Analysis of Novel Psychoactive Substances and Metabolite Discovery in Authentic Biological Samples Mandi Mohr, M.S.
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