Common Drug-Testing Methodologies

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

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

Approach The Standards The methodologies Where does synthetic cannabinoid testing sit? Toxicology

AS/NZS 4308:2008 for urine AS 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

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

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

Immunoassay: Thresholds (mg/L) differ from Country to Country Recommended by: Standards Australia SAMHSA (AS/NZS 4038) Cannabinoids Cocaine metabolites Sympathomimetic Amines Opiates Benzodiazepines 200 Phencyclidine Toxicology

Toxicology

Courtesy of Bio-Rad Laboratories Ltd
Toxicology

Toxicology

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

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

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

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

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

Urine Creatinine - average concentration 10-12 mmol/L in males 8-10 mmol/L in females “usual range” 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/ 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

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

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

GCMS ASSAY - URINE THC CONFIRMATION Date Lab No d9-THCA U.Creat Ratio ug/L mmol/L 06/11/ 13/11/ screen neg 20/11/ Large fluctuations in the urinary creatinine will complicate interpretation of absolute values. Toxicology

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/ 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

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 < < (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 ug/L Methamphetamine >1500 ug/L ug/L MDMA Not detected ug/L MDA Not detected ug/L Phentermine Not detected ug/L Ephedrine Not detected ug/L Pseudoephedrine Not detected ug/L Creatinine mmol/L Amphetamine ug/L Methamphetamine ug/L Toxicology

Sympathomimetic Amines – the faces
Toxicology

MS ASSAY - URINE SYMPATHOMIMETIC AMINE CONFIRMATION Cut-off Amphetamine >1500 ug/L ug/L Methamphetamine Not detected ug/L MDMA Not detected ug/L MDA Not detected ug/L Phentermine Not detected ug/L Ephedrine Not detected ug/L Pseudoephedrine Not detected ug/L Creatinine mmol/L Assayed to AS/NZS 4308:2008. Subject prescribed Dexamphetamine Toxicology

MS ASSAY - URINE SYMPATHOMIMETIC AMINE CONFIRMATION Cut-off Amphetamine Not detected ug/L Methamphetamine Not detected ug/L MDMA Not detected ug/L MDA Not detected ug/L Phentermine >1500 ug/L ug/L Ephedrine Not detected ug/L Pseudoephedrine Not detected ug/L Creatinine mmol/L Phentermine ug/L Toxicology

MS ASSAY - URINE SYMPATHOMIMETIC AMINE CONFIRMATION Cut-off Amphetamine ug/L ug/L Methamphetamine ug/L ug/L MDMA Not detected ug/L MDA Not detected ug/L Phentermine Not detected ug/L Ephedrine Not detected ug/L Pseudoephedrine Not detected ug/L Creatinine mmol/L Subject has Parkinson disease. Toxicology 30 30

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

Metabolite patterns of benzodiazepines (Source – taken & amended from Most simply inactivated by demethylation/hydroxylation/amination/conjugation but: Toxicology

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

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 < < 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

MS ASSAY - URINE BENZODIAZEPINE CONFIRMATION Cut-off Oxazepam Not detected ug/L Temazepam Not detected ug/L Diazepam Not detected ug/L Nordiazepam Not detected ug/L 7-Aminoclonazepam Not detected ug/L 7-Aminoflunitrazepam Not detected ug/L 7-Aminonitrazepam Not detected ug/L Alpha OH-alprazolam Not Detected ug/L Creatinine mmol/L Assayed to AS/NZS 4308:2008. Subject reports prescribed Zoloft. Toxicology

Toxicology 39 39

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

GCMS ASSAY - URINE THC CONFIRMATION Date Lab No d9-THCA U.Creat Ratio ug/L mmol/L 10/12/ 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

Oral fluid testing (AS 4760:2005) Important considerations: History of the Standard Pros & Cons AS 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 44 44

2005 – Draft Standard DR released for public comment 2005 – Victorian police commence trial of random roadside testing of oral fluid for 4 classes 2006 – 1st November, AS published 2006 – random road-side testing of oral fluid for drugs became widespread – 2 classes only AS 45 45

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 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 47 47

Monday, 05 December :13pm FWA backs "role" for urine testing, finds saliva tests flawed AS 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 49 49

Wednesday, 28 March :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 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 51 51

Oral fluid testing (AS 4760:2005) Important considerations: History of the Standard Pros & Cons AS 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 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 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 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 56 56

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 57 57

Amphetamine Type Stimulants – looking at 2010 & 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 58 58

Cocaine <1.0% of all referred non-negative samples 1 insufficient to confirm 4 false positive but (one) false negative AS 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 60 60

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

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

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

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

Chemical nature – The Classes Toxicology

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

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

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

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

New synthetic cannabis dodges Aussie ban Fiona Willan, ninemsn- 12:30 AEDT Wed Oct 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

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

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

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

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

Analytical considerations – laboratory screening Now available Toxicology

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

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

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

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

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

Common Drug-Testing Methodologies

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