Introduction
• New psychoactive substances (NPS) are compounds that
mimic effects of illicit drugs and are produced by
introducing slight modifications to chemical structures of
controlled illicit drugs to by-pass law enforcement.
• Their detection is a challenge due to their transience on
the drug scene.
• Currently more than 450 NPS in the market, synthetic
cathinones and cannabinoids being the most common.
• Which NPS are being used? Which do we monitor?
• LC-QTOFMS provides sensitive full-spectrum MS data for
the identification of known and previously unknown
compounds.
•e
Results and Discussion
I. Sample Preparation
• 20 pooled urine samples collected from various locations in London, UK
• Overnight hydrolysis at 37 °C using β-glucuronidase
• Protein precipitation with acetonitrile (1:2)
• Evaporated and reconstituted in 60 µl H2O/ acetonitrile(98:2, v/v)
Methodology
• More than 30 NPS and metabolites detected. • ≈ 5 hits in each 20 samples after fragment elucidation of which
phenylethylamines are the most common. • Possible metabolite biomarkers were tentatively identified. • Retention time and product ion data can be added to the
database for future use. • Pooled urine analysis (PUA) can be useful to narrow down
which NPS to be purchased for further studies and monitoring. • All-Ions MS/MS combined with a structured workflow is useful
for qualitative screening purposes. • Retrospective analysis of acquired data is possible.
Future Directions
• Include retention time prediction model (Bade,
Bijlsma, Sancho, & Hernández, 2015) to the data
processing workflow.
• Reference standards can be purchased for
frequently detected NPS to confirm retention
time.
• Identified metabolites can be monitored in
influent sewage to detect use in a community
population.
• Data from pooled urine analysis can be used to inform the early warning systems.
Detection and identification of new psychoactive substances in pooled urine using
liquid chromatography coupled to high-resolution mass spectrometry
Juliet Kinyua1, Noelia Negreira
1, Bram Miserez
2, John Ramsey
2, Adrian Covaci
1, and
Alexander L.N. van Nuijs1
1University of Antwerp, Toxicological Center, Wilrijk Belgium
2 TICTAC Communications Ltd. St George's University of London. London, UK.
Acknowledgements We would like to thank the SEWPROF (European Commission and Marie Curie Actions ) Project Grant No.317205 for their fellowship and funding support , the Flanders Scientific Funds for Research (FWO) , University of Antwerp (UA) for their institutional support and particularly the members of the Toxicological Center for their role in this project.
Table 1: Instrumental conditions
Instrument Agilent 1290 Infinity LC, Agilent 6530 QTOFMS
Column Phenomenex Biphenyl (100 x 2.1 mm, 2.6 µm)
Mobile Phase A: 0.04 % formic acid in H2O B: 0.04 % formic acid in (80:20 v/v) acetonitrile/H2O
Gradient 0 min, 2 % B; 2 min, 2 % B; 18 min, 40 % B; 25 min, 90 % B; 29 min, 90 % B; 29.5 min, 2 % B; 33 min, 2 % B
Flow rate 0.4 ml/ min; run time 33min
Injection volume
2 µl
Source Drying gas 350 °C, gas flow 10 L/min, nebulizer 40 psi, Capillary 4000 V , ESI, positive and negative ionization
Acquisition 2.5 spectra/s; scans: 0, 15 and 35 eV with fragmentor at 100 V
II. Instrumentation
III. Data processing workflow
x106
0
0.2
0.4
0.6
0.8
1 Cpd 4: Methiopropamine: +ESI EIC(156.0841)
x105
0
2
4
6
Cpd 4: Methiopropamine: EIC-Frag(58.0663)
5.46
x105
0
2
4
6
8
Cpd 4: Methiopropamine: EIC-Frag(97.0112)
5.46
Counts vs. Acquisition Time (min)
5.2 5.3 5.4 5.5 5.6 5.7 5.8
5.45
Sampling of anonymous urine from urinals placed in target areas
Contact: [email protected]
Phenylethylamines
Synthetic cannabinoids Classic drugs
Compound Hits Identification URB-602 2 Tentative
O-1602/O-1821 1 Tentative
JWH-049/JWH-182/JWH-262/JWH-213/JWH-011 1 Tentative
JWH-368/JWH-307 1 Tentative XLIR-11 1 Tentative O-1918 2 Tentative
Compound Hits Identification
5-MeO-MiPT 3 Tentative
Dimethyltryptamine (DMT)/ α-ethyltryptamine (AET)
1 Tentative
4-OH-MET/4-MeO-DMT/5-MeO-DMT
1 Tentative
O-acetylpsilocin (4-acetoxy-DMT) 8 Tentative
Tryptamines
Compound Hits Identification
Benzoylecgonine 8 Confirmed
Cocaine 3 Confirmed
Dehydronorketamine 1 Confirmed
Ketamine 7 Confirmed
Nor-ketamine 1 Confirmed
Hydroxynorketamine 1 Tentative
EDDP 3 Confirmed
Methadone 2 Confirmed
MDMA 5 Confirmed
Heroin 3 Tentative
Data-independent acquisition on LCQTOF-MS (All-Ions MS/MS)
Data processing algorithm Find by formula (FbF)
Suspect screening Library with (formulae + no MS/MS spectra)
EICs for product ions in spectra at 15eV and 35eV
Qualified product ion elucidation
Tentative confirmation
Qualified product ion elucidation
Compound Hits Identification
3-methoxy-4-methylamphetamine (MMA ) 10 Tentative
2,3-methylenedioxymethcathinone (2,3 MDMC) 2 Tentative
Dibutylone / Eutylone 1 Tentative
Cathinone 1 Tentative
Methylone 2 Confirmed
Ethylone 1 Confirmed
5-(2-aminopropyl) benzofuran (5-APB) 1 Confirmed
Methedrone 1 Confirmed
Methiopropamine 1 Confirmed
M_436_MDPV* 1 Tentative
M_264_α-PVP* 1 Tentative
M_250_α-PVP* 3 Tentative
Pyrrolidinopropiophenone (PPP) 1 Tentative
3,4-dihydroxymethamphetamine 1 Tentative
• Using data-independent acquisition mode, all ions are
fragmented without a specific isolation of a precursor ion
in the first mass analyzer.
• In a single injection, different collision energies can be
applied, providing accurate fragmentation spectra for each
precursor ion.
• This acquisition mode allows retrospective analysis using
the accurate mass full-acquisition and “MS/MS”
information even years after data are acquired.
Objectives:
1. To demonstrate a suspect screening approach based on
data-independent acquisition (All-Ions MS/MS).
2. Demonstrate utility of pooled urine in determining
commonly used NPS.
•e
Identification of methiopropamine and it’s fragment ions with the workflow
Data-independent acquisition on LC-QTOFMS (All-Ions MS/MS)
Data processing algorithm Find by formula (FbF)
Suspect screening Library with (formulae + no MS/MS spectra)
EICs for product ions in spectra at 15 eV and 35 eV
Qualified product ion elucidation
Tentative confirmation
Confirmation with reference standards
*Generated in vitro (Negreira et al., 2015) *Detected in pooled human urine for the first time
