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: Juliet.Kinyua@uantwerpen.be

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