Identification of Major Metabolites in Human Blood and Urine Associated with the Ingestion of Alpha PVP

Melissa Friscia* MSFS, Sarah Wolf BS, Amanda LA Mohr MSFS, Francis X. Diamond BS, Jillian K Yeakel MSFS, Barry K Logan PhD DABFT

FSF Emerging Forensic Scientist Award

Oral Presentation

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

• Novel psychoactive substances (NPS) refers to a broad category of products containing unregulated phenethylamines, cathinones, and synthetic cannabinoids

• NPS market is in constant flux

– Limited ability to develop analytical methods in a timely fashion

– Limited research on metabolism

NPS Use at 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

• Users believe that they are using common club drugs like MDMA – Many participants are ingesting NPS drugs

– Increased potency leads to increased health concerns

Project Design and Objectives

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

• Project goals included:

– Characterize potential metabolites of alpha-PVP using in vitro studies with human liver microsomes (HLM)

– Evaluate the extent to which these metabolites are present in authentic blood and urine samples

Alpha-PVP

• Belongs to the “bath salts” group of substances

– PVP, O-2387, alpha

• 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

α-Pyrrolidinopentiophenone

Human Liver Microsome Incubations

• Samples were prepared by adding: – 100 mM Phosphate Buffer (pH 7.4) with 10 mM MgCl2

– 5 µL of 1 mg/mL drug of interest – 50 µL of 10 mM NADPH – 25 µL of pooled human liver microsomes

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

• Samples were analyzed on a Waters ACQUITY UPLC ® I Class coupled with a Waters Xevo ® G2-S QTOF – Using the Forensic Toxicology Screening Solution with

UNIFITM

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

In silico and in vitro metabolism studies support identification of designer drugs in human urine by liquid chromatography/quadrupole-time-of-flight mass spectrometry Elli Tyrkkö, Anna Plander, Raimo A. Ketola

Metabolite Name

Parent Alpha-PVP

M1 5-OH-PVP

M2 OH-Alkyl-PVP

M3 2”-oxo-PVP

M4 Reduced 2”-oxo-PVP

M5 Amino-PVP

M6 Carboxyamino-PVP

M7 Carboxy-PVP

α

RESULTS

HLM Incubations

Alp

ha-P

VP

Binary Comparison in UNIFI®

Name: 2”-oxo-PVP MF: C15H19NO2

[M+H]+: 246.1489

Control Incubation

Alpha-PVP Incubation

2”-oxo-PVP in MSMS Mode

Carboxyamino-PVP

Alpha-PVPOH-Alkyl-PVP5-OH-PVP

2"-oxo-PVP

OH-Phenyl-PVP

Butylamino, OH-Alkyl-PVP

Amino-PVP

5-OH-PVP

• Reduction of the ketone to an alcohol

• Commercially available standard to confirm identification

5-OH-PVP

5-OH-PVP Standard

Sample MS075

5-OH-PVP

5-OH-PVP Standard at 4.78 minutes

Major Peak at 4.64 minutes

Minor Peak at 4.78 minutes

5-OH-PVP

Enantiomeric Pair Enantiomeric Pair

Diastereomers

• Collected samples from 145 subjects

– IRB Approved Study for Human Subjects

– 105 urine samples

• 13 alpha-PVP cases

– 65 blood samples

• 8 alpha-PVP cases

Samples from Human Subjects

Alpha-PVP Metabolites: Urine Samples (n=13)

0

2

4

6

8

10

12

14

Alpha PVP Metabolites: Blood Samples (n=8)

0

1

2

3

4

5

6

7

8

Conclusion

• 8 metabolites were identified using HLM incubations

• 6 metabolites had previously been reported in rat urine and human urine

– Determined two additional metabolites in human samples previously not detected • Positional isomer of OH-Phenyl-PVP

• Butylamino, OH-Alkyl-PVP

Primary Urine Metabolites Primary Blood Metabolites

• 5-OH-PVP • Butylamino,OH-Alkyl-PVP • 2”-oxo-PVP • OH-Alkyl-PVP

• 5-OH-PVP • 2”-oxo-PVP

Acknowledgements

• The National Institute of Justice

• Waters Corporation

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

• AAFS Toxicology Section

FSF Emerging Forensic Scientist Award

Oral Presentation

Contact Information

Questions?

Melissa.Friscia@frfoundation.org