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Robert A. Middleberg, Ph.D., DABFT, DABCC(TC) NMS Labs Willow Grove, PA

Specialty Toxicology Testing:  The Interface with the Clinician and the

Response to Emerging Drugs of Abuse

The laboratory response to the brave new world

• I have no known conflicts of interest • Any mention of a given laboratory is to

reference only • Many pictures are from google images

Our chicken and egg issue

• Laboratories need to develop tests to stay relevant

• Clinicians need tests that are relevant, so …

Should a laboratory develop tests before clinicians need them, or should clinicians tell labs what is needed?

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Obvious answer

Both are needed!

Are all toxicology laboratories created equal?

• The basic stuff – Hospitals – Some reference labs

• The basic stuff and more – Specialized reference labs

Are all toxicology laboratories created equal?

Hospital/Basic Specialized

Adavantages TAT STAT Automated Good enough Cost

Number of analytes Agility Instrumentation Specificity Sensitivity Staff/Skill set Flexibility

Disadvantages Number of analytes Not agile Instrumentation Specificity Sensitivity Staff/Skill set Inflexible

TAT STAT Not automated Detailed Cost

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The Problem • Laboratories proactively develop tests based

on perceived need – Clinical input – Trends – Research – FDA approvals – Etc.

• In a toxicology laboratory, a typical test costs on average ~$40-60K to develop and takes ~3 months

How is a new test developed?

• Specification – Physico-chemical information – General relevancy – PK data – Proper specimen types – Critical concentrations – Reference comments – Analytical considerations – References – http://toxwiki.wikispaces.com/duloxetine

How is a new test developed?

• R&D – Obtaining reference materials – Analytical egg-heads

• Method Validation – Parameters include,

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VALIDATION STUDY NAME Required/ Optional

Chromatography/ Spectrophotometry/

Immunoassay Exogenous/ Endogenous

A. Specimen(s) of Choice Required All Both B. Primary Standard Validation Required All Both C. Standard Curve / Calibration Required All Both D. Linearity Study AMR Required All Both E. Limit of Detection (LOD) Required All Both F. Lower Limit of Quantitation (LLOQ) Required Chromatography Both G. Lower Limit of Quantitation (LLOQ) Required Spectrophotometry Both H. Precision – Within Run Required All Both I. Precision – Between Run Required All Both J. Precision - Total Required All Both K. Accuracy – Bias Estimate Required All Both L. Accuracy – Method Correlation Required All Both M. Interfering Substances Required All Exogenous N. Interfering Substances Required All Endogenous O. Recovery Optional All Both P. Stability Required All Both Q. Maximum Allowable Dilution Required All Both R. Dilution of Samples Required All Both S. Matrix Matching Optional All Both T. Matrix Effect Required All Both U. Carryover Required All Both V. Standard Addition Optional All Both W. Robustness and Ruggedness Required All Both X. Reference Intervals Required All Endogenous Y. Clinical Sensitivity and Specificity Optional All Endogenous Z. Hook Effect Required Immunoassay Both AA. Cross-Reactivity Required Immunoassay Both BB. Uncertainty Required All Both

So, what can go wrong?

• The build it and they will come risk

So, what can go wrong?

• Great intentions, but …

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So, what can go wrong?

• I shoulda’ thought of that

So, what can go wrong?

• The regulators – Accrediting bodies – CLIA vs. FDA and LDTs

A laboratory-based toxicologist’s view of specialized tox testing in support of Medical Toxicology

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• Will it help with patient treatment – Maybe yes, maybe no – Will results be able to help the Dx or Tx?

A laboratory-based toxicologist’s view of specialized tox testing in support of Medical Toxicology

Clinical Correlate

• Unexplained hypoglycemia – Pathology vs. Toxicology – Toxicological agents of interest

• Insulin • Oral hypoglycemics • Natural products

– Woman comes back from Jamaica – Develops a severe, life-threatening hypoglycemia – No known pathology

Source: Google images

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Source: Google images

• Will it help the patient – Maybe yes, maybe no – Will results be able to help the Dx or Tx? – Risk (Cost) vs. Benefit

• Reimbursement/ACOs/Hospital lab-based pressures

A laboratory-based toxicologist’s view of specialized tox testing in support of Medical Toxicology

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• Will it help the patient – Maybe yes, maybe no – Will results be able to help the Dx or Tx? – Risk (Cost) vs. Benefit

• Reimbursement/ACOs/Hospital lab-based pressures

• Are there medicolegal implications • Trends • “Gee, I wish I had done that!” • “It’s not what we do!”

• Academic interest

A laboratory-based toxicologist’s view of specialized tox testing in support of Medical Toxicology

So, what should a clinician do?

• Consult with the laboratory toxicologist –  It should be a partnership not “who knows

more” or “who has the right …” – Make sure the test will provide the information

you need • The value of case history

– Make it clear the expectations, e.g., TAT – Understand the sample parameters

• Evaluate the cost vs. benefit • Be prepared for the worst

The evolution of specialty toxicology testing

• What allows us to do what we do? • What is the expectation today of such tests? • Where are we going?

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The Analytical Part - 1840

Source: Google Images

The progression of analytical tools

• Color/Precipitation – Marsh/Gutzeit for arsenic

• Plating – the Reinsch Test (1842) • Spectrophotometry -1940 (Beckman) • Chromatography

– GC – 1952 (Martin and James) • Spectrometry (mass spec) - ~1900 (Thompson) •  Immunoassays – 1960 (Yalow)

• Hyphenated Techniques – GC-MS (1968) – LC-MS (1977) –  ICP-MS (1975) – LC-MS/MS, LC-TOF, LC-MSn, GC-MS/MS

• Sample preparation

The progression of analytical tools

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What might the future bring?

Emerging drug trends

Why do we need these tools?

December 12, 2012

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December 23, 2012

November 9, 2012

April 2013

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Designer Drugs, in a nutshell

Synthetic cannabinoids Known on the street as K2 or “fake weed”, these drugs are very real. The newest compounds on the street are chemically different than the first generation compounds. They are potent, impairing and addictive, but their chemistry makes them invisible to older designer drug screens and traditional drug tests.

Bath salts Data becoming available shows “bath salts” are deadly. Of 472 samples recently submitted to one laboratory, 30% confirmed positive for 1 or more analytes in our Expanded Bath Salts & Stimulants Panel. Other next generation compounds, 25I-NBOMe, 25C-NBOMe, 25H-NBOMe, are commonly found in “bath salts” and have been recently linked to deaths in the United States. Although labeled as “not for human consumption,” these substances often come as a powder to be snorted, injected or smoked by users.

K2/Spice

What is K2/Spice? •  Synthetic chemicals that look to

the brain like marijuana. •  Sprayed onto plant material for

smoking. •  Give marijuana-like high •  Sold online and in head-shops •  Legal grey area

Synthetic Cannabinoids 2010 2011 2012

JWH-018 AM-2201 AM-2201 JWH-073 AM-694 AM-694 JWH-019 JWH-018 JWH-018 JWH-250 JWH-019 JWH-019

JWH-073 JWH-073 JWH-081 JWH-081 JWH-122 JWH-122 JWH-200 JWH-200 JWH-210 JWH-210 JWH-250 JWH-250

RCS-4 RCS-4 RCS-8 RCS-8

JWH-203 JWH-022

JWH-018 Cl-analog UR-144 XLR-11

AM-2233 AM-1248 A-796260

NMS Labs Data

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History

•  1960s – Analogues of THC first developed – HU-210, dronabinol, etc.

•  1970s – Pfizer developed CP series of compounds – CP 47,497

•  1990s – John W. Huffman (Clemson University) synthesized a new series of compounds- e.g., naphthoylindoles (JW-015, JW-018, etc); naphthylmethylindoles; naphthoylpyrroles; naphthylmethylindenes; and phenylacetylindoles

•  2000s – Other compounds are synthesized 37

*1988/1990- Cannabinoid receptor identification – CB1 and CB2

•  First appeared in Europe approximately 2004 – The Psyche Deli

•  First appeared in U.S. around 2008

•  Believed to be synthesized in China, India, and other growing economies

•  Originally believed to be a blend of herbal products

•  Considered here to stay

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History

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A: Jan 2014: New Lenox, IL. $2 million mail order synthetic weed operation gets busted by Feds. Hosey, Joseph.“Feds Charge New Lenox Father & Son with Running $2M Synthetic Pot Biz”New Lenox Patch January 31, 2014.

B: Dec 2013: Denver, CO. Synthetic marijuana is linked to the sickness of 221 people during a month long outbreak in Colorado “Synthetic Marijuana has sickened more than 200 people in Colorado”The Huffington PostDecember 13, 2013.

C: Nov 2013: Waverly, NE. 18 year old smokes synthetic cannabis, goes to sleep and never wakes up., Reece “Nebraska teen’s death brings synthetic marijuana battle to forefront.”Daily NebraskanNovember 15, 2013.

D: Jan 2014: Wasilla, AK. Teenager found dead in his room next to a pipe full of Spice, official cause of death is unknown but spice is suspected culprit. Doogan, Sean. “In the wake of a teen’s unexplained death, answers about Spice are hard to come by”Alaska DispatchJanuary 10, 2014.

E: Feb 2014 Dalton, GA. Officials discover new liquid form of synthetic marijuana during a tobacco store raid. Regan, Tom. “Liquid synthetic pot new drug threat”Action News JaxFebruary 7, 2014.

F: Aug 2013: Asheboro, NC. Police confiscate 3.5 pounds of synthetic weed from three stores valued at $12,000. “3.5 Pounds of Synthetic cannabinoids seized from stores in Asheboro.”WFMY News 2 August 9, 2013.

Cannabinoids Adverse Effects

• Major Effects • Cardiovascular

•  Hypertension Tachycardia

•  Gastrointestinal •  Vomiting

•  Neurological •  Agitation •  Confusion •  Hallucinations •  Seizures

•  Death?

•  Other Effects •  Headache •  Muscle •  Numbness •  Slurred speech

Syncope •  Vomiting •  Tremors •  Drowsiness

Texas Poison Center Networks

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Marijuana/K2 Effects

Red  eyes  /  bloodshot  Burning  of  the  eyes    

Xerostomia  (dry  mouth)    Tachycardia  

Changes  in  percep=on/mood    Balance  and  Coordina=on  

Hallucina=ons    Seda=on    

Subjec=ve  thought  disrup=on/loss  of  concentra=on  

Impaired  sense  of  =me  

Self  assessed  impairment    Arrhythmias  

Seizures/Convulsions  Panic  AFacks  

Paranoia  and  Anxiety  Sickness  

Specific laboratory challenges

• Matrix – Blood vs. Urine

• Both are of value –  ED – if under the influence, most likely still detectable in

blood

• Metabolites – what are they?

• Analytical –  Immunoassays – Reference materials – Validation

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Delta-9 THC

Eicosanoid

C5H11

Anandamide

JWH-018 Naphthoylindole

CP-47,497 Cyclohexylphenol

AKB - 48 Adamantylcarboxamide indole

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PB-22 (QUPIC) 5-F-PB-22

The latest and greatest, until …

Bath Salts

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Media Stories—Bath Salts Usage is Growing in Your Backyard

A: Apr 2013: Orlando, FL. 17 year old dies after an overdose on bath salts. “Drug in bath salts caused teen’s overdose death, Dr. G says.” Click Orlando, WKMG Local 6 April 9, 2013.

B: Nov 2013: Anchorage, AK. Matt Scott becomes the first person to die from bath salts in Alaska. Hollander, Zaz. “Alaska case first federal prosecution involving a methylone death” Anchorage Daily NewsNovember 27, 2013.

C: Nov 2013: Springfield, MO. Bath salts user attacks police dog and officer. Herzog, Stephen. “Man on ‘bath salts’ assaulted officer and dog”News Leader November 30, 2013.

D: Dec 2013: Bakersfield, CA. One thousand pounds of bath salts and other synthetic drugs are found in a smoke shop owner’s warehouse along with $2.7 million in cash. Jauregui, Andres.“1000 Pounds of Bath Salts, Other Synthetic Designer Drugs Seized in California“ The Huffington Post 17, 2013.

E: Jan 2014: Luling, LA. A man high on bath salts rapes his ex-girlfriend. Karoliszyn, Henrick.“Luling man convicted of rape, admits to using bath salts during assault” The Times-Picayune 17, 2014.

F: Feb 2014: Sherwood, OR. New LSD-like drug 25I, or N-bomb, caused seizures in Sherwood, deaths around nation. Hammill, Luke. “New LSD-like drug 25I, or N-bomb, caused seizures in Sherwood, deaths around nation” The Oregonian February 3, 2014.

One year ago, DMAA was the culprit with a 33%

positivity rate. Alpha PVP wasn’t even on the charts.

Containing

!  Cathinones !  Phenethylamines (Shulgin/Nichols)

!  2C-X !  NBOMe’s

!  Pyrovalerones !  Pyrrolidophenones

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Synthetic Stimulants Adverse Effects

•  Agitation •  Insomnia •  Mydriasis •  Myoclonus •  Tachycardia •  Hypertension •  Chest pain •  Paranoia •  Delusions/hallucinations •  Excited Delirium

•  Combative behavior •  Hyperthermia •  Rhabdomyolysis •  Kidney Failure •  Seizures •  Death

Scope: Hallucinogens & Stimulants

Drug Count DMAA 66 alpha-PVP 62 MDPV 39 Methylone 32 4-MEC 11 Buphedrone 10 Pentylone 8 MBZP 7 DMA 7 Ethylone 7 Butylone 6 3,4 DMMC 5 2C-I 4 1,4 DBZP 3 25I-NBOMe 3

Screen: Basic Extraction EI-GC-MS or LC-TOF

Confirmation: GCMS LC-TOF LC-MS/MS

Analytical Challenges

• Not as severe as synthetic cannabinoids • Blood vs. Urine

– Metabolites? – Reference materials

• Often appear in screens of basic compounds using GC-MS, LC-MS/MS, LC-TOF, if you know what to look for

• Questionable cross-reactivity with amphetamine-based immunoassays

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Other emerging (emerged) drugs

• Synthetic opioids – Doxylam (AH-7921) – Krokodil (desomorphine) – Acetylfentanyl – ????

Clinicians role with emerging drugs •  Understand the signs and symptoms •  Understand that these substances can kill •  Ask questions of the patient or friends

–  Gather as much information as possible •  Collect specimens (blood/urine) – Why?

–  It’s the only way to identify trends –  It’s the only way to identify new compounds –  It’s the only way to accumulate PK data

•  Consider being part of studies •  Speak to the lab, be part of the solution …