The Analytical & Interpretive Challenges of Keeping Up with - - PowerPoint PPT Presentation

Barry K Logan PhD, F‐ABFT

Executive Director, Center for Forensic Science Research and Education Chief Scientist, NMS Labs

The Analytical & Interpretive Challenges

• f Keeping Up with Fentanyl Analogs &

Related New Opioids

Michigan Forensic Pathology Conference April 2018

The First Opioid Crisis…

• The British East India

Company promoted sales of

• pium into China.
• By 1825 1 in 4 Chinese had

an opium habit

• China tried to stop British importation of
• pium, which led to 2 opium wars in 1839‐

1842, and 1856‐1860.

The First Opioid Crisis…

• In 1810, the Daoguang Emperor issued a decree:
• Opium has a harm. Opium is a poison,

undermining our good customs and morality. Its use is prohibited by law…

• Recently the purchasers, eaters, and consumers
• f opium have become numerous…
• If we confine our search for opium to the

seaports, we fear the search will not be sufficiently thorough...

• We should also order the general commandant
• f the police and police‐ censors at the five

gates to prohibit opium and to search for it at all gates…

Forensic Toxicology Did Alcohol or Drugs, Cause or Contribute, to this Person’s Death

• r Intoxication?

Challenges for Forensic Toxicologists

• Analysis:
• Knowing what to test for
• Access to the appropriate

technology

• Interpretation:
• The significance of the

presence of novel compounds in cases

• Capacity:
• Keeping up with the workload

Challenges for Forensic Toxicologists

• Analysis:
• Knowing what to test for
• Access to the appropriate

technology

• Interpretation:
• The significance of the

presence of novel compounds in cases

• Capacity:
• Keeping up with the workload

Opioid Evolution

• Morphine and the
• pium alkaloids.
• Semisynthetic opioids

synthesized 1914 ‐ 1920

• Meperidine,

Alphaprodine synthesized in 1930’s

• Anileridine 1940’s

Fentanyl

• Structure of morphine

elucidated in 1925

• SAR inspired by

meperidine, led to Dextromoramide 1956, Piritramide then fentanyl (R4263)

• Fentanyl invented in

1960 by Dr Paul Janssen

Fentanyl Analogs

• More potent or longer

acting analogs Alfentanil, Sufentanil, Carfentanil, synthesized by Paul Janssen in the mid 1970’s

• Analgesic potencies in

mouse models of 0.25 to 500 times that of fentanyl

Other μ Opioid Agonists

• Opioid analgesic drugs developed

at Upjohn in the 1970s.

• Cyclohexyl‐N‐methylbenzamides
• Mixed mu and kappa agonism
• Derived from AH‐7921
• More potent than Morphine, less

potent than fentanyl

Right: U‐47700, U‐49900, methylenedioxy U‐47700, … U‐50488, U‐48753, U‐51754

Explosion in Prescription Opioids

• 1980 Porte and Jick letter

in NEJM

• 1990’s Backlash to the

undertreatment of pain

• Availability of MS Contin

and Oxycontin

• Led to massive increase in

painkiller prescribing

• Peaking in 2011 at 219M

prescriptions

Porter J, Jick H. Addiction rare in patients treated with narcotics. N Engl J Med. 1980 Jan 10;302(2):123.

Explosion in Prescription Opioids

• By 2014, the problem

was apparent

• Reduction in medical
• pioid prescribing led to

users seeking heroin

• 2014 Fentanyls

emergence as a cheaper alternative

• Proliferation and

diversification through 2018

Time, Jun 2015

NFLIS Trend Data ‐ Fentanyl

https://www.deadiversion.usdoj.gov/nflis/2016_annual_rpt.pdf

Our Latest Opioid Crisis

https://www.nytimes.com/interactive/2017/06/05/upshot/opioid-epidemic-drug-overdose-deaths-are-rising-faster-than- ever.html?mcubz=0&_r=0

• New York Times, June 5th 2017
• Drug overdose deaths in 2016 most likely

exceeded 59,000, the largest annual jump ever recorded in the United States.

• Although the data is preliminary, the

Times’s best estimate is that deaths rose 19 percent over the 52,404 recorded in

• 2015. And all evidence suggests the

problem has continued to worsen in 2017.

Our Latest Opioid Crisis

https://www.nytimes.com/interactive/2017/09/02/upshot/fentanyl-drug-overdose-deaths.html

• New York Times, Sept 2nd 2017
• Fentanyl Deaths in 2016: Up 540% in Three Years
• Drug overdoses killed roughly 64,000 people in

the United States in 2016.

• Drug deaths involving fentanyl more than

doubled from 2015 to 2016

• Synthetic opioids

— primarily fentanyl and its analogues — continue to push the death count higher.

Fentanyl Analogs

Phenylethyl piperidine Phenyl/anilinyl Propanamide

• ‐Fluorofentanyl
• p‐Fluorofentanyl
• …
• Carfentanil
• Alfentanil
• Sufentanil
• Remifentanil
• 3‐methylfentanyl
• α‐methylfentanyl
• β‐hydroxyfentanyl
• β‐hydroxy/Thiofentanyl
• Ethylfuranylfentanyl
• Acetylfentanyl
• (Fentanyl)
• F/Butyrylfentanyl
• F/Isobutyrylfentanyl
• Valerylfentanyl
• Tetrahydrofuranylfentanyl
• 2/3‐Furanylfentanyl
• Acrylfentanyl
• Crotonylfentanyl
• Methoxyacetylfentanyl
• Cyclopropylfentanyl
• Tetramethylcyclopropylfentanyl
• Cyclopentylfentanyl
• Cyclohexylfentanyl
• …

A B C D E

TACTICS

• Limited real time data on seized drug trends.
• No national repository for either morbidity or

mortality data consolidation (toxicology or medical examiner).

• No NPS Testing in ER admissions.
• No standardized testing menu between forensic

toxicology laboratories.

• Significant inter‐laboratory gaps in scope for

fentanyl analogs.

• Highly variable turnaround times on fentanyl

reporting.

Challenges – Knowing What to Test For

TACTICS

• Seized drug casework
• Toxicological Casework
• Government Data/NFLIS
• Electronic Databases and

Subscriptions

• Peer review literature monitoring
• Conference proceedings
• Collaborations with vendors
• International conferences and

networking

• Drug user forums, chat rooms and

events

• Metabolomics Lab work

Knowing What to Test For

Opioids Testing Scope 2018

Opiates Morphine, codeine Semi-synthetics Heroin, oxycodone, oxymorphone, hydrocodone, hydromorphone Synthetics (meperidine, propoxyphene) methadone Fentanyl derivatives and precursors Fentanyl, Remifentanil, Sufentanil, Carfentanil, Alfentanil, Lofentanil, Ocfentanil, Acetylfentanyl, F/Butyrylfentanyl, F/Isobutyrylfentanyl, Valerylfentanyl, Tetrahydrofuranylfentanyl, 2/3-Furanylfentanyl, Acrylfentanyl, Crotonylfentanyl, Methoxyacetylfentanyl, Cyclopropylfentanyl, Tetramethylcyclopropylfentanyl, Cyclopentylfentanyl, Cyclohexylfentanyl, 3-methylfentanyl, α-methylfentanyl, β- hydroxyfentanyl, β-hydroxy/Thiofentanyl, Ethylfuranylfentanyl, Fluorofentanyl, 4-ANPP, Benzylfentanyl… Cyclohexyl-N- methylbenzamides U-47700, U-49900, U-48800… Arylcyclohexylamines Tramadol, Tapentadol, Bromadol…

TACTICS

Knowing What to Test For

Challenges for Forensic Toxicologists

• Analysis:
• Knowing what to test for
• Access to the appropriate

technology

• Interpretation:
• The significance of the

presence of novel compounds in cases

• Capacity:
• Keeping up with the workload

Analytical Approach

Sample Received Review History Blood Alcohol Immunoassay Screening Chromatography MS Screening Confirmatory/ Quantitative Testing

+ + +

Report

• Special testing

Interpret

Cross‐reactivity: Fentanyl Analogs

Analyte % Cross Reactivity Acetylfentanyl 111% Fentanyl 100% Butyrylfentanyl 88% Furanylfentanyl 74% p‐Fluorofentanyl 61% +trans‐3‐methylfentanyl 50% α‐methylfentanyl 19% +cis‐3‐methylfentanyl 3% Alfentanil <1% 4‐ANPP (precursor/met.) <1%

Phenylethyl piperidine Propanamide

Analytical Challenges

 Fentanyl/Opioid Immunoassay Limitations

Limitation Outcome  Immunoassays for opioids do not cross‐react with fentanyl, its analogs or any emerging opioids. False Negative Screens  Immunoassays for fentanyl do not cross‐ react with all fentanyl analogs or any emerging

• pioids.

Some False Negative Screens  Confirmatory LCMSMS assays may not include most current fentanyl analogs. Unconfirmable Positive Screens

Analytical Approach

Sample Received Review History Blood Alcohol Immunoassay Screening Chromatography MS Screening Confirmatory/ Quantitative Testing

+ + +

Report

• Special testing

Interpret

Technology ‐ The Past

 Gas Chromatography/Mass Spectrometry  Non‐Targeted: Flags unknowns, but limited tools for their identification.  Forensically significant concentrations of NPS drugs are often too low to detect by traditional GCMS screening methods.  Out of date libraries and databases do not include the most current NPS drugs.  The chemistries of many NPS compounds are not amenable to GCMS.

Technology ‐ The Present

 Targeted analysis assumes we know what we are looking for.  Metabolites of most novel compounds are not known.  Sensitivity is appropriate for typical NPS drug categories.  Diversity of chemical structures limits scope in any given analysis.  Liquid Chromatography/Tandem Mass Spectrometry

Technology ‐ The Near Future

 Non‐targeted data acquisition of accurate mass, retention time, and fragmentation  Targeted reporting but with data‐mining capabilities.  Full spectrum data acquisition.  QTOF adds structural elucidation tools.  Retrospective analysis of data files allows for pharmacoepidemiology.  Liquid Chromatography/TOF/QTOF

Q1 Q1 CID CID TO TOF Analyzer Analyzer

The Future…

 Non‐Targeted acquisition of all masses/fragments throughout the entire run.  In real time, targeted querying of data for currently known drugs and metabolites for which we have a standard (time and mass).  Retrospectively query the archived data for newly emerging compounds to see backwards in time…  Analytical Time Travel to see backwards in time…

TACTICS

• Turnaround time for Commercial Standards
• Sometimes 3 to 6 months following identification
• Or never…
• Custom Synthesis
• Expensive
• 1‐2 months, not commercial scale
• Importation
• Delays of 2 to 4 months
• Lack of Resources Internationally is a Problem for the US

Challenges ‐ Analytical Standards and ISTDs

Challenges for Forensic Toxicologists

• Analysis:
• Knowing what to test for
• Access to the appropriate

technology

• Interpretation:
• The significance of the

presence of novel compounds in cases

• Capacity:
• Keeping up with the workload

 With every new substance there is a lag in collecting reference range data to aid in interpretation  Over time, collection and publication of reference range data assist the profession  The investment of resources in developing quantitative ranges for comparative purposes is worthwhile  Online Collaborative Databases are a solution

Challenges – Available Interpretive Data

Challenges – Available Interpretive Data

• Estimates of Analgesic Potency

Compound Analgesic Activity ED50 (mg/kg) Analgesic potency relative to morphine Source

Morphine 13.9 1 Ujvary, 2017 Isobutyryl fentanyl .048-0.261 1.3-6.9 Higashikawa et al, 2008 Butyryl Fentanyl 0.047-0.220 7 Higashikawa et al, 2008 Furanyl Fentanyl 0.02 7 Huang, 1986 Acetyl Fentanyl 0.021 16 Higashikawa et al, 2008 Para-fluoro fentanyl 0.021 16 Higashikawa et al, 2008 Alpha methyl fentanyl 0.0058 56.9 Higashikawa et al, 2008 Alfentanil 0.044 75 Vardanyn.2015 Acryl fentanyl 0.082 170 Ujvary, 2017 Remifentanil 0.73 220 Wax, 2003 Fentanyl 0.062 224 Ujvary, 2017 Sufentanil 0.00071 4,520 Van Beyer1976 (+) cis 3-methyl fentanyl 0.00058 5,530 Van Bever WF 1976 Carfentanil 0.00032 10,030 Van Beyer,1976

Recommended Methods for the Identification and Analysis of Fentanyl and its Analogues in Biological Specimens Laboratory and Scientific Section UNITED NATIONS OFFICE ON DRUGS AND CRIME Vienna Oct 2017 In Press

Interpretation

• Quantitative Analysis?

Wh Why Bo y Bother? ther? Yes! es!

Lack of resources for method development and validation. More information is always better. Unknown pharmacology. You have to start somewhere. No reference range data exist. More potent compounds may warrant different approaches to scheduling. Lack of internal standards make quantitation difficult. Relative amounts of drug may address issues in “Burrage” cases. Trend towards more specificity in death certification.

Analyte Median Concentration (ng/mL) Average Concentration (ng/mL) Range (ng/mL) N para-Fluorofentanyl 0.17 0.23 (±0.19) 0.1-1 37 Carfentanil 0.33 0.81 (±4.6) 0.1-120 827 Valerylfentanyl 0.44 0.44 N/A 1 Acrylfentanyl 0.69 1.8 (±3.2) 0.1-29.0 316 U-49900* 1.5 1.5 N/A 1 Furanylfentanyl 1.9 7.4 (±30.8) 0.1-710 1311 Butyrylfentanyl/isobutyryl fentanyl 2.2 25.2 (±66.5) 0.1-760 175

• rtho-Fluorofentanyl

2.4 2.4 N/A 1 para-Fluorobutyrylfentanyl/FIBF 3.2 17.6 (±45.9) 0.1-760 839 Cyclopropylfentanyl 7.5 11.5 (±19.5) 0.075-230 178 Methoxyacetylfentanyl 6.7 32.2 (±56.4) 0.06-300 86 U-47700 9.9 134 (±374) 0.2-3800 651 4-Methoxybutyrylfentanyl 79 79 N/A 1 Tetrahydrofuranylfentanyl* 339 339 N/A 1

Blood Drug Concentrations

TACTICS

• Legal or Illegal?
• State patchwork scheduling is inconsistent and

confusing.

• Federal Scheduling is based on emergency,

temporary, then permanent scheduling.

• Even Temporary Scheduling is time consuming.
• Analog Laws
• Nobody agrees on definitions.
• Litigation is time consuming and has to be re‐

litigated in every case

Challenges – Scheduling

Fentanyl Analogue Scheduling

• Core Structure Scheduling
• In February 2018, the DEA implemented core structure

scheduling for the fentanyl class.

• Scheduling based on the key elements of fentanyl

structure, whether substituted to whatever extent

 

Phenylethyl piperidine Anilinyl/Phenyl Propanamide

https://www.deadiversion.usdoj.gov/fed_regs/rules/2018/fr0206_4.htm

Challenges for Forensic Toxicologists

• Analysis:
• Knowing what to test for
• Access to the appropriate

technology

• Interpretation:
• The significance of the

presence of novel compounds in cases

• Capacity:
• Keeping up with the workload

The Toll

Analyte Number Reported Fentanyl 19851 Acetylfentanyl 2520 Furanylfentanyl 1620 para‐Fluorobutyryl/Fluoroisobutyryl Fentanyl 899 Carfentanil 813 Cyclopropylfentanyl 486 Acrylfentanyl 322 Methoxyacetylfentanyl 247 Butyrylfentanyl/Isobutyryl Fe 172 3‐methylfentanyl 97 para‐Fluorofentanyl 39 Fluorofentanyl 5 Tetrahydrofuranfentanyl 4 Valerylfentanyl 3

• rtho‐Fluorofentanyl

3 4‐Methoxybutyrylfentanyl 3 * Total cases with at least one positive result for fentanyl and/or an analog = 23,576

Fentanyl and Analogs in blood reported by NMS Labs (Jan 2016 ‐ Jan 2018)*

The Future of the Opioid Crisis?

• The Crisis is Here to Stay:
• We have created a system of immense

demand.

• The market has responded with

dramatically increased supply.

• The more potent drugs are easier to

clandestinely import into the United States.

• Role of Naloxone on the course of the

epidemic is unclear.

• Deaths and autopsy rates will increase and

laboratories will continue to be challenged to keep up.

Acknowledgements

• NMS Labs
• Donna Papsun
• Sherri Kacinko
• Dan Isenschmid
• Brian Holsey
• Joseph Homan
• Stephanie Kumor
• CFSRE
• Mandi Mohr
• Melissa Friscia
• Alex Krotulski

barry.logan@nmslabs.com www.nmslabs.com www.forensicscienceeducation.org