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


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Grant Moore
Section Head Toxicology
Canterbury Health Labs,
PO Box 151, Christchurch 8014

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

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