Analytical challenges in the forensic toxicological analysis of ......Maurice Wilde1,2 and Volker...
Transcript of Analytical challenges in the forensic toxicological analysis of ......Maurice Wilde1,2 and Volker...
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
[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
*
e-poster version:
Please find spectra flipchart
in print version or download
from our homepage via QR-code