Mass spectrometry of efaproxiral
333
4
-[[(3,5-dimethylanilino)carbonyl]methyl]phenol. The solid
O
O
O
O
was filtered and washed with 20 ml of 0.1 M aqueous HCl,
0 ml of water, extracted into 30 ml of 1 M aqueous NaOH
O
OH
OH
2
N
N
and washed with 10 ml of t-butyl methyl ether. The desired
product was precipitated by acidification of the aqueous
layer using 6 M aqueous HCl, collected by filtration and
dried in a desiccator over phosphorus pentoxide in vacuo.
To 1.5 g (5 mmol) of 4-[[(3,5-dimethylanilino)carbonyl]
methyl]phenol, 25 ml of acetone and 2.75 g of powdered
sodium hydroxide were added followed by a dropwise
addition of 1.25 ml of chloroform yielding crude efaproxiral.
The product was purified by flash chromatography on
silica gel and fractions containing the desired compound
were evaporated to dryness. The resulting residue was
subsequently precipitated from acetone/n-hexane yielding
an oily semisolid, which was dried in vacuo over phosphorus
pentoxide to give the pale-yellow crystalline product.
H
1
O
H
2
3
O
O
O
*
OH
OH
N
H
O
In order to prepare structurally related or isotopically
labeled compounds, acetone was substituted by 2-butanone,
chloroform by C-chloroform and 3,5-dimethylaniline by
m-toluidine, yielding the compounds 2, 3 and 4 respectively,
as depicted in Scheme 1.
O
O
1
3
N
H
4
Scheme 1. Chemical structures of synthesized and char-
acterized compounds: efaproxiral (RSR13, 1, m.w. D 341),
Electrospray ionization-tandem mass spectrometry
ESI-MS(/MS) was performed on a Thermo LTQ Orbitrap
mass spectrometer employing positive as well as negative
ionization. The instrument was calibrated using the manu-
facturer’s calibration mixture, which allowed for mass accu-
racies <3 ppm in positive and <6 ppm in negative ionization
analyses. Analytes were dissolved in acetonitrile/water (1 : 1,
v : v) at concentrations of 2 µg/ml and introduced into the
mass spectrometer using a syringe pump at a flow rate of
2
-f4-[(3,5-dimethylphenylcarbamoyl)methyl]phenoxyg2-methyl-
13
butyric acid (2, m.w. D 355),
C-labeled efaproxiral (3,
m.w. D 342) – the labeled carbon is marked with an aster-
isk – and 2-methyl-2-[4-(m-tolylcarbamoylmethyl)phenoxy]pro-
pionic acid (4, m.w. D 327).
addition, the implementation of efaproxiral into an existing
screening procedure based on liquid chromatography (LC)
and MS/MS in doping controls is presented.
5
µl/ min. The ionization voltages were 4.5 and ꢀ3.3 kV for
positive and negative ionization, respectively. The capillary
temperature was set to 270 °C, and protonated or depro-
EXPERIMENTAL
tonated precursor ions were dissociated using normalized
collision energies between 25 and 35. Damping gas in the lin-
ear ion trap was helium 5.0, and gas supplied to the curved
linear ion trap (CLT) was nitrogen obtained from a Peak
Scientific NM30L nitrogen generator.
Chemicals and reagents
3
,5-Dimethylaniline(98%), m-toluidine(99%), 4-hydroxyphe-
1
3
13
nylacetic acid (98%), C-chloroform (99 atom% C), thionyl
chloride (97%, distilled before use), 2-butanone (99%) and
sodium hydroxide (powder, 97%) were obtained from
Sigma (Deisendorf, Germany). Acetone, t-butyl methyl
ether and n-hexane were purchased from KMF (St.
Augustin, Germany). Deionized water was of MilliQ grade.
Hydrochloric acid (32%, p.a.), chloroform (p.a.), silica gel
Liquid chromatography–tandem mass
spectrometry
Routine LC–MS/MS analyses were performed on an Agilent
1
100 Series HPLC interfaced by atmospheric pressure
chemical ionization (APCI) to an Applied Biosystems
API2000 triple-quadrupole mass spectrometer. The LC
was equipped with a Macherey–Nagel Pyramid column
6
0 (70–230 mesh) and xylene (p.a.) were obtained from
VWR (Darmstadt, Germany), and Serdolit PAD1 was pur-
chased from Serva Electrophoresis GmbH (Heidelberg,
Germany).
(
4 ð 70 mm, 3-µm particle size), and the eluents used were
5
mM ammonium acetate containing 0.1% acetic acid (mobile
Synthesis of efaproxiral and related compounds
The synthesis of efaproxiral and structural analogs was
accomplished according to established procedures described
phase A) and acetonitrile (mobile phase B). After 3 min
of isocratic flow at 100% A, a gradient was employed to
100% B within 7 min, and the column was re-equilibrated at
100% A for 2.2 min. The flow rate was set to 800 µl/min
with a postcolumn split of approximately 1 : 5. The ion
6
,8
elsewhere.
7 mmol) was converted into the corresponding acetyl
chloride by reflux in 10 ml of thionyl chloride for
0 min. Excess of thionyl chloride was evaporated, 3,5-
Briefly, 4-hydroxyphenylacetic acid (2.6 g,
1
source was operated in the negative mode at 350
°
C, and the
3
analyte efaproxiral was detected by means of characteristic
fragment ions generated from the deprotonated molecule
by CID utilizing the multiple reaction monitoring (MRM)
dimethylaniline (5.7 g, 35 mmol) was added and the mix-
ture was refluxed in 50 ml of xylene for 3 h to give
Copyright 2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 332–338