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Workplace Drug Screening Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MSMS)
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Workplace Drug Screening Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MSMS)

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Grant Moore …

Grant Moore
Section Head Toxicology
Canterbury Health Labs,
PO Box 151, Christchurch 8014
grant.moore@cdhb.govt.nz

(P27, Thursday 27, Ilott Theatre, 2.00)

Published in: Health & Medicine, Technology

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  • The material was manufactured by Australian Scientific Enterprises as Human urine specimens 6 linear concentration levels – a low and a high pool with 4 intermediate levels In setting the specifications for all the drugs we used the AS/NZS:4308 standard cut-off limits to offer a range of concentrations from 0ug/L through to significantly above the cut-off value for each drug This year and again in 2011 laboratories can purchase either a 1ml or 10ml vial depending on the sample volume required for analysis The program consists of a single 12 month cycle January through to December and 1 sample per month The program has a specifically designed report for each participant to evaluate their results according to the cut-offs for their device The target values for each drug for every concentration level were set using the standard QAP target setting statistical system suing GCMS and LCMSMS methods
  • Transcript

    • 1. Workplace Drug Screening Using LCMSMS Grant Moore Workplace Drug Screening Canterbury Health Labs
    • 2. Overview
      • AS/NZS 4308:2008 Testing Standard
      • Testing Options available
      • LCMSMS Drug Screening
      • POCT EQA Programme
    • 3. A Spouse ’ s Perspective
      • “ Drugs are terrorists. They ’ re brilliant. They create the perfect need for themselves and trick you into thinking they ’ re your friend and everything will be okay as long as your with them. It ’ s Stockholm Syndrome by chemical. You defend your captor and adopt their way of thinking ”
      • “ And even a drug like marijuana, that ’ s supposedly non-addictive, has an enormous dependency factor ”
          • - Eliza Roberts, wife of Eric Roberts, Actor
    • 4. Workplace Drug Testing Standard
      • First edition 1995
      • Subsequent revisions 2001, 2008
      • 2008 Revision allowed for POCT devices as front line screen
    • 5. AS/NZS-4308 Testing Standard
      • “ … The objective of this Standard is to ensure that the detection of drugs in urine meets the expectations for testing of specimens for medico-legal, workplace or court-directed purposes… ” - AS/NZS 4308:2008
      • Describes a set of acceptable analytical procedures and documentation that gives surety about the results reported by an accredited screener or laboratory.
      • Provides ‘ peace of mind ’ to client/customer regarding the quality of the work performed.
    • 6. Additions to the Standard - 2008
    • 7. Additions to the Standard
      • All POCT devices should have a certificate stating their compliance with the Standard
      • In theory all new batches of testing devices should be re-certified
    • 8. Workplace Testing Standard
      • Testing standard allows use of POCT devices
      • Screening by immunoassay
      • Confirmation by LCMSMS (or GCMS)
    • 9. POCT devices iScreen One step drug card Inverness Medical Sure Step6 Panel cassette Thermo Fisher Scientific Micro Tox Multi Drug Screening Test Branan QuickTox Bio-Rad Tox/See
    • 10. Workplace Testing Standard – Immunoassay cut-offs
    • 11. Immunoassay Screening
      • Only 5 drug classes defined for screening
      • Other drugs of abuse present in community
        • Phencyclidine (USA)
        • Methadone
        • Oxycodone (Increasing problem)
        • Propoxyphene
        • Synthetic Cannabinoids
        • Ethanol
      • Not all compounds within drug classes are able to be confirmed
    • 12. Immunoassay analysers
      • Typical chemistry/immunoassay analyser
    • 13. Workplace Testing Standard – Confirmation cut-offs
    • 14. Confirmation Compounds
      • Amphetamine-type substances - 8 compounds
      • Benzodiazepines - 8 compounds
      • Cannabinoids - 1 compound
      • Cocaine metabolites - 2 compounds
      • Opiates - 3 compounds
      • In most cases there are significantly more compounds that will give ‘ positive ’ results that are not implicitly stated in the Standard.
    • 15. ABI 3200 Qtrap LCMSMS
    • 16. … and after the Earthquake! … it still worked!
    • 17. LCMSMS – Some terminology!
      • Chromatography – separating a mixture of compounds so each can be identified individually
      • LC – Liquid based chromatography system
      • MS – detector used to identify compounds based on molecular weight
      • Ion source – area where the liquid stream initially enters MS and compounds are ionised ready for identification
      • Quadrupole – part of MS, uses changes in voltages and current to allow transport of ionised compounds of a pre-determined molecular weight
    • 18. LCMSMS – more terminology!
      • 3 Quadrupoles in detector
      • Q1 – allows initial mass identification and screens out masses not being measured
      • Q2 – provides charged area to ionise or break down compounds of interest
      • Q3 – provides mass selection of fragments produced in Q2
      • Detector – collector that detects ions that are allowed through the quadrupole
    • 19. MS Components
    • 20. So how does it work?
      • Morphine
      • Formula C 17 H 19 NO 3
      • MW =285.34 da
      • Member of the opiate family
      • At Q1 allow MW 285 through
      • At Q2 fragmentation of MW 285
      • At Q3 allow defined fragments 165 and 153 through to the detector
      • Detector recognises a signal and responds
      • Referred to as Multiple Reaction Monitoring (MRM)
    • 21. So how does it work? Q1 Q2 Q3
    • 22. So how much sample do we need?
    • 23. So how much sample do we need?
      • … about 100 μ L!
      • This is hydrolysed with an enzyme
      • Internal standard is added
      • 800 μ L is added
      • 20 μ L is injected onto the column.
    • 24. So what can we detect/confirm?
      • Amphetamine-type substances
      • Benzodiazepines
      • Cannabinoids
      • Cocaine metabolites
      • Opiates
      • Methylphenidate(Ritalin®)
      • Synthetic Cannabinoids
      • Buprenorphine
      • Others
    • 25. Amphetamine-type substances
      • Currently screening for 8 different compounds
        • Amphetamine
        • Methamphetamine
        • MDMA
        • MDA
        • Ephedrine
        • Pseudoephedrine
        • Phentermine
        • 1-Benzylpiperazine
      • Current AS/NZS-4308:2008 list
      Amphetamine MDMA
    • 26. Amphetamine-type substances
      • Should we also be testing for:
        • Methcathinone
        • Methedrone
        • Flephedrone
        • Methylethcathinone
        • Butylone
        • Methylone
        • Methylmethcathinone (Mephedrone)
        • N-ethylamphetamine
        • MDEA
        • PMA
        • PMMA
        • ? Others
      • How prevalent are these compounds?
      • We ’ re not sure….!
      Methcathinone p-Methoxyamphetamine
    • 27. Benzodiazepine metabolites
      • Screening for over 20 different compounds
      • AS/NZS-4308:2008 compounds
        • Diazepam, Nordiazepam, Oxazepam, Temazepam
        • 7-aminoclonazepam, 7-aminonitrazepam
        • 7-aminoflunitrazepam, α -hydroxyalprazolam
      • Other common benzodiazepines
        • Midazolam, Lorazepam, Triazolam, Clobazam, Flurazepam
      • Other sedatives
        • Zopiclone, Zolpidem
      Diazepam Oxazepam
    • 28. Benzodiazepines
      • Commonly screen for 8 compounds described in testing standard
      • Other benzodiazepines available
      • Some sedatives not even being measured but could be causing impairment in the workplace
      • Possible to have ‘ not-negative ’ POCT or Immunoassay screen but not able to confirm by MS
      • Presence of a number of metabolites additive in their effect for screening, i.e. Diazepam metabolites
      • May not have one present at sufficient concentration to be above cut-off value.
    • 29. Tetrahydrocannabinol (THC) Δ 9 -Tetrahydrocannabinol
      • Our most common drug of abuse!
      • The dried flowering or fruiting tops of the pistillate plant of Cannabis sativa (Cannabinaceae).
      • Cannabis contains over 60 derivatives of 2-(2–isopropyl–5–methylphenyl)-5–pentylresorcinol, known as cannabinoids.
      • Hence the difference in cut-off values between the screen and confirmation processes (50 ng/mL cf 15 ng/mL).
    • 30. Opiates
      • AS/NZS 4308:2008 compounds
        • 6-monoacetylmorphine, codeine and morphine
      • Other common opiates also available
        • Dihydrocodeine, oxycodone, diacetylmorphine, hydrocodone, hydromorphone, pholcodine, ethylmorphine
    • 31. Opiate metabolism
    • 32. Synthetic Cannabinoids
      • ‘ Kronic ’ , ‘ Spice ’
      • Currently, JWH-018 and JWH-073
      • Other known ones are:
        • JWH-007
        • JWH-016
        • JWH-020
        • JWH-030
        • JWH-098
        • JWH-122
        • JWH-147
        • JWH-203
        • JWH-210
        • JWH-250
        • JWH-307
        • HU-210
        • CP-47,497
    • 33. What is the significance of JWH?
      • John W Hufman, an organic chemist at Clemson University, synthesized analogues and metabolites of Δ 9 -tetrahydrocannabinol (THC), the principal active component of cannabis.
      • JWH-018 is one of these analogues, with studies showing an affinity for the cannabinoid brain (CB1) receptor five times greater than that of THC.
      • JWH-073 has been shown to produce behavioural effects very similar to Δ 9 -tetrahydrocannabinol (THC) in animals.
      • JWH-073 binds to CB1 with a higher affinity than THC
    • 34. HU-210
      • HU-210 is a synthetic cannabinoid that was first synthesized in 1988 by the group led by Professor Raphael Mechoulam at the Hebrew University.
      • HU-210 is 100 to 800 times more potent than natural THC and has an extended duration of action.
      • HU-210 is the (–)-1,1-dimethylheptyl analog of 11-hydroxy- Δ8- tetrahydrocannabinol
    • 35. Structural Similarities? Tetrahydrocannabinol
    • 36. JWH-018 metabolites
    • 37. Are there Cross-Reactivity issues?
      • Probably!
      • Some samples received for Carboxy-THC confirmation
      • Given ‘ not-negative ’ result on POCT device
      • MS confirmation detects absence of carboxy-THC!
      • There is nothing there!
      • Subsequent analysis for synthetic cannabinoids gives positive result.
      • Usually present in the 100 ’ s of ng/mL.
    • 38. Synthetic Cannabinoids Method
      • Validated standards purchased from Chiron
      • Diluted standard (parent compound) infused into LCMS for compound optimisation
      • Q1 ->Q3 transitions established for monohydroxy metabolites
      • Stored into CLiquid library
      • Method parameters stored in CLiquid
      • Blank and potential positive samples run through method
    • 39. Establishing a method
    • 40. Procedure
      • Samples hydrolysed with glucuronidase
      • Deuterated internal standards added
      • Dilute with H 2 O
      • Inject into LCMS using appropriate method
    • 41. Results – blank samples
    • 42. Kronic negative
    • 43. Kronic positive
    • 44. How prevalent is Kronic use?
      • Snapshot of 30 samples
      • Sample from standard drug clientele
      • Mix is predominantly Methadone Programme patients
      • One sample was a ?Kronic use
      • All samples negative by LCMS method
    • 45. Where to from here?
      • Validation of method
      • Incorporating new synthetic cannabinoids into screen as there will surely be others released onto the market!
      • DMAA screening – already seeing some significant cases turning up in ED
      • There will always be new drugs coming on to the market!
    • 46. Kronic selection
    • 47. POCT EQA Programme
      • Availability from Royal College of Pathologists Australasia
      • Test ability of screening staff to get the right result
      • Tests ability of screening device to detect the correct compound
      • Allows informed decision regarding what device to purchase
      • Allows clients to have the best testing available
      • Minimises potential liability around testing
    • 48. OUTS Program
        • Human urine specimens
        • 6 linear concentration levels
          • A low and high pool (L1 and L6) & 4 intermediate levels
          • Used the AS/NZS:4308 Standard cut off limits to offer a range of concentrations from 0 ug/L through to significantly above the cut-off value for each drug
        • Participants purchase 10ml vials
        • 1 sample per month
    • 49. Cannabis End of Cycle report
    • 50. Cannabis EOC Report
      • Graphical representation of participants results
      • Shows concentration of sample
      • Shows ‘outliers’
    • 51.
      • At 109 ng/mL 4/43 not detected
      • At 87 ng/mL 2/43 not detected
      • At face value OK, i.e. 10% not detected at 2 x cut-off!
      • Only 5% not detected at cutoff +70%
      • Shouldn’t be a cross-reactivity issue
      • Perhaps a training issue
      • More than one manufacturers device
      • Standard allows up to 10% of devices inaccurate at +30%.
      Cannabis EOC
    • 52. Methamphetamine EOC
    • 53.
      • 0 ng/mL - 100% not detected
      • 378 ng/mL - 10/17 and 12/23 not detected - >50%
      • 474 ng/mL - 5/17 and 5/21 not detected
      • Top OUTS sample is >50% above cut-off.
      • 25-30% of testing devices didn’t detect Methamphetamine at this concentration
      • AS/NZS-4308:2008 Appendix A – ‘Testing device should be able to detect positive at 30% above cut-off’
      Methamphetamine EOC
    • 54. POCT EQA
      • Companies using POCT devices should be involved in a quality assurance programme
      • POCT should be subject to the same requirements as laboratory based screening
      • Working to a legal testing standard therefore should have systems in place to cover this
      • This should include an external quality assurance programme
    • 55. POCT EQA Scheme
    • 56. Some reflections on the past year!
    • 57. Acknowledgements
      • Canterbury Health Labs
      • Toxicology Staff