Analysis of Novel Psychoactive Substances and Metabolite …€¦ · Metabolite Discovery in...
Transcript of Analysis of Novel Psychoactive Substances and Metabolite …€¦ · Metabolite Discovery in...
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?