reaction vessel was kept in an ice bath (0°C). Samples were taken
[18 H, s(m), HG-I + P-T + O or J]; ␦ 1.61 (2 H, m, HJ or O); ␦ 1.81 (2 H,
tt, J = 7.2 Hz and 7.6 Hz, HF); ␦ 1.97 (4 H, m, HN + K); ␦ 2.41
[2 H, apparent t, J = 7.4 Hz (average value), HE]; ␦ 3.78 (2 H, dt,
J = 5.6, HC); ␦ 4.03 (2 H, t, J = 5.6 Hz, HB); ␦ 4.31 (2 H, m, J =
5.6-7.2, HL + M); ␦ 8.43 (1 H, broad s, HD). As for the spectrum of
OEA, minor peaks at ␦ 2.93, ␦ 7.26, ␦ 7.63, ␦ 8.48, and ␦ 8.74 and
a broad peak at ␦ 5.33 were attributed to the solvent.
from the reaction vessel at 0 min (before addition of chlorine
gas), 15 min, and 60 min, before being diluted 400 times with
MeCN and subjected to LC-MS analysis for the expected reaction
product (9,10-dichloro-SEA) and remaining OEA.
LC-MS and LC-MS/MS. LC analyses of samples were per-
formed on a Thermo Finnigan Surveyor HPLC-system coupled to
a TSQ Quantum ultra AM triple quadrupole (Thermo Finnigan,
San Jose, CA). The mobile phases consisted of milli-Q water with
0.05% formic acid (mobile phase A) and MeCN with 0.05% for-
mic acid (mobile phase B). Elution was performed using a flow of
200 µl/min and running a gradient of 60% B to 100% B over 6
min, followed by 1 min at 100% B before returning to 60% B over
0.5 min. The column compartment was kept at 40°C. All MS anal-
yses were run using ESI in the positive ionization mode. The ion-
ization settings were as follows: spray voltage, 4000 V; sheath gas
pressure, 50 (arb); ion sweep gas pressure, 0 (arb); Aux gas pres-
sure, 20 (arb); capillary temperature, 350°C; capillary offset, 1
(arb); and skimmer offset, 5 (arb).
For isotopic pattern spectra of OEA and the reaction product
with m/z 396 (9,10-dichloro-SEA), the MS was set to perform a
full scan in Q1 with a scan speed of 0.7 s, resolution of 0.25
FWHM, and scan ranges of m/z 320–335 or m/z 390–405 for OEA
and the reaction product, respectively. Simulated isotopic pat-
tern spectra were calculated using Xcalibur Qual Browser soft-
ware version 2.0 (Thermo Electron Corporation).
RESULTS
Validation of commercial SPE columns
Four commercially available SPE columns with silica sor-
bents were investigated with respect to recoveries of 2-AG
and seven different NAEs at clinically relevant concentra-
tions (Fig. 2). The results showed significant differences in
retention and recoveries between SPE columns despite
the fact that the columns were filled with the same type of
sorbent (i.e., silica). For most of the compounds, the col-
umns showed average recoveries of 95–120%. However,
the recoveries were lower than 100% for EPEA and DHEA
on the Phenomenex columns (58% and 83%, respectively)
and generally for OEA on all columns (81–93%). When
looking at overall retention during the wash procedure,
the Phenomenex columns showed the best performance
for all the studied compounds, whereas the poorest per-
formance was with the Isolute columns that lost between
14% (PEA) and 93% (2-AG) during the wash procedure.
For AEA, EPEA, and DHEA, the losses during the wash
step were considerable on the Supelco, Waters, and Iso-
lute columns (average losses: 34% for AEA; 33% or EPEA;
41% for DHEA). None of the columns were able to satisfy-
ingly retain 2-AG, because most of the compound was lost
in the wash step on all columns (losses: 39% on Phenom-
enex; 71% on Waters; 86% on Supelco; 93% on Isolute).
The results for 2-AG notwithstanding, the average results
for the investigated compounds on the respective SPE col-
umns were (wash loss % SD; total recovery % SD): Su-
pelco (11.7% 11.4%; 110.3% 17.1%), Waters (18.9%
10.2%; 95.9% 8.4%), Isolute (35.3% 19.4%; 108.4%
9.5%), and Phenomenex (2.2% 1.6%; 86.5% 14.8%).
Most interesting were the results showing the abilities,
or lack thereof, of the individual columns to retain the poly-
unsaturated compounds, which are normally present in
low concentrations in biological tissues [e.g., brain (7, 8)],
i.e., AEA, EPEA, DHEA, and LEA. For these compounds,
the Isolute SPE columns already lost on average 55% during
the wash step (range 21–62%), whereas the average losses
on the other columns for these compounds during the wash
step were 24% (Supelco; range 3–29%), 27% (Waters; range
12–33%), and 4% (Phenomenex; range 1–4%).
For MS/MS analyses of OEA and 9,10-dichloro-SEA, the set-
tings were: OEA: isolation m/z, 326.250; collision energy, 25 (arb);
q2 gas pressure, 2.0 (arb); Q1 = 0.50 FWHM and Q3 = 0.5 FWHM;
Q3-scan m/z, 30–335. Settings for 9,10-dichloro-SEA: isolation
m/z, 396.250; collision energy, 25 (arb); q2 gas pressure, 2.0 (arb);
Q1 = 0.50 FWHM and Q3 = 0.5 FWHM; Q3-scan m/z, 30–405.
NMR analyses. 1H-NMR spectra were acquired using a
Bruker Avance 400 WB (Bruker Biospin, Karlsruhe, Germany)
operating at 400.13 MHz for 1H, equipped with a 1 mm TXI (1H
observe, 13C, 77Se decouple). Samples were dissolved in d5-pyridine
in a standard 1 mm NMR tube at T = 310.2. Sixty-four transients
for OEA or 128 for 9,10-dichloro-OEA were added. The 90° pulse
was 5.75 µs at 9 dB dampening. A relaxation delay of 1.0 s was
used, and the FID was collected using 32,768 data points in the
time domain. A 1 Hz exponential line broadening was applied,
and the FID was Fourier transformed using 131,072 data points
for the real part of the transformed spectrum. For 9,10-dichloro-
OEA, a phase-sensitive COSY spectrum was acquired using a stan-
dard pulse program from the Bruker library (cosyph). The 90°
pulse was 5.75 µs at 9 dB dampening. A relaxation delay of 1.0 s
was used. A total of 512 increments in t1 each of 96 added tran-
sients were acquired using 1,024 points in t2. A squared unshifted
sinebell (QSINE, SSB = 0) was applied in both dimensions before
2D Fourier transformation. Then 2,048 points (real part of the
transformed spectrum) were used for both dimensions allowing
cross peaks for small couplings to be observed. (All NMR spectra,
except the H-NMR spectrum of 9,10-dichloro-SEA, are presented
in the supplementary data section).
1H-NMR of OEA gave the following result: ␦ 0.9 (3 H, s, HU); ␦
1.31–1.40 (20 H, broad s, HG-J+O-T); ␦ 1.83 (2 H, tt, J = 6.8 Hz and
J = 7.2 Hz, HF); ␦ 2.12 (4 H, tt, J = 6.4 Hz and J = 6.8 Hz, HN+K);
␦ 2.42 (2 H, t, J = 7.4 Hz, HE); ␦ 3.75 (2 H, apparent dt, J = 5.6 Hz,
HC); ␦ 4.02 (2 H, t, J = 5.6 Hz, HB); ␦ 5.50 (2 H, broad s, HL+M);
␦ 8.39 (1 H, broad s, HD). Minor peaks at ␦ 2.93, ␦ 7.26, ␦ 7.63,
␦ 8.48, and ␦ 8.74 and a broad peak at ␦ 5.33 were attributed to
the solvent.
Identification of SEA and PEA in solvents used for
sample preparation
Based on previous, unpublished experiments, where
PEA and SEA were found in allegedly blank samples, it was
investigated whether PEA and SEA could be found in blank
CHCl3, ethyl acetate, or hexane used for sample prepara-
tion. After evaporation of increasing volumes (15, 30, and
60 ml) of the solvents and subsequent reconstitution in 100
1H-NMR of 9,10-dichloro-SEA gave the following result: ␦ 0.91
[3 H, apparent t, J = 6.8 Hz (average value), HU]; ␦ 1.28–1.64
Pitfalls in the analysis of N-acylethanolamines
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