Post on 06-Mar-2018
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