Dr Charles Appleton

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 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


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 ( m g/L) differ from Country to Country

Recommended by:

Cannabinoids

Cocaine metabolites

Sympathomimetic Amines

Opiates

Benzodiazepines

Phencyclidine

Standards Australia SAMHSA

(AS/NZS 4038)

50

300

50

300

300

300

200

1000

2000

25

Toxicology

Toxicology 8

Courtesy of Bio-Rad Laboratories Ltd

Toxicology 9

Toxicology 10


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 11

Toxicology 12


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”- 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 18


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





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 21


Urine testing - Legitimate “innocent” positives

– Over-the-counter medications

– Prescribed medications

– Food constituents

– Products of metabolism of other drugs

Toxicology

Cases – Opiates


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 23

The metabolic pathway of the opiates.

XII-Biotech-C-Opiate Chemistry-3

Toxicology 24

Cases – Opiates





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 25

Cases – Sympathomimetic Amines


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 26

Sympathomimetic Amines – the faces

Toxicology 27




Cut-off

Amphetamine >1500 ug/L 150 ug/L

Methamphetamine Not detected 150 ug/L







Assayed to AS/NZS 4308:2008.

Subject prescribed Dexamphetamine

Toxicology 28




Cut-off

Amphetamine Not detected 150 ug/L

Methamphetamine Not detected 150 ug/L



Phentermine >1500 ug/L 500 ug/L




Phentermine 31300 ug/L

Toxicology 29




Cut-off

Amphetamine 917 ug/L 150 ug/L

Methamphetamine 3040 ug/L 150 ug/L







Subject has Parkinson disease.

Toxicology 30

Cases – Benzodiazepines


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



Subject reports prescribed Valium.

Toxicology 31

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 32


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





13/01/10 <50 61 11.9 <4 5

No trace of codeine is detected.

Subject reports taking Duro-Tuss.

Toxicology 34

Sympathomimetic Amines – the faces

Toxicology 35

Sympathetic Amines – the family

Toxicology 36

Sympathetic Amines – the family

Toxicology 37



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



Subject reports prescribed Zoloft.

Toxicology 38



Toxicology 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





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 41


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 4760-2006

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


• 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


• 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

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


• Monday, 05 December 2011 2:13pm

• FWA backs "role" for urine testing, finds saliva tests flawed

AS 4760-2006

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


• 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

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


Oral fluid testing

(AS 4760:2005)

Important considerations:

• History of the Standard

• Pros & Cons

AS 4760-2006

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

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

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

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


•

•

Opiates – looking at 2010 & 2011 only


•




• all (3) codeine

• 0 codeine with morphine

• 0 codeine with heroin

• 0 morphine

AS 4760-2006


• Amphetamine Type Stimulants – looking at 2010 &

2011 only





• 31.2% methamphetamine

• 2.3% amphetamine

• (1%) MDMA

AS 4760-2006


• Cocaine

– <1.0% of all referred non-negative samples

– 1 insufficient to confirm

– 4 false positive

– but (one) false negative

AS 4760-2006


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


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


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


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


Scope:

• History

• Chemical nature


• Legality



Toxicology


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


Scope:

• History

• Chemical nature

• Manufacture considerations

• Legality



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


• Legal considerations



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 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


Scope:

• History

• Chemical nature


• 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 d

9-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


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



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

Dr Charles Appleton

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