Maurice Wilde1,2 and Volker Auwärter1 1 Forensic Toxicology, Institute of Forensic Medicine, Medical Center – University of Freiburg, Freiburg, Germany 2 Hermann-Staudinger-Graduate School, University of Freiburg, Freiburg, Germany

Institute of Forensic Medicine Forensic Toxicology

IRM

Analytical challenges in the forensic

toxicological analysis of novel synthetic opioids

from the class of ‘U-drugs’

In total, nine novel synthetic opioids from the class of so-called ‘U-drugs’ (developed by Upjohn), another class of new opioids besides fentanyl

analogues, have been reported to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) until the end of 2018. The unambiguous

identification of these compounds or their metabolites is often hampered by structural similarities (e.g. isomeric metabolites). The aim of this study was

to investigate the phase I in vitro metabolism of eight compounds using pooled human liver microsomes (pHLM) and to identify isomeric and specific

metabolites.

Structural isomeric compounds (parent compounds)

METHODS

reaction mix

(buffer…)

pHLM

Incubation

1 h

37°C

Protein

precipitation

AC

N

Centrifugation

10 min

15.500 x g

Extract evaporation &

Concentrating step

LC

s

olv

en

t

4x

Conc. N2

Substance acquisition (online monitoring) and analyses (structure

verification)

LC-qToF-MS LC parameters

C18 column

Mobile phase:

A H2O (1% ACN, 0.1% HCOOH,

2 mM NH4+HCOO-)

B ACN (0.1% HCOOH, 2 mM NH4+HCOO-)

Gradient elution

MS parameters

MS full scan/bbCID scan; scanrate: 1 Hz, CE: 6/30 (±6) eV

Calibration in HPC mode (external/internal)

Data acquisition: HyStar® (Bruker)

Data processing: DataAnalysis® (Bruker)

0

1

2

3

4

5

0

20

40

60

80

100

0 5 10 15 20 25 30

Flow rate [ml/min]

Mob. Phase B

[%]

Time [min]

Bruker Daltonik

Elute UHPLC system

impact II qToF

instrument

AIMS

RESULTS

CONCLUSION

Sufficient chromatographic separation is essential for valid identification

of ‘U-drugs’. Furthermore, the extensive metabolism of these compounds

may cause interferences by isomers which need to be considered when

interpreting analytical findings, especially when analysing biological

samples.

Maurice Wilde

Medical Center – University of Freiburg

Institute of Forensic Medicine

Albertstraße 9

79104 Freiburg, Germany

maurice.wilde@uniklinik-freiburg.de

[1] European database on new drugs

(EDND) provided by the EMCDDA

https://ednd.emcdda.europa.eu/html.cfm/i

ndex7246EN.html

Literature Contact

Cl

Cl

N H

O N

Cl

Cl

N

O

N Cl

N

O

N

Cl

Br

N

O

N

Cl

Cl

N

O

N Br

N H

O

N Cl

N

O

N Cl

Cl

Cl

N

O

N

AH-7921 Isopropyl-U-47700 N-Methyl-U-47931E U-47700 U-47931E

(Bromadoline)

U-48800 U-49900 U-51754

(Methene-U-47700)

Substrates

In vitro phase I metabolism studies using pooled human liver

microsomes (pHLM)

N

O

O

O N

Cl

Cl

N

O

N

3,4-Methylendioxy-U-47700

U-50488

Six of the investigated ‘U-drugs’

form pairs of constitutional

isomers showing the same exact

masses and partly forming

identical fragment ions in HRMS-

bbCID analysis.

Evaluation of analytical targets

for LC-MS/MS screening method

of opioids

Max. 2.1e6 cps.

4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 Time, min

0.0

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

5.9e6

Intensity, cps

Agilent

6890 series gas chromatograph

5973 series mass detector

7683 B series injector

Bruker Daltonik

Elute UHPLC system

impact II qToF instrument

Bruker Physik

Avance III HD 400 MHz

Sampler: BASC 120

1000 2000 3000 4000 5000 6000 7000 8000 9000

10000

Abundance

100 200 300 360 0 m/z

3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 Time

[min]

0

1

2

3

4

5 6 x10

Intens.

GC-MS spectra

library

Compounds – used for pHLM assay to investigate (in vitro) phase I metabolites Compounds – reported to the EMCDDA until the end of 2018

While U-49900 and Isopropyl-

U-47700 only showed few

identical fragment ions, AH-7921

and U-47700 exhibited a very

similar spectrum of fragment

ions, and were distinguishable

only by low abundant fragment

ions. Furthermore some of the

‘U-drugs’ are regio-isomers, e.g.

U-48800 and U-51754, differing

in the substitution pattern of the

two chlorine substituents (2,4-

vs. 3,4-position), which are not

distinguishable by the MS-

fragment ions (Fig. 2).

Cl

Cl

N

O

N

U-47700

Fig. 1: Fragmentation reactions commonly observed for the

class of ‘U-drugs’, examplified for U-47700.

312 284

232 204

Cl

Cl

N

O

+ Cl

Cl

N

O

+

Cl

Cl

NH2

O

+ Cl

Cl

NH2

O

+

HN +

N

+

2HN +

N

+

110

154

140

126

173

190

81

Cl

Cl

O +

+

Cl

Cl

NH3

O

+

Identical fragment ions

Unique fragment ions Unique fragment ions

Distinguishable by MS fragmentation

Cl

Cl

N

O

N

Isopropyl-U-47700

Cl

Cl

N

O

N

U-49900

Molecular formula: C18H26Cl2N2O Molecular formula: C16H22Cl2N2O

Cl

Cl

N H

O N

AH-7921

Cl

Cl

N

O

N

U-47700

+

+

81 95

Unique

fragment ions

Unique

fragment ions

Identical fragment ions

190 Cl

Cl

O +

Cl

Cl

NH3

O

+ 173

204

Cl

Cl

NH2

O

+ Cl

Cl

N H

O

+ 202/

204

Cl

Cl

N

O

+

Cl

Cl

N H

O

+

284 284

Distinguishable by MS fragmentation, but loss of sensitivity

Molecular formula: C17H24Cl2N2O

Cl

N

O

N Cl

Cl

N

O

N

Cl

U-51754

(Methene-U-47700) U-48800

NOT distinguishable by MS fragmentation

Identical fragment ions

Cl

N

O

+ Cl

+

81

+

81

Cl

N

O

+ Cl

Cl

NH2

O

+ Cl

Cl

NH2

O

+ Cl

Cl O

+

Cl

Cl O

+

Cl

2HN +

2HN +

112 112

218

298

158

298

158 218

Fig. 2: Pairs of isomeric compounds. Unique fragment ions

for each compound are shown within the green box; the

fragment ions in the yellow box are unique by monoisotopic

mass, but analytical interference with a heavier chlorine-

isotopolgue occurs; fragment ions highlighted by the red

boxes are identical by constitution and/or by exact mass.

In vitro phase I metabolites – complexity of analytical findings

driven by ‘second level‘ isomerism

More than a hundred in vitro

phase I metabolites and

potential degradation

products were identified for

the eight investigated

compounds. Besides N-

dealkylation, several

hydroxylated metabolites

either of the parent

compound or the N-

dealkylated metabolites

were detected. Hydrolysis of

the amide function seems to

play a minor role for this

The compounds showed extensive metabolism in the pHLM assay. The main phase I metabolic

reaction observed was N-dealkylation resulting in several isomeric metabolites (Fig. 3).

detected for most of the parent

compounds or their corresponding

N-dealkylated metabolites.

Flipbook: HRMS (fullscan/bbCID) spectra of the investigated parent

compounds and their 5 most abundant metabolites (and suggestions of

the structutal formulae) identified for each of the investigated compounds.

Isomers

Isomers Isomers Isomers

Cl

N2

O

N1 Cl

Cl

N2

O

N1

Cl

Cl

N2

O

1NH Cl

Cl

N2

O

1NH

Cl

Cl

N2

O

1NH2 Cl

Cl

N2

O

1NH2

Cl

Br

N2

O

N1

Br

2N H

O

N1

Br

N2

O

1NH

N-Methyl-

U-47931E

U-47931E (Bromadoline) N-Demethyl-N-Methyl-

U-47931E

Identical metabolites

Br

2N H

O

1NH

N-Demethyl-U-47931E

U-48800 U-51754 (Methene-

U-47700)

N-Demethyl-U-48800 N-Demethyl-U-51754

N,N-Didemethyl-U-48800 N,N-Didemethyl-U-51754

N-d

ealk

yla

tion

N-d

ealk

yla

tion

N-d

ealk

yla

tion

N-d

ealk

yla

tion

N-d

ealk

yla

tion

Cl

Cl

N2

O

N1

Isopropyl-

U-47700

N-d

ealk

yla

tion

Cl

Cl

N2

O

1NH2

N,N-Didemethyl-isopropyl-

U-47700

Cl

Cl

N2

O

N1

U-49900

N-Demethyl-U-49900

N-d

ealk

yla

tion

Cl

Cl

N2

O

HN1

Isomers

Cl

Cl

N2

O

1NH

Cl

Cl

2N H

O 1NH

N-Demethyl-U-47700 N-Demethyl-AH-7921

Cl

Cl

N2

O

N1

U-47700

Cl

Cl

2N H

O N1

AH-7921

N-d

ealk

yla

tion

N-d

ealk

yla

tion

Fig. 3: Map of isomeric in vitro phase I metabolites for the class of ‘U-drugs’ formed by the

most predominant metabolic reaction (N-dealkylation, possibly at N1 position), and the

investigated parent compounds (bold). Yellow boxes frame pairs of isomers, whereas red

boxes highlight the formation of identical metabolites from different parent compounds.

Marked (*) metabolites may show an additional isomer formed by dealkylation, which is not

depicted here (see spectra flipbook). Isomers

Cl

Cl

2N H

O 1NH2

N,N-Didemethyl-AH-7921

N-d

ealk

yla

tion

Identical metabolites

Cl

Cl

N2

O

1NH2

N,N-Dideethyl-U-49900 =

N,N-Didemethyl-U-47700

N-d

ealk

yla

tion

*

* * *

* *

*

N-d

ealk

yla

tion

class of opioids considering

the in vitro data. Dehalo-

genation was detected only

for the bromine-substituted

analoguess U-47931E & N-

methyl-U-47931E, though

the debrominated meta-

bolites showed relatively

low abundances. Moreover,

further oxidation products

such as ketones, diketones,

N-oxides or carboxylic acids

– possibly formed by ring

opening – were also

0

1

2

3

4

5 5

x10

50 100 150 200 250 300 350 m/z

Intens.

Meas. m/z Ion Formula m/z err [ppm] mSigma

81.0696 C6H9 81.0699 3.7 1.1

126.1275 C8H16N 126.1277 1.6 10.5

172.9551 C7H3Cl2O 172.9555 2.7 8.3

189.9815 C7H6Cl2NO 189.9821 2.9 4.8

232.0284 C10H12Cl2NO 232.0290 2.6 13.3

270.0442 C13H14Cl2NO 270.0447 1.9 5.8

312.0911 C16H20Cl2NO 312.0916 1.9 7.5

357.1492 C18H27Cl2N2O 357.1495 0.8 113.3

*

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