Forensic Toxicology
In vitro incubation
immediately to 10% (over 1 min) prior to re-equilibration.
The total analysis time was 10 min.
The HLM method was based on that previously described
Heated electrospray ionisation (HESI-II) settings were:
sheath gas ꢁow-rate 70; auxiliary gas ꢁow-rate 10; spray
voltage 3.75 kV; capillary temperature 320 °C; S-lens radio
frequency level 55.0; auxiliary gas temperature 350 °C. Full-
scan acquisition parameters were: positive ionisation mode;
scan range m/z 100–1000; resolution 70,000 full width at
half maximum (FWHM); automatic gain control (AGC) tar-
[
(
19]. Brieꢁy, solutions containing 10 µg of each substrate
1 mg/mL, 10 µL) were added to Eppendorf LoBind™
tubes (Stevenage, UK), and evaporated to dryness. Buꢃer
solution (0.1 mol/L aqueous phosphate buffer, pH 7.4,
5
00 µL), NADPH-regenerating solutions [50 µL solution
+
A: 26 mmol/L NADP , 66 mmol/L glucose-6-phosphate,
6
2
and 66 mmol/L MgCl (aq); 10 µL solution B: 40 U/mL
get 1×10 . MS acquisition parameters were: parallel reac-
tion monitoring mode (based on an inclusion list containing
the appropriate precursor ions); scan range m/z 100–750;
2
glucose-6-phosphate dehydrogenase in 5 mmol/L sodium
citrate], and deionised water (390 µL) were added. The tubes
were capped, vortex-mixed, and preincubated (37 °C) with
continuous gentle shaking for 10 min using an Eppendorf
6
resolution 35,000 FWHM; AGC target 1 × 10 . The isola-
tion width was 1.2 amu. The normalised collision energy
(NCE) was optimised manually using available standards
and in-house synthesised products. For data review, total ion
chromatograms were extracted using a± 2.5 ppm extraction
window based on the exact m/z values.
®
ThermoMixer (Stevenage, UK). Reactions were initiated
by the addition of HLM solution [protein content 20 mg/mL
in 250 mmol/L sucrose (aq), 50 µL], gently mixed by hand
®
and were incubated using an Eppendorf ThermoMixer (2 h,
total reaction volume 1000 µL). A negative control was pre-
pared by incubating each of the compounds with buꢃer solu-
tion and water, omitting the HLM and NADPH solutions.
For incubations with ethanol, the initial volume of deionised
water added to the incubation mixture was reduced to 340
µL, and aqueous ethanol solution [5.86% (v/v), 50 µL] was
added. The ꢀnal concentration of ethanol was 50 mmol/L
Cannabinoid receptor assay
To assess the biological activities of the compounds, live
cell-based reporter assays that monitor protein-protein inter-
actions via the NanoLuc Binary Technology were used.
Here, the receptor activation is evaluated via the interac-
tion between β-arrestin 2 (βarr2), a cytosolic protein, and
the cannabinoid receptor CB or CB . Both βarr2 and CB /
(
0.23 g/dL). In addition, incubations were carried out in the
presence of speciꢀc carboxylesterase (CES), and nonspe-
ciꢀc esterase inhibitors (BNPP and saturated NaF solution,
respectively). For both experiments, the initial volume of
deionised water added to the incubation mixture was reduced
to 290 µL, and aqueous ethanol solution [5.86% (v/v), 50 µL]
and inhibitor solution [either 1 mmol/L BNPP, or 10% (w/v)
NaF in water, 50 μL] was added. The ꢀnal concentration of
ethanol was maintained at 50 mmol/L.
1
2
1
CB are fused to an inactive part of nanoluciferase. When
2
CB or CB are activated by a ligand, βarr2 is recruited to
1
2
the receptor, allowing interaction of the complementary
nanoluciferase subunits, yielding a functional enzyme that
generates a bioluminescent signal in the presence of the sub-
strate furimazine. Details regarding the development of the
stable CB and CB cell lines used here have been reported
1
2
After incubation, all reactions were quenched by the
addition of ice-cold acetonitrile (250 μL). Quenched solu-
tions were vortex-mixed and centrifuged (13,000 g, 15 min).
Supernatants were transferred to glass autosampler vials for
analysis by LC–HRMS (5 μL injection volume).
elsewhere [36]. The original human embryonic kidney
(HEK) T293 cell line was provided by Prof. O. De Wever
(Laboratory of Experimental Cancer Research, Department
of Radiation Oncology and Experimental Cancer Research,
Ghent University Hospital, Belgium).
The cells were routinely maintained at 37 °C, 5% CO ,
2
LC–HRMS
under humidiꢀed atmosphere in Dulbecco’s modiꢀed Eagle’s
medium (GlutaMAX™; Thermo Fisher Scientiꢀc, Waltham,
MA, USA) supplemented with 10% heat-inactivated fetal
bovine serum (FBS), 100 IU/mL penicillin, 100 mg/L strep-
tomycin and 0.25 mg/L amphotericin B. Stability of the cell
lines was followed by ꢁow cytometric analysis. For experi-
ments, cells were deposited on poly-d-lysine coated 96-well
An UltiMate 3000™ HPLC system coupled to a Q
Exactive™ high-resolution mass spectrometer (both Thermo
Scientiꢀc, San Jose, CA, USA) was used. Xcalibur™ soft-
ware (version 14.0) was used for instrument control and
data analysis. Chromatographic separation was performed
using an Acquity™ BEH C18 column (50 × 2.1 mm i.d.,
4
plates (Thermo Fisher Scientiꢀc) at 5 × 10 cells/well and
1
0
.7 μm) (Waters, Borehamwood, UK). Eluents consisted of
incubated overnight. The cells were washed twice with Opti-
®
.3% (v/v) formic acid in deionised water (eluent A) and
MEM I Reduced Serum Medium (Thermo Fisher Scien-
®
in acetonitrile (eluent B). Column temperature was main-
tained at 40 °C. The elution gradient started at 10% eluent
B (2 min), ramped to 95% (over 5 min), and then decreased
tiꢀc) to remove any remaining FBS, and 100 μL Opti-MEM
I was added. The Nano-Glo Live Cell reagent (Promega,
Madison, WI, USA), a nonlytic detection reagent containing
1
3