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Drugs of Abuse TestingDrugs of Abuse TestingDr Charles Appleton
Q l d M di l L bQueensland Medical Laboratory
RCPA AACB Chemical Pathology Conference 2013RCPA AACB Chemical Pathology Conference 2013
Toxicology
Where does drug testing “pop up” in laboratory practice?laboratory practice?
• Drug‐rehab programs, suspected overdose or misused d k k• Suspected drink spiking
• Mum finds “tablet” in son’s room O ti l t ti d i id t l t d• Occupational testing – random, incident‐related, pre‐employment
• Court‐ordered testingCourt‐ordered testing• DoCS‐ordered testing• Sports drug testingSports drug testing
Toxicology
Drugs of Abuse Testingg g
Learning outcomes• Briefly review major considerations of testing in this field• Allude to challenges of reporting to medical and non‐medical requestors
• Recognise shortcomings of current testing
Toxicology
Drugs of Abuse Testingg g
What matrix?• Urine• Oral fluid• Hair• Blood• Sweat• Breath
Toxicology
• others
Drugs of Abuse Testingg g
Relevant Standards define procedures for – Collection– Transportation– Analysis, and– Reporting
AS/NZS 4308:2008 for urine/
Toxicology
Drugs of Abuse Testing
Toxicology
Drugs of Abuse Testingg g
Methods of analysis:• Immunoassay – ward/workplace/laboratory• Extraction, chromatography and detection• Other separative techniques• Chemical methods e.g. ethanolg
Toxicology
Drugs of Abuse Testingg gImmunoassay:• Generally a “class” method
– Cannabinoids 4308, 4670
– Opiates 4308, 4670
S th i ti A i 4308 4670– Sympathomimetic Amines 4308, 4670
– Cocaine 4308, 4670
– Benzodiazepines 4308p– Barbiturates– Phencyclidine
h d– Methadone– and many others
Toxicology
Drugs of Abuse Testingg gImmunoassay: Thresholds differ from Country to Country
Recommended by: Standards Australia SAMHSA(AS/NZS 4038)
Cannabinoids 50 50Cocaine metabolites 300 300S th i ti A i 300 1000Sympathomimetic Amines 300 1000Opiates 300 2000Benzodiazepines 200pPhencyclidine 25
Toxicology
Toxicology
Drugs of Abuse Testingg g
Chromatographic procedures• Sample cleanup/extraction• Chromatographic separation of different drugs and
metabolites of those drugs– gas, high performance liquid, thin layer
• Identification (and possibly quantitation) of the individual parent and metabolite peaks– Mass spectrography
Toxicology
Drugs of Abuse Testingg g
Limitations of MS• specificity• isomers
Toxicology
Drugs of Abuse Testingg g
Cannabis• Derived from plant – Cannabis sativa• Sedation, euphoria, hallucinations, temporal distortion• Historical positives ‐ detection window depends on nature of use • False positives ‐ uncommon• Innocent positives ‐ uncommon• Passive positives ‐ exceptionalp p
Toxicology
Fig. 1 Chemical structure of main cannabinoids in Cannabis sativa.
ToxicologyCopyright © 2008 The Royal College of Psychiatrists
ASHTON, C. H. Br J Psychiatry 2001;178:101-106
Cases – Cannabinoids Non negative initial urine screenNon‐negative initial urine screen
GCMS ASSAY - URINE THC CONFIRMATION
Date Lab No d9-THCA U.Creat Ratio
ug/L mmol/L
08/12/09 223 33.2 7
d9-THCA: 11 nor delta tetrahydrocannabinol-9-carboxylic acid (assayed according to AS/NZS 4308:2008 requirements)according to AS/NZS 4308:2008 requirements)
The AS/NZS 4308:2008 cut-off for this compound is 15 ug/L.
Toxicology
Cases – Cannabinoids Non negative initial urine screenNon‐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
Th bj t i fli ht tt d t h l i th t h tt d d t i hi h bi h bThe 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
Cases – Cannabinoids Non negative initial urine screenNon‐negative initial urine screen
GCMS ASSAY - URINE THC CONFIRMATION
Date Lab No d9-THCA U.Creat Ratio
ug/L mmol/L
23/12/09 45 14.8 3.0
Th i iti l it tiThe initial on‐site screen was non‐negative.The subject reports use of hemp oil skin care products.
Toxicology
Drugs of Abuse Testingg gOpiates• Natural, semisynthetic and synthetic derivatives of morphine
i ll f ifcommercially from Papaver somniferum • Morphine and its close relatives• Analgesic cough suppressantAnalgesic, cough suppressant• Detection window 3‐4 days • No historical positives• False positives – common seasonally• Innocent positives – common • Passive positives rare• Passive positives – rare
Toxicology
Drugs of Abuse Testingg gOpiate/opioid family – detection on “standard” immunoassay
InvisibleHigh dose only“Standard” InvisibleHigh dose onlyStandard
buprenorphinenaltrexone codeine4308methadoneoxycodonemorphine4308
fentanylpholcodinepethidinenaloxoneheroin4308 (6‐mam)
p p
propoxyphenedihydrocodeinehydromorphone
Toxicology
The metabolic pathway of the opiates.XII‐Biotech‐C‐Opiate Chemistry‐3
Toxicology
Cases – Opiates Non negative initial urine screenNon‐negative initial urine screen
MS ASSAY - URINE OPIATE CONFIRMATION
Date Codeine Morphine U Creat Cod/Crea Mor/CreaDate Codeine Morphine U.Creat Cod/Crea Mor/Crea
(ug/L) (ug/L) (mmol/L) Ratio Ratio
18/12/09 315 556 18.7 17 30
Toxicology
Cases – Opiates Non negative initial urine screenNon‐negative initial urine screen
MS ASSAY - URINE OPIATE CONFIRMATIONDate Codeine Morphine U Creat Cod/Crea Mor/CreaDate Codeine Morphine U.Creat Cod/Crea Mor/Crea
(ug/L) (ug/L) (mmol/L) Ratio Ratio17/11/08 1576 >2000 3.0 525 >667
GCMS confirms the positive opiate screen and also reveals the presence of a small amount of 6-monoacetyl morphine (13 ug/L).
The presence of 6 MAM is indicative of probable use of heroin within eightThe presence of 6-MAM is indicative of probable use of heroin within eight hours of sample collection.
Additional to this, the subject appears to have used a codeine-containing medication as well. This may be done in an attempt to "mask" the use of ed cat o as e . s ay be do e a atte pt to as t e use oheroin but the presence of 6-MAM defeats this.
Toxicology
Cases – Opiates Non negative initial urine screenNon‐negative initial urine screen
MS ASSAY - URINE OPIATE CONFIRMATIONC d i hi C C d/C /CDate 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 mayAlthough 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
Toxicology
Toxicology
Drugs of Abuse Testingg gSympathomimetic amines (now referred to as “Amphetamine‐
type Stimulants” or “ATS”)type Stimulants or ATS )• Natural and synthetic relatives of epinephrine (adrenalin)• Stimulant, decongestant, appetite suppressantg pp pp• Detection window 3‐4 days but depends on pH of urine • No historical positives• False positives – interesting • Innocent positives – frequent (depends of source population)
Passi e positi es rare• Passive positives – rare
Toxicology
Drugs of Abuse Testingg gSympathomimetic amine family – immunoassay detection
InvisibleLess sensitive“Standard”
methylphenidatepseudoephedrine4308amphetamine4308phenylephrinephentermine4308methamphetamine4308
tyramineMDA4308
ephedrine4308MDMA4308methylphenidatepseudoephedrine4308amphetamine4308
phenylethylaminebenzylpiperazine*4308
4‐bromo‐2,5‐dimethoxy amphetamine and other
Toxicology
amphetamine and other “designers”
Sympathomimetic Amines – the facesy p
Toxicology
Sympathetic Amines – the familyy p y
Toxicology
Toxicology 30
Cases – Sympathomimetic Amines Non negative initial urine screenNon‐negative initial urine screen
MS ASSAY - URINE SYMPATHOMIMETIC AMINE CONFIRMATION
Cut-off
Amphetamine 1600 ug/L 150 ug/L
Methamphetamine 6100 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
P d h d i N t d t t d 500 /LPseudoephedrine Not detected 500 ug/L
Creatinine 14.8 mmol/L
Toxicology
Cases – Sympathomimetic Amines Non negative initial urine screenNon‐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
P d h d i N t d t t d 500 /LPseudoephedrine Not detected 500 ug/L
Creatinine 12.6 mmol/L
Subject has Parkinson disease
Toxicology
Subject has Parkinson disease.
Cases – Sympathomimetic Amines Non negative initial urine screenNon‐negative initial urine screen
MS ASSAY - URINE SYMPATHOMIMETIC AMINE CONFIRMATION
Cut-off
Amphetamine Not detected 150 ug/L
Methamphetamine 232 ug/L 150 ug/L
MDMA Not detected 150 ug/L
MDA Not detected 150 ug/L
Phentermine Not detected 500 ug/L
Ephedrine >1500 ug/L 500 ug/L
P d h d i >1500 /L 500 /LPseudoephedrine >1500 ug/L 500 ug/L
Creatinine 6.1 mmol/L
Ephedrine 62700 ug/LEphedrine 62700 ug/L
Pseudoephedrine 490000 ug/L
Toxicology
Drugs of Abuse TestingBenzodiazepines• Chemically derived from fusion of benzene and diazepine• Sedative, anxiolytic, anticonvulsant, muscle relaxant• Detection window depends on nature of use ‐ historical positives• False positives – relatively common but depends on population• Innocent positives – prescription usep p p• Passive positives – not a consideration• This group typically has complex metabolite patternsThis group typically has complex metabolite patterns
Toxicology
Metabolite patterns of benzodiazepines(Source – taken & amended from www nature com/clpt/jpurnal/v76/n6/fig tab/clpt2005539ft html)(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
Drugs of Abuse Testingg gBenzodiazepine family – MS confirmation
Not listed in StandardListed in 4308:2008
ClobazamTemazepamLorazepamDiazepam
BromazepamNitrazepamMidazolamClonazepamTriazolamOxazepamoba ae a epa
FlunitrazepamBromazepamNitrazepam
Alprazolam
Toxicology
Cases – Benzodiazepines Non negative initial urine screenNon‐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 A i it N t d t t d 100 /L7-Aminonitrazepam Not detected 100 ug/L
Alpha OH-alprazolam Not Detected 100 ug/L
Creatinine 11.0 mmol/L
Subject reports prescribed Valium.
Toxicology
Cases – Benzodiazepines Non negative initial urine screenNon‐negative initial urine screen
MS ASSAY - URINE BENZODIAZEPINE CONFIRMATION
Cut-off
Oxazepam 470 ug/L 200 ug/L
Temazepam 4900 ug/L 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 A i it N t d t t d 100 /L7-Aminonitrazepam Not detected 100 ug/L
Alpha OH-alprazolam Not Detected 100 ug/L
Creatinine 10.9 mmol/L
Subject reports prescribed Temaze.
Toxicology
Cases – Benzodiazepines Non negative initial urine screenNon‐negative initial urine screen
MS ASSAY - URINE BENZODIAZEPINE CONFIRMATION
Cut-off
Oxazepam 7390 ug/L 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 A i it N t d t t d 100 /L7-Aminonitrazepam Not detected 100 ug/L
Alpha OH-alprazolam Not Detected 100 ug/L
Creatinine 15.2 mmol/L
Subject reports prescribed Serepax.
Toxicology
Cases – Benzodiazepines Non negative initial urine screenNon‐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 80 ug/L 100 ug/L
7 A i it N t d t t d 100 /L7-Aminonitrazepam Not detected 100 ug/L
Alpha OH-alprazolam Not Detected 100 ug/L
Creatinine 6.6 mmol/L
Brought by parent to doctor after party on weekend
Toxicology
Cases – Benzodiazepines Non negative initial urine screenNon‐negative initial urine screen
Toxicology
Drugs of Abuse Testingg gCocaine• Naturally derived from Erythroxylon coca• Naturally derived from Erythroxylon coca• Stimulant, local anaesthetic• Detection window 2 3 days• Detection window 2‐3 days • No historical positives
False positi es rare• False positives – rare • Innocent positives – rare
P i iti i ti• Passive positives – rare in practice
Toxicology
Toxicology
Courtesy of Bio‐Rad Laboratories Ltd
Cases – Cocaine Non negative initial urine screenNon‐negative initial urine screen
MS ASSAY - URINE COCAINE METABOLITE CONFIRMATION
Cut-offCut off
Cocaine Not detected Unspecified
Benzoylecgonine 1390 ug/L 150 ug/L
Ecgonine methyl ester 320 ug/L 150 ug/LEcgonine methyl ester 320 ug/L 150 ug/L
Creatinine 17.9 mmol/L
Assayed to AS/NZS 4308:2008Assayed to AS/NZS 4308:2008.
Toxicology
Toxicology
Toxicology
Drugs of Abuse Testingg gThe “invisible” substances of abuse• Opioids – fentanyl, pethidine, oxycodone, methadone, buprenorphine p y , p , y , , p p• Some designer sympathomimetic amines• Ethanol • ‐hydroxybutyrate• ‐hydroxybutyrate• Ketamine• Propofol
P il bi d th l t h ll i• Psilocybin and other plant hallucinogens• Lysergic acid diethylamide• Phencyclidine
And the “new” drugs
Toxicology
Drugs of Abuse Testingg gRecent “invisible” substances of abuse• Synthetic cannabinoids – over 800 developedSynthetic cannabinoids over 800 developed• Synthetic cathinones e.g. MDPV, mephedrone• Synthetic hallucinogens e.g. 25I‐NBO Me, Bromo‐Dragonfly, PMA
S th ti i• Synthetic cocaines
Reference source – European Monitoring Centre for Drugs and Drug p g g gAddiction (EMCDDA)
Toxicology
Toxicology 49
Synthetic CannabinoidsSubstances which• Are at least partially man made• Are at least partially man‐made• Bind to CNS CB1 receptor with a medium to high
ffi iaffinity• When binding to CB1, act as agonist• Minimally interact with CB2 receptor• Are commonly marketed so as to avoid detection
Toxicology
Are commonly marketed so as to avoid detection of unlawful drug use
Synthetic CannabinoidsyScope:• History• Chemical nature• Chemical nature • Manufacture considerations• Legality• Analytical aspects
Toxicology
Synthetic Cannabinoids
History:• 1965 THC synthesised• 1965 THC synthesised• 1980 Cannabinoid receptors recognised• 1984 John W Huffman’s group commenced synthesising substances for medical usey g
• 2004 “Spice” herbal mixtures sold in Germany2008 CP 47 497 & JWH 018 id tifi d i “S i ”• 2008 CP‐47,497 & JWH‐018 identified in “Spice”
Toxicology
Synthetic Cannabinoids
History ‐ Australia:d• 2010 use increasing in WA mining industry
• January 2011 my first Australian enquiryJanuary 2011 my first Australian enquiry • 7 Australian laboratories offering MS testing• Antibody screening test available 2012
Toxicology
Synthetic Cannabinoids
Toxicology
Synthetic CannabinoidsyScope:• History• Chemical nature• Chemical nature• Manufacture considerations• Legality• Analytical aspects
Toxicology
Synthetic CannabinoidsyChemical nature – The Classes• Classical cannabinoids e.g. THC, HU‐210• Nonclassical cannabinoids e g CP 47 497• Nonclassical cannabinoids e.g. CP‐47,497• Hybrid cannabinoids e.g. AM‐4030• Aminoalkylindoles e.g. JWH‐018, JWH‐073• Eicosanoids e.g. methanandamide
Toxicology• Others
Synthetic CannabinoidsyChemical nature – The Classes
Toxicology
Synthetic CannabinoidsTh N G iThe Next Generation
Toxicology
Synthetic CannabinoidsyScope:• History• Chemical nature• Chemical nature • Manufacture considerations• Legality• Analytical aspects
Toxicology
Synthetic CannabinoidsyManufacture 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 detectionC ith f t i difi ti
Toxicology
• Concerns with manufacturing modifications
Synthetic CannabinoidsyScope:• History• Chemical nature• Chemical nature • Manufacture considerations• Legal considerations• Analytical aspects
Toxicology
Synthetic CannabinoidsyLegal considerations:• Worldwide variation in banning these• In Australia• In Australia......
Toxicology
Synthetic CannabinoidsyScope:• History• Chemical nature• Chemical nature • Manufacture considerations• Legal considerations• Analytical aspects
Toxicology
Synthetic CannabinoidsyThe Standard –• Collection and handling• On site screening• On site screening• Within laboratory screening• MS confirmation/characterisation/quantitation
Toxicology
Synthetic Cannabinoidsy
Toxicology
Synthetic Cannabinoidsy
Analytical considerations –– JWH‐018 JWH‐073 JWH‐122 AM‐2201– JWH‐018, JWH‐073, JWH‐122, AM‐2201, UR‐144 & potentially others
h h ld /– Detection threshold 10 ug/L
Toxicology
Synthetic CannabinoidsyAnalytical considerations – laboratory screening– No consistency with the metabolites sought– No consistency with detection thresholds– No certainty that confirmation laboratories are testing for the same substances
– All test for JWH‐018 & JWH‐073 metabolites if no others– Do not cross‐react with cannabis metabolites
Toxicology
Synthetic CannabinoidsyAnalytical considerations – MS testing• At least 7 Australian laboratories offering MS detection and quantitationq
• Apply considerations from AS/NZS 4308• Standards expensive and difficult to obtain• Standards expensive and difficult to obtain• Deuterated standards remain difficult to obtain
Toxicology
• Commercial controls not available yet
Synthetic CannabinoidsyAnalytical considerations – MS testing (cont)• Laboratories test for a limited number of substances – range differs from lab to labg
• Sensitivities determined by individual laboratory decision which is based at least in part ondecision which is based at least in part on analytical considerations
• “Menu” continually expandingToxicology
• Menu continually expanding
Synthetic CannabinoidsyAnalytical considerations – what is QML finding?• Testing for JWH‐018, JWH‐073, JWH‐122, JWH‐200 & JWH‐250 parent and metabolitesp
• Detection threshold 5 ug/L• Positive finding in approx 2% of samples• Positive finding in approx. 2% of samples• JWH‐018 metabolite and JWH‐073 metabolite
i h l fi diToxicology
remain the prevalent findings
Synthetic CannabinoidsyAnalytical considerations – recent developments• Importation of AM‐2201 found in mid‐2012
• subsequently added to MS panelq y p
• Importation of UR‐144 and XLR‐11 found in late 20122012• Subsequently added to MS panel
Toxicology
Synthetic Drug ConclusionsFuture considerations – where are we heading?
y g
• 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
Synthetic Drug ConclusionsFuture considerations – what’s missing?
y g
• Detailed pharmacokinetics and detection characteristics• Clinical toxicology
• Short term clinical effects• Long term clinical effects• Acute toxicity
Toxicology
Toxicology