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Analysis of Novel Psychoactive Substances and Metabolite Discovery in Authentic Biological Samples

Mandi Mohr, M.S.

Disclaimer

The project was supported by Award No. 2013-DN-BX-K018, awarded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice. The opinions, findings, and conclusions or recommendations expressed in this publication/program/exhibition are those of the author(s) and do not necessarily reflect those of the Department of Justice.

Novel Psychoactive Substances

• Emergence of “designer drugs,” “legal highs,” and/or “club drugs” has seen a steady increase since early 2008

• NPS refers to a broad category of products containing unregulated phenethylamines, cathinones, and synthetic cannabinoids

• Market is in constant flux – Limited ability to develop analytical methods in a

timely fashion

– Limited research on into metabolism

Electronic Dance Music Festivals

• U.S. Electronic Dance Music (EDM) is characterized by techno-rock music and rave culture

• Literature sources indicate as many as 70% of attendees may be using recreational drugs

• Emphasis on harm reduction, public health and awareness

– Dancesafe.org

– Bunk Police

NPS at EDM Festivals in the U.S.

• Ultra Music Festival – Miami, Florida

– 2013: 1 death

– COD: Acute Methylone Toxicity – 1.2 mg/L

• Electric Zoo Festival – New York, New York

– 2013: 2 deaths

– COD: Acute MDMA intoxication with hyperthermia

• Bonnaroo Music Festival – Manchester, TN

– 2013: 2 deaths

– COD: Information unavailable

Project Design and Objectives

• Collection and analysis of paired blood, urine, and oral fluid samples

• Project goals included:

– Identify novel drugs on the market

– Determine prevalence of designer drugs

– Characterize potential metabolites using in vitro studies and authentic samples

SAMPLE COLLECTION

Location

• Approximately 100 meters from entrance gate

• Able to obtain volunteers from heavy foot traffic

– Location between entrance and public transportation stop

Sample Collection

• Step 1: Consent forms/Survey

• Step 2: Oral Fluid Collection – Immnualysis

Quantisal

– Alere DDS2 Cartridge

• Step 3: Urine

• Step 4: Blood

Disclosure: Participants were not required to donate all 4 samples, and only donated samples based on their comfort level. The

gift card incentive was only given if the participant donated a blood sample.

Sample Set

• Collected samples from 145 subjects

– 105 urine samples

– 65 blood samples

– 125 oral fluid samples screened with the Alere DDS2

– 136 oral fluid samples collect with the Immunalysis Quantisal

DEMOGRAPHICS AND SURVEY RESULTS

Demographics

• Samples were collected from 76 males and 60 females – 9 subjects did not indicate M/F

• Average age: 23.7 (±6.4) – Range: 18-57 years old

• 72% of participants indicated they had taken a recreational or medicinal substance in the last week – THC was most common answer, followed by

Cocaine

57% THC

13% MDMA

14% Molly

5% Ecstasy

16% Cocaine

13% Alcohol

Responses of Drug Use

n=104

Novel Psychoactive Substance Use

• 33 participants reported taking MDMA, “Molly,” or “Ecstasy” within the last week

• Typically routes of administration were orally via a pill or capsule

• Self-reported dosages:

– MDMA: 0.3 grams up to 25 grams

– Molly: 0.2 mg up to 2.3 grams

SAMPLE PREPARATION & ANALYSIS

Sample Preparation

• Blood samples were extracted using a basic liquid-liquid extraction

– Samples were made strongly basic using 0.1M borax buffer (pH 10.4) and extracted into a 70:30 mix of n-butyl chloride/ethyl acetate

– Samples were dried to completion and reconstituted in 5 mM ammonium formate (pH 3) and 0.1% formic acid in acetonitrile (90:10)

Waters ACQUITY UPLC® I Class

Waters Xevo® G2-S QTOF

UPLC-QTOF Parameters

• ACQUITY UPLC® I-CLASS Conditions:

Time (min) %A %B

Initial 87 13

0.5 87 13

10.0 50 50

10.75 5 95

12.25 5 95

12.5 87 13

15.0 87 13

– Mobile phase A: 5mM ammonium formate (pH 3.0)

– Mobile phase B: 0.1% formic acid in acetonitrile

– Column: ACQUITY UPLC® BEH C18 2.1 mm x 150 mm, particle size 1.8 micron

– Flow rate: 0.4 mL/min – Column Temperature:

50°C – Injection Volume: 5µL

UPLC-QTOF Parameters

• Xevo ® G2-S QTOF Conditions: – Ionization: Positive electrospray

• Capillary voltage: 0.8 kV

• Sample Cone Voltage: 25 V

• Extraction Cone Voltage: 4 V

• Source Temperature: 150°C

• Desolvation Temperature/Flow: 400°C/800 L/h

• Reference Lockspray: Leucine Enkephalin

– Resolution Mode: 50-1000 m/z • Collision Energy (Function 1) – 6eV

• Collision Energy (Function 2) – 10-40eV

Acceptability Criteria (UPLC-QTOF)

• Chromatographic peak must be clearly identifiable, as well as internal standard peak

• Chromatographic peak must be within ±0.25 min of analyte in database

• Observed mass of molecular ion must be within ± 5ppm of mass in database

• Observed mass of fragment ion must be within ± 2mDa

• Response greater than 1500 (3D data)

IN VITRO METABOLITE DISCOVERY

Human Liver Microsome Incubations

• Method adopted from Tiller et. al. (2008) – Rapid Communications in Mass Spectrometry

• Samples were prepared by adding: – 100 mM Phosphate Buffer (pH 7.4) with 10 mM

MgCl2

– 5 µL of drug of interest (1 mg/mL – reconstituted in 1:1 ACN:Phosphate Buffer)

– 50 µL of 10 mM NADPH • Not added to control samples

– 25 µL of pooled human liver microsomes

Microsome Incubation

• Samples were incubated in a water bath for 2 hours at 37⁰C

• Reaction was stopped with 500 µL of acetonitrile

• Samples were centrifuged at 10000 RPM for 5 minutes – Supernatant was dried at 33⁰C for 10 minutes and

then filtered using a centrifugal filter (10000 RPM for 5 minutes)

BLOOD SCREENING RESULTS

Screening Results Overview

• 58% (38 subjects) of the blood samples screened positive for a common drug of abuse/metabolite or NPS substance

– Most common drug of abuse: Cocaine*

• 6 subjects screened positive for cocaine

• Of the 38 subjects screening positive, 55% had at least one NPS substance detected

*Samples have not been screened for acidic compounds

NPS Analytes

• NPS analytes detected were commonly discussed stimulants on EDM forum websites

• Typical effects of these analytes include: – Euphoria, alertness, sexual arousal, focused mind,

and overall positive feeling

NPS Analyte

4-FA/ 2-FA

Alpha-PVP

Dimethylone/Ethylone/ Butylone

Methylone MDMA/

Methedrone

Number of Positive Samples (n=21)

1 10 6 9 5

“Molly”

• Several participants indicated they had taken “Molly” within the last week

• Samples of subjects who reported taking “Molly” contained: – MDMA/Methedrone

– Alpha-PVP

• Samples of subjects who reported taking MDMA contained: – Methylone

– Dimethylone/Ethylone/Butylone

– Alpha-PVP

Alpha-PVP

• Belongs to the “bath salts” group of substances

– PVP, O-2387, alpha-pyrrolidinopentiophenone, alpha

• Generally sold as tablets or as a powder to be taken orally, insufflated, or dissolved in solution to be injected

– Effects occur within 30 to 45 minutes of administration

• Seizures of “gravel” were reported in late 2013

– Typically found to be a combination of clonazepam, methamphetamine, and alpha-PVP

METABOLITE CHARACTERIZATION

Metabolite Name

1 Alpha-PVP

2 Amino-PVP

3 2”-oxo-PVP

4 OH-Alkyl-PVP

5,6 OH-Phenyl-Amino-PVP

7 OH-Phenyl-PVP

8 OH-Alkyl-oxo-PVP

9-11 Carboxy-oxo-PVP

12 OH-Phenyl-2”-oxo-PVP

13 di-OH-PVP

14 OH-Phenyl-Carboxy-

oxo-PVP

HLM Incubations

Alp

ha-P

VP

Fragment Matching

Alpha-PVP-5-OH

HLM Metabolite

MS124 – Blood Metabolite

Alpha-PVP-5-OH

MS124 – Blood Metabolite

OH-Phenyl-Amino-PVP

Conclusions

• Broad-based screening using HRMS provided successful identification of common drugs of abuse and NPS analytes

– Confirmed high rates of drug use among attendees

• Structural elucidation toolset is useful in characterizing unknown metabolites

– Fragment matching using mol files

– Use of Metabolite Identification (MetID) processing

– Running in “true” Q-TOF mode

Acknowledgements

• The National Institute of Justice

• Waters Corporation

– Michelle Wood, Jeff Goshawk, Jim Bulgarelli, Scott Freeto, Peter Harrsch, Will Harris

• Dr. Barry Logan, Melissa Friscia, Jill Yeakel

• Fran Diamond, Sarah Wolf, Helen Piper

The Research Team

Questions?

mmohr@frfoundation.org