ECL Pressentation: Screening of new psychoactive ... Pressentation: Screening of new psychoactive...

Juliet Kinyua

Friday, June 02, 2017

Screening of new psychoactive substances in biological matrices and sewage to monitor

community-level consumption

Environmental Chemistry Lab Seminar July 12, 2017

1

Outline

1. Conceptual Framework

2. Aims of the project

3. Approaches (Part I-III)

4. Summary and Conclusions

2

Illicit Drugs

Induce dependency

Crime and violence

Morbidity and Mortality

Conceptual Framework

3

Traditional methods used to estimate illicit drug use

1. Interviews 2. Surveys3. Statistics


Estimate drug consumption in communities Collect and measure the untreated influent sample Apply back-calculation models= g/day

Sewage-Based Epidemiology (SBE)

Source: EMCDDA

http://www.emcdda.europa.eu/topics/pods/waste-water-analysis#panel2

Complimentary approach

6

Ring test (round robin); Interlab Annual sampling campaign (> 60 European Cities)

7

SEWPROF PROJECT

11 EU teams working in the emerging field of SBE

Advance knowledge and bridge gaps in SBE

Funded by the European Commission, Marie Curie Actions, Seventh Framework Program, Initial Training Network.

http://sewprof-itn.eu/

http://cordis.europa.eu/fp7/people/initial-training_en.html

designer drugs; Legal Highs

mimic effects of classic drugs

(cocaine, amphetamine, MDMA, LSD)

Minor chemical modifications

Forgotten or failed pharmaceuticals

New Psychoactive SubstancesNPS

9

Bypass drug control

Purchased on dark-net and in smart

shops

Routine methods for illicit drugs

dont always detect them

Fatal intoxications

New Psychoactive Substances

Why NPS? Unknown use in general population; fatal

intoxications reported

Routine methods of illicit drug detection do not detect them

Explore novel uses of SBE

When we started in 2013 (4 SBE studies on NPS)


11

Aims of the Project

1. Develop analytical methods for analysis of NPS

in sewage and biological matrices

2. Identify potential biomarkers of NPS use

3. Conduct monitoring studies to evaluate

feasibility of SBE and Pooled Urine Analysis

12

ANALYTICAL METHODSPart I

Quantitative method

Quantify NPS in sewage MCX cartridge for SPE LC-MS/MS (Agilent 6410) Target 7 NPS LOD and LOQ < 2 ng/L

Part I: Analytical Methods

(Kinyua et al., DTA 2015)

1st application study

(Kinyua et al., DTA 2015)


Site MXT Butylone Ethylone Methylone MPA PMMA PMA

Antwerp North 1.8 ND ND ND ND ND ND

Ruisbroek ND ND ND ND ND ND ND

Zele ND ND ND ND ND ND ND

Boechout 1.9 D ND ND ND ND ND

Boechout 2 1.7 ND ND ND ND ND ND

Antwerp South 3.1 ND D ND ND ND ND

Swiss sample1 2.5 ND D 2.5 ND D ND

Swiss sample 2 1.8 D D 0.6 ND D ND

Swiss sample 3 1.5 ND ND ND ND ND ND

BE and CH

LOD ~0.2 0.5 ng/LLOQ ~0.5 - 2 ng/L

Are they consumed? Are concentrations

too low? Wrong biomarker ?

- Metabolites?- In sewer

transformation by microorganisms?

Site

MXT

Butylone

Ethylone

Methylone

MPA

PMMA

PMA

Antwerp North

1.8

ND

ND

ND

ND

ND

ND

Ruisbroek

ND

ND

ND

ND

ND

ND

ND

Zele

ND

ND

ND

ND

ND

ND

ND

Boechout

1.9

D

ND

ND

ND

ND

ND

Boechout 2

1.7

ND

ND

ND

ND

ND

ND

Antwerp South

3.1

ND

D

ND

ND

ND

ND

Swiss sample1

2.5

ND

D

2.5

ND

D

ND

Swiss sample 2

1.8

D

D

0.6

ND

D

ND

Swiss sample 3

1.5

ND

ND

ND

ND

ND

ND

Challenges

The constantly moving target

Availability of reference standards Target analysis Cost of standards (incl.

metabolites)

What biomarker? Parent or metabolite?


Modify goals:- Detection frequencies of NPS

Analytical methods:- Qualitative screening (suspect and non-target)

Move closer to source- Urine/ blood of users- Sample at festivals/events

Build a biomarker database- In vitro and In vivo experiments - In sewer experiments

Different approach

17

Qualitative method

LC-QToFMS (Agilent 6530)

Based on data-independent acquisition (DIA)

Broad screening

- LC method wide LogP range

- (+ in-house library >2000 entries)

- Strong biomarker database

Developed data analysis workflow


18

Target List: Ref. standards

available

MS/MS spectra and

tR:

In vitro

metabolites

Previously

confirmed

intoxication

MS/MS Spectra at different CE

tR

In-house library development

19

Suspect List: No ref. standards available

Known compounds:

Molecular formula

Name

Source:

Published literature

EMCDDA, TICTAC London,

EWS, UNODC

In-house library development

Qualitative methodPart I: Analytical Methods

(Kinyua et al., ABC 2015)

Schymanski et al. Environmental Science and Technology (2014) 48(4):2097

Confidence communicationPart I: Analytical Methods

22

IDENTIFICATION OF BIOMARKERS OF EXPOSURE

Part II

23

In vitro studies

Part II: NPS Biomarkers (In vitro)

Incubations of NPS with human liver fractions (microsomes, cytosol) + rCYPs + co-factors for Phase I and II metabolism

Analysis and elucidation of metabolic pathways by LC-QToFMS- High resolution MS: accurate mass ~ molecular formula fragmentation pattern ~ molecular structure

(Van den Eede et. al.,TAAP 2015;Lai et al., JPBA 2015; Negreira & Kinyua ABC 2016)

Nitracaine (N-):deethylation, di-

deethylation, hydroxylation, and de-esterification

CYP2B6 and CYP2C19-main enzymes in Nitracainemetabolism

Phase II: Glucuronidation

Added metabolites to our library

O

O

N

NO

O

O

O

NH

NO

O

O

O

NH2

NO

O

HO N

HO NH

Nitracaine

M4

M3

M1

rCYP2B6rCYP2C19

rCYP2B6rCYP2C19

HO N

M2rCYP2B6

rCYP2B6rCYP2C19

OH

O

NO

O

O

OH O

HOOH

N

O

HO

GLU

not CYP-mediated metabolism

not CYP-mediated metabolism

O-

OH

O

H2N

O

O

NO

O

OHO

OH

OH

O

OH

NH

O

NO

O

NH2

O

OHO

NH

O

NO

O

O

OH

Nitro-reduction Glutamineconjugation

Glucuronideconjugation

TP-GLU

p-aminobenzoic acid

p-nitrobenzoic acid

TP-Glutamine

TP-Glutamine derivedp-nitrobenzoic acid-GLU

* not detected in urine# only detected in urine

##

#

(Negreira & Kinyua, ABC 2016)

Part II: NPS Biomarkers (In vitro)

25

In sewer

Microorganisms in the biofilm In sewer degradation of NPS? Explore stability in presence of biofilm (Eawag) Identify transformation products (TPs) formed

(UA)

Biofilm lining sewer walls

(McCall et al., WR 2016)

Part II: NPS Biomarkers (In sewer)

26

* % Degradation

Experiment I:

Mixed NPS spike + biofilm reactors

LC-MS/MS analysis

High Stability

(0-20 %)*

Medium Stability

(20-60 %)*

Low Stability

(60-100 %)*

Experiment II:

Individual NPS spike +

Individual biofilm Reactor

Quantitative

Relevant NPS Relevant NPS

Tentatively identified TPs +

Proposed biotransformation

Qualitative

Proposed biomarkers

(Level 1 and 2 confirmation)

TP identification

(Suspect and Non-target screening)

LC-QTOFMS analysis


(McCall et al., WR 2016)

0

20

40

60

80

100

120

0 1 2 4 6 8 10 12 14 16 18 20 22 24

% C

once

ntra

tion

Incubation time (hrs)

MXE

MPA

PMMA

PMA

MDPV

Mephedrone

KET

NK

Reactor I- nanopure H2O Reactor III- big sewer biofilm

0

20

40

60

80

100

120

140

0 1 2 4 6 8 10 12 14 16 18 20 22 24

% c

hang

e in

con

cent

ratio

n


MXE

MPA

PMMA

MDPV

Mephedrone

KET

NK

Reactor II- small sewer biofilm

0

20

40

60

80

100

120

140

0 1 2 4 6 8 10 12 14 16 18 20 22 24

% C

once

ntra

tion

Incubation time (hours)

MXE

MPA

PMMA

PMA

MDPV

Mephedrone

KET

NK

Reactor IV- sewage (no biofilm)

0

20

40

60

80

100

120

140

160

0 1 2 4 6 8 10 12 14 16 18 20 22 24

% C

once

ntra

tion


MXE

MPA

PMMA

PMA

MDPV

Mephedrone

KET

NK

28

Experiment I:

Mixed NPS spike + biofilm reactors

LC-MS/MS analysis

High Stability

(0-20 %)*

Medium Stability

(20-60 %)*

Low Stability

(60-100 %)*

Experiment II:

Individual NPS spike +

Individual biofilm Reactor

Quantitative

Relevant NPS Relevant NPS

Tentatively identified TPs +

Proposed biotransformation

Qualitative

Proposed biomarkers

(Level 1 and 2 confirmation)

TP identification

(Suspect and Non-target screening)

LC-QTOFMS analysis


(Kinyua et al., Submitted)

Synthetic Cathinones: MDPV: Four transformation

products (TPs)- Reduction, O-

demethylation, dihydroxylation reactions

Methylone: Five TPs- Reduction, (N, O)-

demethylation, N-hydroxylation

Mephedrone: Three TPs- Reduction, N-

demethylation


(Kinyua et al., Submitted)

NH2 O

OH

O NH2 O

O

OHTP-210 a

C11H16NO3+

Exact Mass: 210.1125

H3N

O

O

O

TP-194C10H12NO3

+


HN

O

O

O

OH

TP-224 C11H14NO4

+


O

NH2

O

O

MethyloneC11H14NO3+Exact Mass: 208.0968

OH

NH2

O

O

TP-210 (b and c)C11H16NO3+Exact Mass: 210.1125

Reduction

OH

NH2

O

O

N -hyd

roxy

lation

N-demethylation

O- d

emet

hyla

tion

Identification of suspicious preparations

Seizures and confiscations from police Found at scene of intoxication/death Amnesty bins at festivals, clubs, events (some countries)

Part II: NPS Biomarkers

Suspicious drug preparations (tablet)

7x10

0

0.2

0.4

0.6

0.8

1

1.2

Counts vs. Acquisition Time (min)2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Proxorphan ?

Vetrabutine ?Oxilorphan?

x105

02.5

5+ESI EIC(300.1948) Precursor ion

x104

0

5

+ESI EIC(107.0491) Product ion

x105

0

1


x104

02.5

5+ESI EIC(135.0813) Product ion

x105

012


Counts vs. Acquisition Time (min)13.2 13.4 13.6 13.8 14 14.2 14.4 14.6

Part II: NPS Biomarkers

32

In vivo samples

User Samples:Urine, blood, serum and plasma

Post-mortem Drugs in traffic Hospital emergency

HybridSPE precipitation IS +100l sample + 300 ACN(0.1%FA) (1:3) 500 l centrifuge filter 8000 rpm for 5 min HybridSPE cartridge

Part II: NPS Biomarkers (In vivo)

6x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

+ESI EIC(180.1384) Scan G2309_POS.d Smooth

8.02

Counts vs. Acquisition Time (min)

2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8 9

5x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

+ESI EIC(166.1221) Scan G2309_POS.d Smooth

3.70

3.197.02

Counts vs. Acquisition Time (min)

2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8

NH3

O

p-methoxyamphetamine C10H16NO+Exact Mass: 166.1226

NH3

OH

p-OH-amphetamine C9H14NO

+


NH2

OH

p-OH-methamphetamine C10H16NO

+


O

NH2

p-methoxymethamphetamine C11H18NO

+


Case Study IUrine sample

PMMA metabolite forms : p-methoxyamphetamine (PMA) p-OH-methamphetamine p-OH-amphetamine(Peters, Schaefer, Staack, Kraemer, & Maurer, 2003) (Baselt, 2014)(Lai et al., 2015)

Compound LevelPMMA 1PMA 1

p-OH-amphetamine 2ap-OH-methamphetamine 2a

3,4-methylenedioxymethamphetamine (MDMA)

13,4-methylenedioxyamphetamine (MDA) 1

4-hydroxy-3-methoxyamphetamine (HMA) 2a4-hydroxy-3-methoxymethamphetamine

(HMMA) glucuronide 2a3,4-dihydroxymethamphetamine (DHMA) 2b

HMMA-sulphate 2a

para-methoxymethamphetamine (PMMA) Part II: NPS Biomarkers (In vivo)

34

MONITORING STUDIESPart III

Target social settings alcohol and drugs used

Themed Festivals

> 20,000 attendees

Part III: Monitoring of NPS

- Sample urine from portable urinals in Target areas

- Higher concentrations

- Target metabolites

- No female samples

Frequently used NPS

> 570 NPS

Pooled Urine Analysis

(Mardal et al., DTA 2016)(Kinyua et al., STOTEN 2016)


Sampling: Pooled Urine City:

London, UK

Festivals: Boom, BE London, UK Copenhagen, DK


Mainstage area

BE Festival July 2015

Friday 6:00 PM; 9:00 PMSaturday-Sunday 6:00 PM; 9:00 PM; 12:00 AM

Sunday 6:00 PM; 9:00 PM

UK City and Festival 2013, 2014Sampling period Site Samples25 -28/10/2013 City 413 -16/12/2013 City 6

17 -20/07/2014 Festival 10

Festivals:

Zurich, CH Boom, BE Amsterdam, NL

Sampling: Sewage

Festival Sampling days Sample type Zurich, CH 2014; 2015

Thursday -Monday 24 h Composite

Boom, BE July 2015 Friday-Monday 24 h Composite

Amsterdam, NL 2012; 2014 Thursday- Sunday24 h Composite


39

Method: Analysis

Pooled urine:- Target + Suspect analysis using LC-QToFMS

(WORKFLOW)

Sewage samples:- Target analysis using LC-MS/MS- Target + Suspect analysis using LC-QToFMS of HLB

extracts (WORKFLOW)


Results: Pooled urine

DOA, NPS, pharmaceuticals and metabolites

UK festival (number of attendees ~ 35,000)- 53 compounds (8 NPS)- incl. MPA, (m)ethylone, MEPH, 5-APB, MXE


(Kinyua et al., STOTEN 2016)

BE festival (number of attendees ~ 80,000)- 28 compounds (3 NPS)- incl. 4-FA, -PVP(a.k.a flakka) and ketamine

Results: Pooled urine


(Kinyua et al., STOTEN 2016)

Results: Sewage


AMS Street Festival- 2012, 2014

We positively identified 8 NPS Classes:

- synthetic cathinones- phenethylamines - opioids

In addition:- benzodiazepine analogues, - classical drugs and - other licit substances with

potential for abuse

1st SBE study to focus on detection frequency- qualitative

(Causanilles & Kinyua, Chemosphere 2017)

Retooling and updating

False positive hits Retention time prediction In silico fragmentation tools

LEVEL 3 CANDIDATES

tR Prediction

2 min

Target MS/MS injection

Search Database: M/Z cloud Massbank Met Frag 2.2

(Specific database)

Met Frag 2.2 (5ppm) Chemspider Pubmed Stoffident

Wastewater Samples

Biological Samples

Blacklist Name tR MS/MS Level 2a or 2b

Yes(Identified)

Yes(Identified)

No No

Purchase reference standard

Purchase reference standard

http://c-ruttkies.github.io/MetFrag/projects/metfragweb/

(Causanilles & Kinyua, Chemosphere 2017)


http://c-ruttkies.github.io/MetFrag/projects/metfragweb/

Summary and Conclusions

Information on biomarkers of exposure - (In vitro, In vivo, In sewer)

Small-scale sampling = tracking current use- Combined approaches PUA + SBE- Events/festivals

Qualitative screening = broader range of NPS- Re-tooling to enhance compound identification- Unavailability reference standards- Retrospective analysis (DIA)

Quantitative screening for established NPS

General Conclusions

Stakeholders

EMCDDA BEWSD Forensic Toxicology Dept. ZNA (Brussels) and UZA (Antwerp) hospitals Law enforcement

General Conclusions

Additional studies

Sewer-scale transformation modeling Analytical method development Back-calculation modeling Drug Survey vs SBE Biomarker identification Drug-user forums

http://sewprof-itn.eu/

Thank you

Seminar attendees

University of Antwerp - Toxicological Center: Environmental and Forensic team

Members of SEWPROF team and Marie Curie ITN SEWPROF Project Grant No.317205

Investigating community use of new psychoactive substances using wastewater-based epidemiology

Juliet Kinyua, Toxicological Centre

University of AntwerpBelgium

January 20,2016 Vrije Universiteit

Full scan + product ions in one injection 3 scan segments:

0 eV for parent compounds 15 and 35 eV for product ions

Retrospective analysis

Data-independent acquisition

(Kinyua et al., ABC 2015)

5050

Qualified Suspects

Correlated Fragments

Fragment elucidation

Tentative/ Probable

Confirmation

Workflow: Suspect Confirmation

Screening of new psychoactive substances in biological matrices and sewage to monitor community-level consumptionOutlineIllicit DrugsTraditional methods used to estimate illicit drug use Sewage-Based Epidemiology (SBE)Slide Number 6Slide Number 7SEWPROF PROJECTNew Psychoactive SubstancesNPSNew Psychoactive SubstancesWhy NPS?Aims of the ProjectAnalytical methodsQuantitative method1st application studyChallengesDifferent approach Qualitative methodSlide Number 19Slide Number 20Slide Number 21Slide Number 22Identification of Biomarkers of exposure In vitro studiesSlide Number 25In sewerSlide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31Slide Number 32In vivo samplesSlide Number 34Monitoring studiesSlide Number 36Slide Number 37Sampling: Pooled UrineSlide Number 39Method: AnalysisResults: Pooled urineResults: Pooled urineResults: SewageRetooling and updatingSummary and ConclusionsStakeholdersAdditional studies Slide Number 48Investigating community use of new psychoactive substances using wastewater-based epidemiologyData-independent acquisitionSlide Number 51

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