's meeting in October. Toxicology

Synthetic Cannabinoids New synthetic cannabis dodges Aussie ban Fiona Willan, ninemsn- 12:30 AEDT Wed Oct 12 2011 Synthetic cannabis is still being sold in Australian stores, three months after a nationwide-ban was introduced. Authorities are now scrambling to outlaw a new strain of Kronic herbal smoking mixture, which a New Zealand company has released specifically for the Australian market. The company, Lightyears Ahead, has managed to dodge Australian laws by releasing a mixture that buyers claim is as potent as marijuana but does not contain any banned chemicals. Toxicology

Synthetic Cannabinoids Scope: History Chemical nature Manufacture considerations Legal considerations What does the Standard bring to bear? Analytical aspects Toxicology

Synthetic Cannabinoids The Standard – urine or saliva Collection and handling On site screening Within laboratory screening MS confirmation/characterisation/quantitation Toxicology

Synthetic Cannabinoids Analytical considerations – On site screening Now at least 3 on site tests available or becoming available but No consistency with the metabolites sought No consistency with detection thresholds No certainty that confirmation laboratories are testing for the same substances Toxicology

Synthetic Cannabinoids Analytical considerations – on site screening All test for JWH-018 & JWH-073 metabolites if no others The least sensitive detects 50 ug/L At this stage, none detect cannabis metabolites Toxicology

Synthetic Cannabinoids Analytical considerations – laboratory screening Now available Toxicology

Synthetic Cannabinoids Analytical considerations – MS confirmation At least 7 Australian laboratories offering MS detection and quantitation Apply considerations from AS/NZS 4308 Standards expensive Standards were difficult to obtain Deuterated standards remain difficult to obtain Commercial controls not available yet Toxicology

Synthetic Cannabinoids Analytical considerations – MS confirmation (cont) Laboratories test for a limited number of substances – range differs from lab to lab Sensitivities not defined in Standard - determined by individual laboratory decision which is based at least in part on analytical considerations “Menu” continually expanding Toxicology

Synthetic Cannabinoids Analytical considerations – what are we finding? WA lab - July 2011 Testing for metabolites of JWH-018, JWH- 073 and JWH-122 only at that time Initially average of 10% positive, up to 50% from some sites Toxicology

Synthetic Cannabinoids Analytical considerations – what is QML finding? Testing for JWH-018, JWH-073, JWH-122, JWH- 200 & JWH-250 parent and metabolites Detection threshold 10 ug/L Positive finding in approx. 2% of samples JWH-018 metabolite and JWH-073 metabolite are the prevalent findings but a single JWH-122 and d9-THCA finding Toxicology

Synthetic Cannabinoids Analytical considerations – what are other labs doing? Testing for JWH-018 & JWH-073 at least Up to 8 others but menu continuously expanding Detection thresholds vary from 50 ug/L to 0.1 ug/L Positive rates vary from 1% to 10% Toxicology

Common Drug-Testing Methodologies Synthetic Cannabinoid Conclusions Future considerations – where are we heading? No sign of diminishing requirement for testing No sign of falling use No court challenge to findings yet No challenge to action taken on positive finding yet Toxicology

Common Drug-Testing Methodologies Synthetic Cannabinoids Future considerations – what’s missing? Detailed pharmacokinetics and detection characteristics Clinical toxicology Short term clinical effects Long term clinical effects Acute toxicity Toxicology

There are traps for the unwary Toxicology

Errors have consequences Toxicology