S-1

ELECTRONIC SUPPLEMENTARY MATERIAL

ANALYTICAL STRATEGY TO INVESTIGATE 3,4-

METHYLENEDIOXYPYROVALERONE (MDPV) METABOLITES IN

CONSUMERS’ URINE BY HIGH RESOLUTION MASS

SPECTROMETRY

María Ibáñez1*, Óscar J. Pozo2, Juan V Sancho1, Teresa Orengo3, Gonzalo Haro4,5,Félix

Hernández1

1Research Institute for Pesticides and Water, University Jaume I, Avda. SosBaynat, E-

12071 Castellón, Spain.

2Bioanalysis Research Group, IMIM, Hospital del Mar Medical Research Institute,

Doctor Aiguader 88, 08003 Barcelona, Spain

3Addictions Treatment Unit Grao-Clínico, Valencia, Spain

4School of Medicine, University CEU-Cardenal Herrera, Castellón

5Psychiatry Department, Consorcio Hospitalario Provincial de Castellón

* Corresponding author ibanezm@uji.es, Tel +34 964 387339, Fax +34 964 387368

S-2

Fig S.1. Extracted Ion Chromatograms (XICs) for all detected metabolites.

S-3

Metabolite M1

Figure S.2. Product ion spectrum of M1 (20 eV) and proposed fragmentation pathway.

m/z 193

m/z 264

m/z 175

M1

- H2O

-

- CH3CH2CH2•

N

H

O

O

m/z 137

m/z 221

N

m/z 264

m/z 126

N H

m/z 126

H

N

H

m/z 84

m/z 193

m/z 123

O

NHO

HO

O

NHO

HO

H

O

NHO

HO

H

O

O

HO

H

O

HO

HO

O

NHO

HO

H

N

HH

m/z 72

HO

HO

O

H

HO

HOm/z 165

- CO

HO

HO

S-4

Metabolite M2

Figure S.3. Product ion spectrum of M2 (20 eV) and proposed fragmentation pathway.

m/z 207

m/z 278

m/z 175

M2

- H2CO

-

- CH3CH2CH2•

N

H

O

O

m/z 151

m/z 235

N

m/z 278

m/z 126

N H

m/z 126

H

N

H

m/z 84

m/z 207

m/z 137

O

NHO

CH3O

O

NHO

CH3O

H

O

NHO

CH3O

H

O

O

CH3O

H

O

HO

CH3O

O

NHO

CH3O

H

HO

CH3O

O

H

HO

CH3O

S-5

Metabolite M3

Figure S.4. Product ion spectrum of M3 (20 eV) and proposed fragmentation pathway.

O

NHO

HO

m/z 193

m/z 278

m/z 175

M3

- H2O

-

- H2O

O

O

m/z 137

m/z 278

m/z 140 m/z 140

m/z 98

m/z 193

m/z 123

O

O

HO

H

O

HO

HO

m/z 86

HO

HO

O

H

HO

HO

m/z 278

O

NHO

HO

OH

H H

m/z 260

O

NHO

HO

O

N

H

O

O

NHO

HO

H

O

NO N HO

H

N

H

O

O

NHO

HO

HO

N

HH

O

S-6

Metabolite M5

O

NHHO

CH3O

H

OHO

O

NHHO

CH3O

OHO

m/z 292

M5

m/z 140 m/z 140

m/z 98

NO N HO

H

N

H

O

-N

H

O

m/z 310

OHO

m/z 87

- H2O

m/z 310

NH

OHO

m/z 158

O

NH2HO

CH3O

OHO

O

NHO

CH3O

HO

m/z 207

m/z 175

- H2CO

O

O

m/z 151

m/z 207

m/z 137

O

O

CH3O

H

O

HO

CH3O

HO

CH3O

O

H

HO

CH3O

OHO

m/z 87

- NH3

m/z 104

OHO

NH3

O

NHHO

CH3O

OHO

NH2

O

NHO

NH

O

OH

O

NO

O

HO

CH3O

N

O

H

HO

CH3O

NH

O

OH

HO

CH3O

NH

OH

O

OH

HO

CH3O

NH

OH

O

OH

O

NHHO

CH3O

H

OHO

M5

- H2O

m/z 310 m/z 310 m/z 310

- H2O

m/z 292

OO-

- H2CO

OO-

- H2CO

- H2O

m/z 206

m/z 260- H2CO

m/z 242

m/z 274

m/z 174

- HCOOH

m/z 214

m/z 232

-

HO

CH3O

NH2

HO

CH3O

N

O

HO

CH3O

NH

m/z 205

S-7

Figure S.5. Product ion spectrum of M5 (20 eV) and proposed fragmentation pathway.

S-8

Metabolite M6

Figure S.6. Product ion spectrum of M6 (20 eV) and proposed fragmentation pathway.

m/z 207

m/z 292

m/z 175

M6

- CH3OH

-

- H2O

O

O

m/z 292

m/z 140 m/z 140

m/z 98

m/z 207

m/z 86

m/z 292

m/z 274N

H

O

NO N HO

H

N

H

O

N

HH

O

m/z 137

O

NCH3O

HO

O

O

NCH3O

HO

H

O

O

NCH3O

HO

HO

O

CH3O

O

H

O

NCH3O

HO

OH

H H

O

NCH3O

HO

CH3O

HO

O

H

CH3O

HO

S-9

Metabolite M7

Figure S.7. Product ion spectrum of M7 (20 eV) and proposed fragmentation pathway.

m/z 207

m/z 292

m/z 175

M7

- CH3OH

-

- H2O

O

O

m/z 292

m/z 140 m/z 140

m/z 98

m/z 207

m/z 86

m/z 292

m/z 274N

H

O

NO N HO

H

N

H

O

N

HH

O

m/z 137

HO

CH3O

O

NHO

CH3O

O

O

NHO

CH3O

H

O

O

NHO

CH3O

HO

O

O

CH3O

H

HO

CH3O

O

H

O

NHO

CH3O

OH

H H

O

NHO

CH3O

S-10

Metabolite M8

Figure S.8. Product ion spectrum of M8 (20 eV) and proposed fragmentation pathway.

m/z 189 m/z 217

m/z 175

M8

- H2O

-

- CH3CH2CH2•

N

H

m/z 159

m/z 278

m/z 260

m/z 207

N

HH

m/z 72

OH

NO

O

OH2

NO

O

m/z 278

NO

O

- H2O

OH

NO

O

H

OH

O

O H

- CH3OH

- H2CO

NO

O

O

O

O

O

O

N

m/z 126

N H

m/z 126

H

N

H

m/z 84

S-11

Metabolite M9

HO

CH3O

O

H

O

NHO

CH3O

H

m/z 205

m/z 173

M9

- H2CO

- N

Hm/z 294

m/z 142

m/z 205

m/z 137

HO

CH3O

O

NHO

CH3O

OH O

NHO

CH3O

H OH

N

OH

m/z 294 - H2O

O

NHO

CH3O

H

OH

- CH3CH=CH2

O

O

CH3O

H

m/z 151

O

HO

CH3O

m/z 276

- N

H

O

NHO

CH3O

m/z 234

O

O

CH3O

H

m/z 205

O

CH3O

H

O

m/z 205

- CH2=CH2 O

CH3O

H

O

m/z 177

- H2O

O

HO

CH3O

OH

m/z 223

- H2O

m/z 216

O

N

O

O

O

M9

O

NHO

CH3O

OH

m/z 294

O

NHO

CH3O

H

OH

- H2O

m/z 276

- CO

N H

m/z 98 m/z 248

- CH4

HN

HO

CH3O

m/z 232

- CH3OH

O

H

N

m/z 200

- CH≡CHN

HH

m/z 72

O

CH3O N

H

H

H

O

NHO

CH3O

H

S-12

Figure S.9. Product ion spectrum of M9 (20 eV) and proposed fragmentation pathway. Only fragmentation corresponding to one of the two possible candidates (concretely, 1-(4-hydroxy-3-methoxyphenyl)-3-hydroxy-2-(1-pyrrolidinyl)pentan-1-one) is shown, but the implied pathways for both candidates will be “identical”.

S-13

Metabolite M10

Figure S.10. Product ion spectrum of M10 (20 eV) and proposed fragmentation pathway. Only fragmentation corresponding to the most feasible candidate (concretely 1-(4-hydroxy-3-methoxyphenyl)-2-(2-hydroxypyrrolidinyl)pentan-1-one is shown, but the implied pathways for both candidates will be “identical”.

m/z 207

m/z 294

m/z 175

M10

- H2CO

-

- CH3CH2CH2•

O

O

m/z 151

m/z 276

m/z 294

m/z 142 m/z 142

m/z 100

m/z 207

m/z 137

O

O

CH3O

H

O

HO

CH3O

HO

CH3O

O

H

HO

CH3O

O

NHO

CH3O

H

OH

NOH

N HHO

H

N

H

OH

N

H

OH

O

NHO

CH3O

HOH

N

HH

OH

m/z 88

- H2O

m/z 70

N

HH

O

NHO

CH3O

OH

- H2O

O

NHO

CH3O

H

m/z 251

O

NHO

CH3O

HOH

m/z 248

N HCH3O

HO