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Common Drug-Testing Methodologies

This article discusses the different methodologies for toxicology and drug-testing in the workplace, including screening and confirmation procedures. It also covers the standards and accreditation for testing, as well as considerations such as adulteration and creatinine levels in urine samples.

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Common Drug-Testing Methodologies

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

  2. 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

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

  4. 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)

  5. 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 Common Drug-Testing Methodologies

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

  7. Toxicology 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 Common Drug-Testing Methodologies

  8. Toxicology

  9. Toxicology Courtesy of Bio-Rad Laboratories Ltd

  10. Toxicology

  11. 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 Common Drug-Testing Methodologies

  12. Toxicology

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

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

  15. 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

  16. 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

  17. 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”

  18. 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.

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

  20. 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 Common Drug-Testing Methodologies

  21. 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.

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

  23. 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.

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

  25. 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.

  26. 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

  27. Toxicology Sympathomimetic Amines – the faces

  28. 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

  29. 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

  30. 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

  31. Toxicology 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.

  32. 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:

  33. 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 Common Drug-Testing Methodologies

  34. 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.

  35. Toxicology Sympathomimetic Amines – the faces

  36. Toxicology Sympathetic Amines – the family

  37. Sympathetic Amines – the family Toxicology 37

  38. Toxicology 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.

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

  40. Toxicology 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 Common Drug-Testing Methodologies

  41. 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.

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

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

  44. 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

  45. 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

  46. 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

  47. 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

  48. 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

  49. 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

  50. 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

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