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

Conceptual Framework

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)

Conceptual Framework

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)

Part I: Analytical Methods

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?

Part I: Analytical Methods

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

Part I: Analytical Methods

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

Part II: NPS Biomarkers (In sewer)

(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

Incubation time (hrs)

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

I