toxicology of designer benzodiazepines and opioids...

WORKSHOP #5

Toxicology of Designer Benzodiazepines and Opioids

Sponsored by the SOFT

Designer Drugs Committee

SOFT 2016

Dallas, TX

Monday, October 17, 2016

Chairs:

Robert Kronstrand and Sumandeep Rana

Workshop #5 Toxicology of Designer Benzodiazepines and Opioids

October 17, 2016

Agenda:

1:30 pm - 1:40 pm Good Afternoon and Welcome Sumandeep Rana

1:40 pm - 2:20 pm A Brief Epidemiology of Designer Benzodiazepines and Opioids Robert Kronstrand

2:20 pm - 3:30 pm Metabolism Studies of Designer Benzodiazepines and Opioids Svante Vikingsson and Ariane Wohlfarth

3:30 pm - 4:00 pm Afternoon Break

4:00 pm - 4:30 pm Detecting Designer Opioids and Benzodiazepines: Understanding the Options Sherri Kacinko and Markus Roman

4:30 pm - 5:15 pm Clinical and Post Mortem Findings in Cases Related To Designer Benzodiazepines And Opioids Dani Mata

5:15 pm - 5:30 pm Panel Discussion

Robert Kronstrand BSc, PhD Robert Kronstrand is currently an associate professor in forensic toxicology at Linköping University and chief toxicologist at the National Board of Forensic Medicine in Linköping, Sweden. He received his BSc degree in analytical chemistry in 1989, and his PhD in human toxicology in 2001, both from the University of Linköping. In 1990, he joined the National Board of Forensic Medicine, Department of Forensic Toxicology located in Linköping, Sweden. Dr Kronstrand has more than 25 years of experience in the fields of postmortem toxicology, DUID, DFSA and drug testing in various matrices. His research has covered a wide range of topics over the years, including opiate toxicity and analytical toxicology with focus on the incorporation of drugs into hair. Over the last 10 years, the toxicology of new psychoactive substances has been another focus of his research. The combination of research and case work has enabled him to contribute to the forensic toxicology community with more than 60 scientific papers, 4 book chapters, and numerous congress presentations, workshops and seminars. Dr Kronstrand is active in several scientific and professional organisations. Among other things, he currently serves as Treasurer of the International Association of Forensic Toxicologists (TIAFT), Vice President for the Society of Hair Testing (SoHT), and President of the Nordic Association of Forensic Toxicologists (NAFT). Dr Kronstrand is a member of Society of Forensic Toxicologists and a fellow of the American Academy of Forensic Sciences (AAFS). Dr Kronstrand has been recognized with several awards including the TIAFT Achievement Award in 2009 and the AAFS Ray Abernethy Award in 2016.

1

A brief epidemiology of designer benzodiazepines and opioids

Robert KronstrandNational Board of Forensic Medicine

Linköping, Sweden

SOFT WS 5 Dallas 2016:Toxicology of Designer Benzodiazepines and Opioids

2

Disclaimer

• The opinions expressed in thispresentation do not necessarilyreflect the views of the NationalBoard of Forensic Medicine

• Neither I nor any member of myimmediate family has a financialrelationship with a company discussed

UNODC

3

Designer benzodiazepines

5

Model structure: 3 or 4 rings

FlubromazepamDiazepam

N

N

Cl

ONH

NBr

O

F

Midazolam

N

N

Cl

N

F

Flubromazolam

N

NBr

F

N

NCH3

FlubromazolamA new life-threatening designer benzodiazepine.Łukasik-Głębocka M et al.Clin Toxicol (Phila). 2016;54(1):66-8.

Detection and identification of the designerbenzodiazepine flubromazepam andpreliminary data on its metabolismand pharmacokinetics.Moosmann B et alJ Mass Spectrom. 2013 Nov;48(11):1150-9.

4 mg dose

3 mg dose

Benzo EU-timeline

7

2007 2016

Phenazepam

Etizolam

Pyrazolam

Flubromazolam

Diclazepam

Nifoxipam

Deschloretizolam

Meclonazepam

Flubromazolam

Clonazolam

Adinazolam

Metizolam

Nitrazolam

Cloniprazepam

3-OH-phena..

Fonazepam

2011 2012 2013 20152014

Nimetazepam

The beginning

8

Benzo timeline

9

2007 2016

Phenazepam

Etizolam

Pyrazolam

Flubromazolam

Diclazepam

Nifoxipam

Deschloretizolam

Meclonazepam

Flubromazolam

Clonazolam

Adinazolam

Metizolam

Nitrazolam

Cloniprazepam

3-OH-phena..

Fonazepam

2011 2012 2013 20152014

Nimetazepam

BZs in autopsy cases

10

2012 2013 2014 2015 2016Etizolam 6 2 1Pyrazolam 10 2Flubromazepam 5 14 3Diclazepam 2 2Flubromazolam 4 34Meclonazepam 43-OH-phenazepam 2

BZs in DUID cases

• Increasing number of false positives– CEDIA screening positive in urine– Confirmed negative in blood

• 87 samples during 3 months– Investigated by TOF

11

BZs in DUID cases

Substance Positives

Flubromazolam 21

Clonazolam 4

Meclonazepam 2

Flubromazepam 1

Bromazepam 2

Lorazepam 16

12

34 %

From diclazepam?

Background overview

13

Designer opioids

14

Model structures

15

NCl

Cl

O

N

Morphine U-47700

N

NN N

O

MT-45 Fentanyl

Opioid timeline

16

2009

ODT

(Iso)butyryl fentanylalpha-Methylfentanylbutanamide analog

(Iso)butyryl-F-fentanylN-benzyl analog

AH-7921

MT-45

Carfentanil

Ocfentanil

Fentanyl butanamide

Acetylfentanyl

4-F-butyrfentanyl

Butorphanol

U-47700

despropionyl-2-F-fentanyl

4-MeO-butyrfentanyl

2012 2013 2014 2015 2016

Furanylfentanyl

Valerylfentanyl

Acrylfentanyl

The beginning

17

OPs in autopsy cases

18

2009 2010 2011 2012 2013 2014 2015 2016

O-dm-Tramadol+mitragynin 2 9 1

AH-7921 9

MT-45 4 24 1Acetylfentanyl 32Butyrfentanyl 14F-butyrfentanyl 14-metoxi-butyrfentanyl 2

Acrylfentanyl 36

Furanylfentanyl 74-F-butyrfentanyl 14-MeO-butyrfentanyl 2

4-Cl-isobutyrfentanyl 2

U47700+mitragynin 1

Acetylfentanyl

19

Scheduled

Not only fentanyl analogs

20

A news flash from Sweden

21

N-desmethyl-Flunitrazepam”fonazepam”

Flunitrazolam

Cyclopentylfentanyl Tetrahydrofuran fentanyl

4F-isobutyrfentanyl 4Cl-isobutyrfentanyl

Svante Vikingsson MSc, PhD Svante Vikingsson is currently an assistant professor at Linköping University. He received his MSc in analytical chemistry in 2007 and his PhD in Medical Science in 2012, both from Linköping University. Specializing in the identification and chromatographic analysis of metabolites of drugs and drugs of abuse, he has authored 20 research articles. He is a member of the International Association of Forensic Toxicologists (TIAFT) as well as the Nordic Association of Forensic Toxicologists (NAFT).

Ariane Wohlfarth, PhD Ariane Wohlfarth is a Guest Researcher at the National Board of Forensic Medicine and an Adjunct Lecturer at Linköping University in Linköping, Sweden. After graduating in pharmaceutical sciences from Freie Universität Berlin, she received her first training in forensic toxicology and analytical chemistry during her PhD years at the Institute of Forensic Medicine in Freiburg, Germany, where she earned a PhD in 2012 for a thesis on natural cannabis. From 2012 to 2015 she worked as a Post-doctoral Fellow at the National Institute on Drug Abuse (NIDA) in Baltimore, MD, focusing on metabolite identification studies for new psychoactive substances, mainly synthetic cannabinoids and synthetic cathinones. During that time she also became interested in in silico metabolite prediction and software-assisted metabolite identification techniques. Since July 2015 she has been continuing this research in Sweden expanding it to different substance classes including designer opioids and benzodiazepines. Her research interests cover different aspects of forensic toxicology and she has published more than 35 research articles in peer-reviewed scientific journals. She is a member in the German Gesellschaft für Forensische und Toxikologische Chemie (GTFCh) and The International Association of Forensic Toxicologists (TIAFT) and also serves in the TIAFT Young Scientists Committee.

Metabolism studies for synthetic opioids & benzodiazepines

Ariane Wohlfarth & Svante Vikingsson

National Board of Forensic Medicine, Linköping, Sweden

Division of Drug Research, Linköping University, Linköping, Sweden

Disclaimer

• The opinions expressed in thispresentation do not necessarily reflect theviews of the National Board of ForensicMedicine

• Neither I nor any member of my immediatefamily has a financial relationship with acompany discussed

Content

Metabolism of designer opioidsFentanyl analogsAH-7921 & U47,700MT-45

Metabolism of designer benzodiazepines3-ring benzodiazepines4-ring benzodiazepines

1st part

2nd part

What type of metabolism studies have been performed?

In vitro studies In vivo studies

HLM HEP ANIMAL CASESHUMAN

EXP

1st partSynthetic opioids

Synthetic opioids for which metabolism data is available

Some fentanyl analogs:AcetylfentanylAcrylfentanyl

FuranylfentanylButyrylfentanyl

Carfentanilß-OH-thiofentanyl

4F-isobutyrylfentanyl

AH-7921U-47700

MT-45

Fentanyl analogs w/ metabolism data# Compound HLM HEP ANIMAL CASES Reference

1 Acetylfentanyl X 30 NBFM data, unpublished

>1 Melent'ev, 2014

1 Cunningham, 2016

X rat Patton, 2014

2 Acrylfentanyl X 7 NBFM data, unpublished

3 Butyrylfentanyl 1 Staeheli, 2016

X NBFM data, unpublished

4 Carfentanil 2 Riches, 2012

X Feasel, 2016

5 Furanylfentanyl X 5 NBFM data, unpublished

6 beta-hydroxy-thiofentanyl

X NBFM data, unpublished

7 4F-isobutyryl-fentanyl

X 2 NBFM data, unpublished

NBFM = National Board of Forensic Medicine, Linköping

Other synthetic opioids w/ metabolism data


# Compound HLM HEP ANIMAL CASES Reference

8 AH-7921 X 1 Wohlfarth, 2015

9 Kronstrand, 2014

1 Vorce, 2014

2 Soh & Elliott, 2014

9 U-47700 4 NBFM data, unpublished

1 Elliott, 2016

10 MT-45 X X NBFM data, unpublished

Note: W‐15 and W‐18, previously described as opioids, seem to have no activity at the opioid receptors – not included!Huang et al. (2016): Pharmacology of W‐18 and W‐15, http://dx.doi.org/10.1101/065623

N N

O

Structures of fentanyl & its analogs

Fentanyl

Acetyl-fentanyl

Acryl-fentanyl

N

O

N

O

Carfentanil4F-isobutyryl-

fentanylFuranyl-fentanyl

Butyryl-fentanyl

N

O

O NN

O

O

O

N

O

N

O

F

= the prototype

beta-OH-thiofentanyl

S OH

N N

O

How is the prototype metabolized?

FentanylNH N

O

N NH

N N

O

OH

NH N

O

OH

N N

O

OH

OH

OH

Norfentanyl = main metabolite

OH-norfentanyl

OH-fentanyl

One or several hydroxylations

Despropionyl-fentanyl

Minor metabolites

N-dealkylationCYP 3A4

Hydroxylation

Amide hydrolysis

Acetylfentanyl metabolism

N N

O

NH N

O

N N

O

OH

N N

O

OH

O

Hydroxylation at phenethyl moiety Di-Hydroxylation &

methylationN-dealkylation

OH+++

NOR+++

OH & MeO+++

2nd isomer: +

AmideHYDR

+

2xOH+

OH+

HLM

HEP

ANIMAL

CASES

N N

O

OH

N N

O

OH

O

Acrylfentanyl metabolism

Hydroxylation at phenethyl moiety

Di-Hydroxylation & methylation

N-dealkylation

OH+++

NOR+++OH & MeO

+++2nd isomer: +

2xOH+++

N N

O

NH N

O

AmideHYDR

+

2xOH+

OH+

HEP

CASES

Excursus: ‘The more, the merrier’7 authentic cases for acrylfentanyl, hydrolyzed --- Major metabolite?

0% 50% 100% 150% 200% 250%

1

2

3

4

5

6

7

0% 50% 100% 150% 200% 250%

1

2

3

4

5

6

7

0% 50% 100% 150% 200% 250%

1

2

3

4

5

6

7

0% 50% 100% 150% 200% 250%

1

2

3

4

5

6

7

hydroxylated metabolite

di-hydroxylated metabolite

nor metabolite hydroxylated & methoxylated

metabolite

N N

O

Butyrylfentanyl metabolism

Hydroxylation at aliphatic side chain

OH+++

Carboxy+++

AmideHYDR

+

NOR+

NH N

O

N N

O

OH

N N

O

OH

OH

O

N NH

N-dealkylation

HEP

CASES

NN

OO

OO

NN

OO

OH

Carfentanil metabolism

Hydroxylation at piperidine ring

OH+++

NOR+++

AmideHYDR

-

Ester HYDR

+

N-dealkylation

NN

OO

O

NHN

OO

O

NN

OO

O

OHCarbonyl++

HEP

CASES

Beta-hydroxythiofentanyl metabolism

NOR+++

AmideHYDR

-

OHminimal

N-dealkylation

Dehydro++

HEP

Piperidine ring opening & reduction

+

N N

OS OH

NH N

O

N N

OS ON N

OS OH

NH N

OS OH

OH

4F-isobutyrylfentanyl metabolismHEP

CASES

Task #1

N N

O

O

This is furanylfentanyl, a synthetic opioid recently emerged on the market. Based on the results of metabolism studies for known analogs, which major metabolites would you suggest?

N NH

N N

O

O

Furanylfentanyl metabolism

Deamination &Hydrogenation

++Amide HYDR

++

Dihydrodiol++

NOR-

OH-

N O

N N

O

O

OH

OH

Amide HYDR & OH

++

Amide HYDR & OH & Sulf

++

HEP

CASES

SummaryFen Ace Acr Fur But Car ß-OH-

thioFibF

NOR +++ +++ +++ - + +++ +++

OH (on phenethyl)

+ +++ +++ - +

Di-OH + + +++ -

OH & methoxy

+++ +++ -

Amide hydrolysis

+ + + ++ & combinations

+

Other abundant metabolites

dihydrodiol,deamino/

hydro

OH at side chain,

carboxy

OH at piperidine, carbonyl

Dehydro++

Parent? Yes Yes Yes Yes Yes Yes Hep only

Hydrolysis useful?

Yes Yes Yes Yes Yes Yes Hep only

prototype

Two close relatives, AH-7921 & U-47700: Similar metabolism too?

NHCl

Cl

O

N

NCl

Cl

O

N

AH-7921C16H22Cl2N2O

U-47700C16H22Cl2N2O

AH-7921 metabolism

desmethyl+++

di-desmethyl+++

OH+

desmethyl & OH++

di-desmethyl & N-hydroxylation

+++

HEP

CASES

NHCl

Cl

O

N

NHCl

Cl

O

NH

NHCl

Cl

O

NH2

NHCl

Cl

O

N

OH

NHCl

Cl

O

NH

OH

NHCl

Cl

O

NHOH

U-47700 metabolism

desmethyl+++

di-desmethyl+++

OH+

desmethyl& OH

++

di-desmethyl & N-hydroxylation

+++

HEP

CASES

NCl

Cl

O

N

NCl

Cl

O

NH

NHCl

Cl

O

NHNH

Cl

Cl

O

NOH

NCl

Cl

O

NH

OH

NHCl

Cl

O

NH

OH

di-desmethyl& OH

++

MT-45

N

N

HLM

HEP

NH

N

N+

N

N

N

OH

hydroxy+++

many isomers

Iminium ion

N-dealkylatedMetabolite

++

2nd partDesigner benzodiazepines

3-ring designer benzos w/ metabolism data


# Compound HLM HEP ANIMAL CASES HumanEXP

Reference

1 Phenazepam rat X Ekonomov, 1979

dog, rat, cat X Zherdev, 1982

Andronati, 1982 (cited by Kopanitsa, 2001)

2 Flubromazepam X 4 mg Moosmann, 2013

3 Diclazepam 1 mg Moosmann, 2014

4 Meclonazepam X Huppertz, 2015

X X mouse 2 NBFM data, TIAFT presentation 2016

X Meyer, 2016

5 Desmethyl-flunitrazepam

3 NBFM data, unpublished

6 Nifoxipam X Meyer, 2016

4-ring designer benzos w/ metabolism data


# Compound HLM HEP ANIMAL CASES HumanEXP

Reference

7 Pyrazolam X 1 mg orally

Moosmann, 2013

8 Flubromazolam X Huppertz, 2015

0.5 mg orally

Huppertz, TIAFT presentation 2015

X X mouse 6 Wohlfarth, 2016, submitted

9 Clonazolam X Huppertz, 2015

X Meyer, 2016

10 Deschloro-etizolam

X Huppertz, 2015

Structures of 3-ring designer benzos

Flubromazepam

Diclazepam Nifoxipam Desmethyl-flunitrazepam

Meclonazepam

Diazepam Flunitrazepam

N

N

Cl

O

N

N

O2N

O

F

N

NCl

Cl

OCH3

NH

NBr

O

F

NH

NN+

Cl

O

CH3

O-

O

NH

N

O

OH

F

N+O

-

O

N

NH O

F

O2N

Phenazepam

N

NH O

Cl

Br

Structures of 4-ring designer benzos

PyrazolamFlubromazolam

Deschloroetizolam

Midazolam

Clonazolam

N

N

Cl

N

F

N

NBr

F

N

NCH3

N

N

N

N

NCH3

Br

N

N

N

N

N+

CH3

ClO-

O

N

N

N

NCH3

S

CH3

How are the prototypes metabolized?

N

N

Cl

O

1

2

N

NH

Cl

O

N

NH

Cl

O

OH

Glucuronides= inactive & major urinary products

Oxazepam

N

N

O2N

O

F

N

N

NH2

O

F

N

N

NH

O

F

O

Diazepam

Flunitrazepam

N-dealkylationHydroxylation

Reduction Acetylation

Basically, three things can happen:

7-amino metabolite acetamidometabolite, inactive

3

N

N

Cl

N

F

Midazolam

N

N

Cl

N

F

OH

OH

Glucuronides= inactive & major urinary products

4

α

N

N

Cl

N

F

OH

HydroxylationHydroxylation

How are the prototypes metabolized? – cont’d

3-ring benzodiazepines

O

NH2

Cl

Br

Phenazepam metabolism

Hydroxylation

3-OH++

(active)OH & MeO

+ benzophenone+

quinazoline+

ANIMAL

HUMANEXP

N

NH O

Cl

Br

N

NH O

Cl

Br

OH

N

NH O

Cl

Br

OH

O

N

NH

O

Cl

Br

Flubromazepam metabolismHUMAN

EXP

Hydroxylation

3-OH++

debrominatedmetabolite

++

HLM

N

NH O

F

Br

N

NH O

F

Br

OH

N

NH O

F

N

NH O

F

OH

Debrominated& OH

metabolite+

minor 2nd OH metabolite

?

Diclazepam metabolism

Hydroxylation

3-OH++

(lormetazepam)

HUMANEXP

HLM

N

NO

Cl

Cl

N

NO

Cl

Cl

OH

N

NH O

Cl

Cl

N

NH O

Cl

Cl

OH

Demethylation

desmethyl+++

(delorazepam)

Desmethyl & OH++

(lorazepam)

Meclonazepam metabolism

amino+++

OH+

HLM

HEP

ANIMAL

CASES

N

NH

O2N

O

Cl

N

NH

NH2

O

Cl N

NH

NH

O

Cl

O

acetamido+++

Reduction

Acetylation

Excursus: When incubating under standard HLM conditions…

When incubating under standard HLM conditions…

ContainCYP,

FMO & UGT

HLM

LackADH & ALDH, MAO, SULT, NAT, GST

NeedCo-factors

When incubating meclonazepam under standard HLM conditions…

amino+++

OH+

N

NH

O2N

O

Cl

acetamido+++

Reduction,catalyzed by

CYP

Acetylationcatalyzed by

NAT

Oxidation,catalyzed by

CYP

No amino metabolite

found!

For further reading: Peng et al. (1984), Nitroreduction of clonazepam in human foetal liver microsomes and hepatocyte cultures

When incubating HLM…

… ensure absence of oxygen to enablereductive reactions – if these are

expected

OR

… use hepatocytes instead

OR

… make sure to double-check resultswith a second system

N

NH

NH

O

F

O OH

Hydroxylation

Desmethylflunitrazepam metabolism

amino+++

CASES

acetamido+++

N

NH

O2N

O

F

N

NH

NH2

O

F

N

NH

NH

O

F

O

N

NH

NH2

O

OH

F N

NH

NH

O

F

O

OH

Reduction

Hydroxylation

amino & OH++

acetamido & OH++

acetamido& OH

+

Nifoxipam metabolism

amino+++

CASES

acetamido++

N

NH

O2N

O

OH

F

N

NH

NH2

O

OH

F

N

NH

NH

O

OH

F

O

N

NH

O2N

O

O

F

O

OOH

OH

OH

OH

Reduction

Acetylation

gluc++

direct glucuronidation

= metabolite of flunitrazepam & desmethyl flunitrazepam

SummaryPhena Flubrom Dicla Meclona Desmethyl-

fluniNifoxi

3-OH ++ ++ ++

Other OH +(phenyl)

+ +where?

Desmethyl +++

OH & MeO +

DebrominatedDebrominated & OH

++ (?)+ (?)

7-amino +++ +++ +++

7-acetamido +++ +++ ++


desmethyl& OH

amino & OH,

acetamido& OH

++

direct glucu-ronida-

tion++

Parent?

nitrobenzodiazepines

4-ring benzodiazepines

Pyrazolam metabolismHUMAN

EXP

HLM

No metabolites detected

N

N

N

N

NCH3

Br

Triazolo ring

Flubromazolam metabolism

α-OH+++

Hydroxylation

N+-gluc+

direct glucuronidation

HLM

HEP

ANIMAL

CASES

HUMANEXP

N

N

F

Br

NN

N

N

F

Br

NN

OH

N

N

F

Br

NN

OH

N

N

F

Br

NN

+

O

OH

OH

OH

OH

O

4-OH++

4

α

N

N

Cl

O2N

NN

Task #2 This is clonazolam, a triazolo nitrobenzodiazepine that recently emerged on the market. Which major metabolites would you suggest?

Clonazolam metabolism

amino+++

CASES

acetamido+++

Reduction

Acetylation

HLM

N

N

Cl

O2N

NN

N

N

Cl

NH2

NN

N

N

Cl

NH

NN

O

N

N

Cl

O2N

NN

OH

N+-gluc+

α-OH++

N

N

Cl

O2N

NN

OH

4-OH+

Hydroxylation

N

N

Cl

O2N

NN

OH

OH-phenyl+

Deschloroetizolam metabolism

NS

N

NN

1

6

3

7

8α

NS

N

NN

OH

NS

N

NN

OH

NS

N

NN

OH

OH OH

OH

HLM

di-OH

Summary

Pyrazolam Flubromazolam Clonazolam Deschloro-etizolam

α-OH +++ ++ Yes

4-OH (6-OH,respectively)

++ + Yes

Other OH Yes(ethyl)

7-amino +++

7-acetamido +++

N+-Gluc + +


Parent? yes yes

Hydrolysis useful? yes

nitrobenzo-diazepine

Conclusions

Be aware how the study was done: Untargeted or targeted search? Suitable system? Number of samples?

Predictions based on metabolism of known analogs often work, but not always!Give it a try, but remain skeptic…

Sherri Kacinko, PhD Sherri Kacinko earned her Bachelor of Science Degree in Chemistry at the University of Pittsburgh in Johnstown, PA and took graduate classes in forensic science at George Washington University. She performed pharmaceutical quality control at Lancaster Laboratories in Lancaster, PA. After spending three years working as a crime laboratory analyst in the chemistry section of the Florida Department of Law Enforcement Orlando Regional Operations Center she returned to graduate school and received her Doctor of Philosophy in Toxicology at the University of Maryland-Baltimore. She is now a toxicologist at NMS Labs in Willow Grove, PA.

Dr. Kacinko is also adjunct faculty in the chemistry department at Arcadia University and serves as an Instructor at the Center for Forensic Science Research & Education. In this role, Sherri lectures in postmortem forensic toxicology and human performance toxicology courses. Her current research interests include the identification and quantification of novel psychoactive substances including synthetic cannabinoids, stimulants, hallucinogens, opioids and benzodiazepines in biological fluids and their toxicological impact.

Sherri is Board Certified by the American Board of Forensic Toxicology (ABFT) and is a member of the Society of Forensic Toxicologists, the International Association of Forensic Toxicologists and the American Academy of Forensic Sciences. Sherri was presented with the American Academy of Forensic Sciences Toxicology Section Irving Sunshine Award in recognition of early career research.

Markus Roman

Markus Roman is currently working as analytical chemist at the National Board of Forensic Medicine in Linköping, Sweden. He received his BSc degree in analytical chemistry in 2000 from the University of Jönköping. In 2002, he joined the National Board of Forensic Medicine, Department of Forensic Toxicology located in Linköping, Sweden. Roman’s main working area has been method development, validation and research on LC-MS-MS instruments and for the last five years high-resolution mass spectrometry. The combination of case work and research has enabled him to contribute to the forensic toxicology community as co-author in more than 10 scientific papers and some congress presentations and posters.

Detecting Designer Opioids and Benzodiazepines:

Understanding the Options

Sherri Kacinko, Ph.D., F-ABFT

NMS LabsWillow Grove, PA


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Disclaimer

• The opinions expressed in this presentationdo not necessarily reflect the views of NMS

• Neither I nor any member of my immediatefamily has a financial relationship with acompany discussed

Screening

• Immunoassay– Enzyme-linked immunoassay

– Use cross-reactivity to discover new designer benzos and opioids

• HRMS

– LC-QTOF targeted toxicological screening*

– LC-TOF screening

– Easy to search for new designer benzos using only molecularformula

3* Liquid chromatography/time‐of‐flight mass spectrometry analysis of post‐mortem blood samples for targeted toxicological screening, M. Roman,L. Ström, H. Tell, M. Josefsson, Analytical and Bioanalytical Chemistry, March 2013

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• Benzodiazepines Direct ELISA Kit 1*• Cutoff: 20 ng/mL (Oxazepam)

200 100 50 20 15 10 5

Pyrazolam + + + + + +/‐ ‐

Etizolam + + + + + ‐ ‐

Phenazepam + + + + +/‐ ‐ ‐

Flubromazolam NT NT NT + +/‐ ‐ ‐

Flubromazepam + + + + ‐ ‐ ‐

Delorazepam + + + + ‐ ‐ ‐

Nimetazepam + + + + NT NT NT

Diclazepam + + + +/‐ ‐ ‐ ‐

Concentration (ng/mL)

Benzodiazepine Blood IA

* Immunalysis Corporation, Pomona, CA

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• EMIT® II Plus Benzodiazepine Assay*• Cutoff: 50 ng/mL (Lormetazepam)

Benzodiazepine Urine IA

* Syva Company/Dade Behring Inc. Cupertino, CA

200 100 50 20

Phenazepam + + + ‐

Flubromazepam + + + ‐

Delorazepam + + + ‐

Nimetazepam + + + ‐

Diclazepam + + + ‐

Etizolam + + ‐ ‐

Pyrazolam + ‐ ‐ ‐


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• Fentanyl Direct ELISA Kit1*• Cutoff: 1 ng/mL (Fentanyl)

Fentanyl Blood IA


10 5 2 1 0.5 0.25 0.1

Furanyl Fentanyl + + + + + ‐ ‐

Butyryl Fentanyl + + + + + ‐ ‐

Para‐fluorobutyryl fentanyl + + + + +/‐ ‐ ‐

Para‐fluoro fentanyl + + + + ‐ ‐ ‐

4‐ANPP +/‐ ‐ ‐ ‐ ‐ ‐ ‐


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• Fentanyl Homogeneous enzyme immunoassay*• Cutoff: 2 ng/mL (Fentanyl)

Fentanyl Urine IA


10 5 2 1 0.5 0.25 0.1

Butyryl Fentanyl + + + ‐ ‐ ‐ ‐

Furanyl Fentanyl + + ‐ ‐ ‐ ‐ ‐

Para‐fluorobutyryl fentanyl + + ‐ ‐ ‐ ‐ ‐

Para‐fluoro fentanyl + + ‐ ‐ ‐ ‐ ‐

4‐ANPP ‐ ‐ ‐ ‐ ‐ ‐ ‐


• Agilent Infinity 1290, Agilent 6550 iFunnel QTOF

• YMC-Triart C18, 2.0 x 50 mm

• A) 0,05 % Formic acid in 10 mM ammonium formateB) Methanol

• Liquid-liquid extraction with ethylacetate at pH 9

• Data-dependent auto MSMS using preferred list

• 36 benzodiazepines in library

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Benzodiazepine – LC-QTOF

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Score > 80

< 5 ppm

< 0.10 min

Score (Lib) > 80

• Data Analysis– Initial identification based on Exact Mass– Obtain standard and inject on LC-TOF for retention

time– Run standard through GCMS Screen panels

(Basic/Neutral & Acid) to determine if they can beconfirmed

• Barriers to success– Standard availability– Isomers– GCMS Screen method extraction

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Benzo & Opioids – LC-TOF

• Analytes currently detected in LC-TOF screen– Diclazepam, Flubromazepam, Flubromazalam,

Delorazepam, Bentazepam, Nifoxipam, Nimetazepam

– Furanyl Fentanyl, fluoro-butyryl fentanyl isomers, FluoroFentanyl isomers, 3-methyl fentanyl, carfentanil, acrylfentanyl

– AH-7921, U-47700, MT-45

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Benzo & Opioids – LC-TOF

• Qualitative confirmation by full scan GCMS– Fentanyl related compounds and opioid compounds

generally extract with basic drugs

– Benzodiazepines extract better in acid fraction, but lowsensitivity

• Quantitative confirmation

– LC-MSMS

Confirmation

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• SHIMADZU Nexera X2, AB SCIEX API 4500

• WATERS Acquity UPLC BEH Phenyl, 2.1 x 50 mm

• A) 0,05 % formic acid in 10 mM ammonium formateB) 0,05 % formic acid in methanol

• Liquid-liquid extraction with tert-butylmethylether(TBME) at pH 11

• 1 – 200 ng/g calibration range, acetylfentanyl0.1 – 10 ng/g calibration range, furanylfentanyl0.01 – 10 ng/g calibration range, acrylfentanyl 13

Fentanyls in blood with LCMSMS

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12 3

4

5

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1) Acetylfentanyl2) Acrylfentanyl3) Fentanyl4) Furanylfentanyl5) 4F-isobutyrfentanyl6) 4MeO-butyrfentanyl7) 4F-butyrfentanyl8) Valerylfentanyl

Chromatographic Separation

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Acrylfentanyl, CE 20 eV

4F-isobutyrfentanyl, CE 20 eV

Furanylfentanyl, CE 20 eV

Spectral Similarities

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Acetylfentanyl n = 30Min 4 ng/gMax 770 ng/gMean 210 ng/gMedian 150 ng/g

Acrylfentanyl n = 31Min 0.01 ng/gMax 5.0 ng/gMean 0.81 ng/gMedian 0.20 ng/g

Furanylfentanyl n = 7Min 0.38 ng/gMax 2.7 ng/gMean 1.0 ng/gMedian 0.90 ng/g

Quantitative Results (post-mortem)

• AH7921, MT-45, U-47700

• Agilent Infinity 1290, Agilent 6460 LCMSMS

• Agilent Zorbax Eclipse Plus C18, 2.1 x 50 mm

• A) 0,05 % formic acid in 10 mM ammonium formateB) 0,05 % formic acid in methanol

• Protein precipitation with 0,075 % formic acid inacetonitrile/ethanol 90/10

• 0.01 – 1.0 µg/g calibration range, AH7921, U-477000.001 – 1.0 µg/g calibration range, MT-45 17

Other opioids in blood with LCMSMS

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AH-7921 n = 9 *Min 0.03 µg/gMax 0.99 µg/gMean 0.37 µg/gMedian 0.30 µg/g

U-47700 n = 2 Min 0.05 µg/gMax 0.58 µg/gMean - µg/gMedian - µg/g

MT-45 n = 30Min 0.004 µg/gMax 1.9 µg/gMean 0.48 µg/gMedian 0.35 µg/g

* Fatal intoxications associated with the designer opioid AH‐7921, R. Kronstrand, G. Thelander, D. Lindstedt, M. Roman, F.C. Kugelberg, Journal of Analytical Toxicology, October 2014

Quantitative Results (post-mortem)

Benzodiazepine Confirm - LCMSMS

• Waters TQD Tandem Mass Spectrometer withWaters Acquity UPLC– Waters Acquity BEH C18 column, 2.1x100mm, 1.7

micron• A) Ammonium Formate Buffer pH 4• B) Methanol

– Liquid-Liquid Extraction• Methyl tert-butyl ether (mtbe)/chlorobutane mixture at pH 9


• Etizolam, Deschloroetizolam, Flubromazolam– Calibration Range: 2-200 ng/Ml

• Clonazolam, Pyrazolam, Diclazepam,Delorazepam, Meclonazepam, Bromazepam– Calibration Range: 5-500 ng/mL

• Flubromazepam, Phenazepam, Nifoxipam– Calibration Range: 20-2000 ng/mL


1) Nifoxipam2) Pyrazolam3) Bromazepam4) Clonazolam5) Flubromazolam6) Meclonazepam

7) Etizolam8) Flubromazepam9) Deschloroetizolam10) Delorazepam11) Phenazepam12) Diclazepam

1 2

3

4 5 6

7

8

910

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Case Report

• 42 year old man died in-custody death

• Expanded post-mortem toxicology:– IA for Barbiturates, Cannabinoids and Salicylates– LC-TOF screen for 230+ drugs

• “Lectopam” listed in case history

Case Report

Bromazepam Positive

Case Report• Lectopam – Bromazepam• Used in Canada (location of case)• Reported concentratinos (Baselt 10th Edition)

Single dose:3 mg = 10 ng/mL; 6 mg = 83 ng/mL; 12 mg = 130 ng/mL

Chronic oral dose (9 mg)Steady-state = 10 – 150 ng/mL (Average 120 ng/mL)

Post-mortem: 2 suspected ODs = 5000 and 6400 ng/mL

Acknowledgements• Robert Kronstrand• Stephanie Kumor – R&D NMS Labs

Dani C. Mata, MSFS, D-ABFT-FT Ms. Mata graduated from Pomona College in 2005 with a Bachelor’s in Chemistry and a Minor in Mathematics. She graduated in 2007 from the University of Illinois at Chicago with a Master’s of Science in Forensic Science. Since 2007, Ms. Mata has worked for the Toxicology Section in Orange County Crime Lab, where she has been Technical Advisor for Antemortem Toxicology, 2011 – 2015, and Method Development Technical Advisor since 2011. Ms. Mata has been certified by the California Department of Health as a Forensic Alcohol Supervisor since 2010 and by the American Board of Forensic Toxicologists as a Diplomate in Forensic Toxicology since 2015. In 2015 and 2016 Ms. Mata served as an affiliate on the Organization of Scientific Area Committee Task Group on Report and Testimony. Ms. Mata has been a member of the Toxicology Section of the American Academy of Forensic Sciences (AAFS) since 2009 and has presented various posters and platform presentations. She won the AAFS Regional Award for Exceptional Contribution to the California Association of Toxicologists (CAT) and the Field of Toxicology in 2013. Ms. Mata has been a member of CAT since 2009, has co-hosted two of the meetings and presented at six CAT meetings. She has co-chaired the Research Grant Committee, 2012 – 2014, and Literature Review Committee since 2012. Ms. Mata has been a member of the Society of Forensic Toxicologists (SOFT) since 2011 and has served on the Young Forensic Scientist Committee for the past three years and is the Volunteer co-Coordinator for the 2016 SOFT meeting. She was awarded the Young Scientist Meeting Award at the 2012 SOFT Meeting.

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Clinical and post mortem findings in cases related to designer

benzodiazepines and opioids

Dani C. Mata, D-ABFT-FTSenior Forensic Scientist

Orange County Crime Lab, Santa Ana, CA


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Disclaimer

• The opinions expressed in thispresentation do not necessarilyreflect the views of the OrangeCounty Crime Lab

• Neither I nor any member of myimmediate family has a financialrelationship with a company

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Presentation Outline• Synthetic Opioids

– Acetyl fentanyl– Ortho-fluorofentanyl– Furanylfentanyl– Mitragynine– Tapentadol

• Synthetic Benzodiazepines– Etizolam– Phenazepam– Diclazepam– Flubromazepam

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Fentanyl and its Analogs

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Fentanyl Trends- Controlled Substances

0

3

6

9

12

15

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2010 2011 2012 2013 2014 2015 2016*

Fentanyl(pharmaceutical)Fentanyl (illicit)

Acetyl Fentanyl

Butyryl Fentanyl

Furanyl Fentanyl

Valeryl Fentanyl

ortho -fluorofentanyl

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Fentanyl Trends- Toxicology

0

3

6

9

12

15

18

21

24

27

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2010 2011 2012 2013 2014 2015 2016*

Fentanyl (DUID)

Fentanyl (PM)

Acetyl Fentanyl

ortho-Fluorofentanyl

Furanyl Fentanyl

Acetyl Fentanyl Case #1

• July 2015• 44 year old male

• Autopsy Findings:– Heart Wt: 630 g

• Has unusual amount ofadipose tissue

– Left Lung: 570 g– Right Lung: 690 g


Drug Postmortem BloodHydroxy-THC 3 ng/mLTHC 7.7 ng/mLCarboxy-THC 38.4 ng/mLAcetyl Fentanyl 280 ng/mL

Mode of Death: NaturalCause of Death: Hypertropic cardiomegly


• August 2015• 28 year old male

• Autopsy Findings:– Heart Wt: 350 g– Left Lung Wt: 650 g– Right Lung Wt: 760 g

Acetyl Fentanyl Case #2 (cont’d)

Drug Postmortem BloodMorphine (Free) 30.7 ng/mLAlprazolam 40.9 ng/mLLorazepam 10.6 ng/mLAcetyl Fentanyl 3900 ng/mL

Mode of Death: Accident Overdose Cause of Death: Acute fentanyl, morphine,

alprazolam and lorazepam intoxication


• December 2015• 19 year old female

• Autopsy Findings:– Heart Wt: 360 g– Left Lung Wt: 710 g– Right Lung Wt: 770 g– Severe pulmonary

edema


Drug Heart Blood

Peripheral Blood

Brain Liver Urine VitreousHumor

Benzoylecgonine 2030 ng/mL

1716 ng/mL

937 ng/g

2130 ng/g

1160 ng/mL

Methamphetamine DetectedAcetyl Fentanyl 370

ng/mL376 ng/mL

612 ng/g

1953 ng/g

9120 ng/mL

262 ng/mL

Ethylone 520 ng/mL

Cocaine 3270 ng/mL

78 ng/mL

Mode of Death: Accident OverdoseCause of Death: Acute polydrug intoxication due to combined effects of acetylfentanyl, ethylone, and methamphetamine


• April 2016• 35 year old male

• Autopsy Findings:– Heart Wt: 360 g– Severe pulmonary

edema– Left Lung Wt: 710 g– Right Lung Wt: 770 g


Drug Heart Blood

Peripheral Blood

Brain Liver Urine VitreousHumor

Acetylfentanyl 1100 ng/mL

261 ng/mL 1330 ng/g

5450 ng/g

500 ng/mL

162 ng/mL

MDMA DetectedMDA DetectedKetamine 68 ng/mL 99 ng/mL 60

ng/gNorketamine DetectedButylone

Mode of Death: To be determined…Cause of Death: To be determined…

ortho-Fluorofentanyl

• April 2016• 21 year old female• Left Lung Wt: 430 g• Right Lung Wt: 560 g

Hospital Urine:Ortho-fluorofentanyl 37 ng/mL morphine (free)

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Mode of Death: Overdose (Abuse)

Cause of Death: Complications of toxicity of ortho-fluorofentanylwith morphine present in the system

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Furanylfentayl

• July 2016• 50 year old female

• Autopsy Findings:– Heart Wt: 330 g– Left Lung Wt: 850 g– Right Lung Wt: 900 g– Severe pulmonary edema

Furanylfentanyl (cont’d)

Drug Heart Blood Peripheral Blood

Liver Stomach Contents

Furanylfentanyl 40 ng/mLU47700 164 ng/mLAlprazolam 350 ng/mL 313 ng/mL 618 ng/g 0.13 mg

Mode of Death: To be determined…Cause of Death: To be determined…

Mitragynine Postmortem Case

• June 2015• 30 year old male

• Autopsy Findings:– Heart Wt: 450 g– Left lung Wt: 640 g– Right Lung Wt: 650 g– Mild pulmonary

congestion and edema– Mild cerebral swelling– Acute gastritis– Obesity 18

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Mitragynine Postmortem Case (cont’d)

Mode of Death: Accident OverdoseCause of Death: Acute poly drug intoxication due to

combined effects of oxycodone, oxymorphone, alprazolam, diazepam, nordiazepam, and mitragynine

Drug Heart Blood (ng/mL)

Peripheral Blood (ng/mL)

Liver (ng/g)

Stomach Contents (mg)

Oxycodone (free) 200 188 197 2.63Oxymophone (free) 9.3 Detected DetectedAlprazolam 147 161 241 0.15Nordiazepam DetectedDiazepam DetectedMitragynine Detected

Mitragynine DUID Case

• 30 year old male• Ran into a car• Dilated Pupils• HGN: 6 cues• Rhomberg: 45 sec• WAT: 5 cues• OLS: 2 cues

Drug Concentration (ng/mL)

Alprazolam 444Cyclobenzaprine ~10Mitragynine ~220Trazodone ~300

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Tapentadol - DUID

• 52 year old male• Ran red light and

hit 2 cars• Constricted Pupils• No HGN nor VGN• Rhomberg: 60 sec• Could not perform

WAT and OLS


Amitritpyline 91.4Codeine (total) 17.5Cyclobenzaprine 105Gabapentin 11100Oxycodone 81.2Oxymorphone (total)

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Tapentadol (total) 790Bupivicaine DetectedDiazepam DetectedNordiazepam DetectedCarboxy-THC Detected

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Tapentadol - Postmortem

• January 2016• 55 year old male

• Autopsy Findings– Heart Wt: 430 g

• Possible hypertensiveheart disease

– Left Lung Wt: 450 g– Rt Lung Wt: 510 g

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Tapentadol – Postmortem (cont’d)

• Mode of Death: Accidental Overdose• Cause of Death: Acute ingestion of diazepam,

zopiclone, citalopram, quetiapine, and tapentadol

Drug Heart Blood (ng/mL)

Peripheral Blood (ng/mL)

Liver (ng/g)

Stomach Contents (mg)

Tapentadol 5000 2020 13600 61.1Quetiapine 3290 1790 18600 158Citalopram 710 333 4510 <3.2Bupropion Detected 113 Detected <1.6Zopiclone 18.2Diazepam 468Nordiazepam 819

The Rise of Etizolam in Orange County, CA

• 2014: 1 cases• 1 DUID

• 2015: 7 cases• 5 DUIDs• 1 Coroner

• 2016: 31 cases• 27 – DUIDs• 3 – Coroner• 1 Sexual Assault

Etizolam Postmortem Case

• March of 2016• 20 year old male

• Autopsy Findings:– Heart wt: 420 g– Left Lung wt: 870 g– Right Lung wt: 1220 g– Sever pulmonary

edema– Hemmorrhage– Atelectasis

Etizolam Postmortem CaseDrug Postmortem Blood Vitreous HumorEthanol 0.336 % (w/v) 0.329 % (w/v)Acetaldehyde DetectedAlprazolam 29.5 ng/mLEtizolam 109 ng/mLTHC 17 ng/mLOH-THC 6.4 ng/mLCarboxy-THC 64.1 ng/mL

• Mode of Death: Overdose (accident)• Cause of Death: Combined effects of

alprazolam, etizolam, THC and ethanol

Etizolam DUID Case

• 22 year old male• Pulled over at 0740

– Swerving over thecenter median

– Almost rear-ended a car

– Went onto the curband brushed a tree

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Alprazolam 7.7Etizolam 55.8THC 2.1Carboxy-THC 14.6MDMA DetectedMDA Detected

Etizolam SFSTDriving behavior Eyes FSTs and Physiological Signs

Hit and Run with tree.

Unaware of accident.

HGN, 6 clues 50 sec Romberg; sway, WAT 6/8 clues; OLS attempted

6 times but unable to perform due to poor balance;

asked to bring down finger during finger to nose and

kept opening eyes

Drove onto curb, ran a

red light, almost hit a

tree

HGN, 6 clues, difficulty

tracking

45 sec Romberg, sway; WAT 5/8 clues, staggered,

walked backward to regain balance; OLS put down foot

6 times, missed finger to nose; pulse (bpm*) 128

Swerving all over the

road and into oncoming

traffic, nodding off,

veered across all lanes

Rapid onset HGN, 6 clues,

room light 5 mm, near total

darkness 7 mm, direct light

4 mm, and LOC. Officer

noted pinpoint pupils

50 sec and 26 sec Romberg, sway, eyelid flutters,

visible carotid pulse, almost fell over; WAT 5/8 clues,

stepped offline 8 times; OLS put down foot 5 times,

arms for balance, started over 4 times; pulse (bpm*)

138, 132, 130, 102; BP† 120/85

Rear ended a stopped

car without slowing

down, backed up and

sped off

Rapid onset HGN, 6 clues,

LOC, VGN, slow reaction to

light, rebound dilation, room

light 7.5 mm, near total

darkness 8.5 mm, direct

light 5 mm

45 sec Romberg, sway, eyelid tremors; WAT slow

robotic movements, deliberate steps; OLS started 3

times before instructed, put down foot, fell; finger to

nose missed 5/6 times, smashed nose; pulse (bpm) 120,

112, 114; BP† 140/8028

Phenazepam DUID

• 20 year old male• Swerving over

center lane• LOC• HGN: 4 cues• Rhomberg: 50 sec• WAT: 6 cues• OLS: did not

perform29


Phenazepam 3Alprazolam 32THC 17Carboxy-THC 84

Diclazepam DUID Case #1

• Male• 31 years old• Going 115 mph and

almost side-swiped atruck

• Constricted Pupils• No HGN nor VGN• OLS: 3 cues• Romberg


Diclazepam DetectedLorazepam DetectedAlprazolam DetectedTHC 5.09Hydroxy-THC 3.66Carboxy-THC Detected

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Diclazepam DUID Case #2

• Male• 30 years old• DUI called in by

citizen• No HGN nor VGN• Refused FST• Pulse: 106 and 88

(2 hr difference)• BP: 110/80


Diclazepam DetectedLorazepam DetectedMethadone DetectedMorphine (free) 20Codeine (free) 2.5

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Flubromazepam DUID

• Male• 20 years old• Stopped for leaving the

scene of an accident• Romberg: 50 seconds• HGN: 6 clues• WAT: 6 clues• OLS: could not perform


Flubromazepam > 500 ng/mLEtizolam 300 ng/mLMorphine 18 ng/mL

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Summary

• Synthetic Opiates andBenzodiazepines are beingabused in driving and deathcases

• Although rarely found alone, themore they are documented, thebetter we understand theirimpact on society

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Acknowledgments

• Stephaine Hopkins and CaliforniaDOJ, Toxicology Laboratory inSacramento, CA

• Orange County Crime Lab, Santa AnaCalifornia

Questions?

Dani C. [email protected]

714-834-4510

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toxicology of designer benzodiazepines and opioids...

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