M. Thevis, G. Opfermann and W. Schänzer, Eur. J. Mass Spectrom.

10, 673–681 (2004) 673

N-methyl-N-trimethylsilyltrifluoroacetamide-
promoted synthesis and mass spectrometric
characterization of deuterated ephedrines

Mario Thevis,* Georg Opfermann and Wilhelm Schänzer

Institute of Biochemistry, German Sport University Cologne, Carl-Diem Weg 6, 50933 Cologne, Germany.
E-mail: [email protected]

Synthesis and mass spectrometric characterization of drugs or metabolites labeled by stable isotopes have been of great interest
in the fields of clinical, forensic and doping control analysis. Deuterated ephedrine and p-hydroxypseudoephedrine were pre-
pared from corresponding amines by a novel procedure utilizing N-methyl-N-trimethylsilyltrifluoroacetamide and deuterated
iodomethane. The mechanism of methylation was studied by mass spectrometry using phenylethylamine as a model compound and
a rearrangement based on an intermediate six-membered ring structure with a trimethylsilyl-enol-ether is proposed giving rise
to a leaving group of trimethyliodosilane and the desired monomethylated product. Deuterated analogs to frequently quantitated
ephedrines were readily synthesized with purities > 90% and mass spectra recorded under different ionization and dissociation
conditions presented distinct fragmentation processes, including eliminations of water and methylamine as well as the generation
of a benzyl cation.

Keywords: MSTFA, alkylation, labeling, isotope dilution mass spectrometry, collision-induced dissociation, fragmentation

Introduction as they provide almost identical physicochemical proper-

ties compared with the analytes of interest.25 In the present
For more than five thousand years, the herb “Ma Huang” study, we demonstrate a novel way to synthesize 2H3-ephe-
(Ephedra vulgaris) has been used in China for the treatment drine as well as 2H3-p-hydroxypseudoephedrine promoted
of various afflictions owing to its stimulating, antipyretic by N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA)
and cough sedating properties, as stated in a Chinese providing an economically priced and facile alternative to
dispensatory in 1596.1 All effects have been mimicked
by modern therapeutic drugs, for example, α-(1-amino-
ethyl)benzyl alcohol derivatives such as ephedrine (α-[1- 3
CH3 CH3 CH3 CH3
(methylamino)ethyl]benzyl alcohol, Scheme 1 (1), and
HO CH3 HO CH3 HO CH3 HO CH3
p-hydroxyephedrine (p-hydroxy-α-[1-(methylamino)ethyl] 1
2 N
H
N
H
N N
H H
benzyl alcohol, Scheme 1 (2), have been extensively utilized
in medical treatment of the common cold and coughs2 as
well as obesity,3,4 based on their stimulating, bronchodi- 1 OH 2 3 OH 4
lating and thermogenic qualities. Desirable properties
include stimulation of adrenoceptors following the release 3
CH3 CH3
of catecholamine noradrenaline. Hence, various procedures HO CD3 HO CD3
2 N N
have been established enabling the extraction of ephedrines 1
H H
from natural sources,5–7 their large-scale synthesis8–10 and
stereospecific separation.11–13 Moreover, stable isotope and
radio-labeled analogs have been prepared14–19 to investi- 5 OH 6
gate metabolism and potency of this class of drugs. Also for
Scheme 1. Chemical structures of ephedrine (1), p-hydroxy-
quantitation purposes in biological matrices, as required for ephedrine (2), pseudoephedrine (3), p-hydroxypseudoephe-
instance in drug testing and doping control,20–24 deuterated drine (4) and corresponding deuterated analogs 2H3-ephedrine
reference compounds are referred to as the “gold standard” (5) and 2H3-p-hydroxypseudoephedrine (6).

DOI: 10.1255/ejms.669 ISSN 1356-1049 © IM Publications 2004

674 Synthesis of Deuterated Ephedrines

classical procedures described in the literature. Experiments and the red-colored solution containing the resulting N-
were performed to elucidate the role of MSTFA in the acetyl-N-trideuteromethyl-O-trimethylsilylated norephe-
acceleration of methylation of amino functions of nore- drines was evaporated under vacuum at 65°C in a water
phedrine and its analogs. Fragmentation pathways of α-(1- bath yielding approximately 3 mL of a viscous residue.
aminoethyl)benzyl alcohol derivatives under various mass Aqueous potassium hydroxide (0.5 M, 300 mL) was added
spectrometric conditions are proposed. to the reaction mixture and incubated at 50°C for 6 h. After
cooling to ambient temperature, aqueous hydrochloric acid
(3 M, 50 mL) and tert-butanol (20 mL) were added slowly
Experimental under constant stirring. The pH of the reaction mixture was
adjusted to 9.6 by the addition of potassium carbonate and
sodium bicarbonate (2 : 1, w : w, 2 g). The aqueous layer was
Chemicals extracted twice with tert-butyl methyl ether (300 mL), the
Phenylethylamine, α-(1-aminoethyl)benzyl alcohol combined organic phases were evaporated to dryness and
hydrochloride (Phenylpropanolamine-HCl), p-hydroxy-α- the resulting dry residue washed twice with cold methanol
(1-aminoethyl)benzyl alcohol hydrochloride (p-hydroxy- (5 mL) yielding 5 or 6, with yields of 45% (75 mg) or 38%
phenylpropanolamine-HCl), 2H6-acetic anhydride and 2H3- (70 mg), respectively.
iodomethane were purchased from Sigma–Aldrich (Steinheim,
Germany). Acetic anhydride, trifluoroacetic anhydride and Characterization and purity of synthesized compounds
ammonium bicarbonate were obtained from Fluka (Buchs, Elemental analysis of synthesized material was performed
Switzerland). N-Methyl-N-trimethylsilyltrifluoroacetamide using a Euro Vector elemental analyzer EA 3000 (Milan,
was supplied by Chem. Fabrik Carl Bucher (Waldstetten, Italy). One to two mg were placed in a tin cup (3.5 × 5.0 mm),
Germany) and distilled before use. tert-Butyl methyl ether was which was closed and introduced into the EA 3000. The
purchased from Kraemer & Martin (St Augustin, Germany), temperatures of the oxidation and reduction reactors were
potassium hydroxide and potassium carbonate from Merck maintained at 1050°C and 650°C, respectively. Helium was
(Darmstadt, Germany) and acetonitrile (HPLC grade) from J.T. utilized as carrier gas at 65 mL min–1 and an oxygen loop
Baker (Deventer, The Netherlands). Hexamethyldisilazane and of a volume of 5 mL was applied during analysis. Nuclear
trimethylchlorosilane were purchased from Macherey–Nagel magnetic resonance (NMR) spectroscopy, including 1H and
13
(Düren, Germany) and deuterated bis-trimethylsilylacetamide C measurements, were carried out on a Bruker Avance
(BSA) from Cambridge Isotope Laboratories (Andover, MA, DPX 200 (Bremen, Germany). Compounds were dissolved
USA). in CD3OD and spectra were recorded at 25°C. Mass spectra
of synthesized material were recorded under various condi-
Synthesis of 2H3-ephedrine (5) and 2H3-p-hydroxypseudo- tions. Electrospray ionization (ESI) followed by single- and
ephedrine (6) dual-stage mass spectrometry (MS) was performed on an
In order to achieve N-monomethylation of norephe- Applied Biosystems QTrap mass spectrometer (Darmstadt,
drines such as α-(1-aminoethyl)benzyl alcohol and its p- Germany) providing information on molecular weights as
hydroxylated analog, starting materials were N-acetylated, well as structures of the investigated compounds. Analytes
hydroxyl functions were subsequently protected by trimethyl- were dissolved in acetonitrile and water (1 : 1, v : v) at a
silylation and trideuteromethylation was carried out using concentration of 5 µg mL–1 and introduced into the analyzer
MSTFA in the presence of 2H3-iodomethane. Protection by a syringe pump at a flow rate of 3 µL min–1. Nitrogen
groups were removed by alkaline hydrolysis and the desired was employed for collision and curtain gases, delivered by
products were extracted and purified. a Whatman K75-72 nitrogen generator. Positive ionization
N-Acetylation of the free bases was accomplished by was utilized at a spray voltage of 5500 V, the declustering
dissolving the starting material (1 mmol) in ammonium potential (DP) was adjusted to 25 V, and the collision energy
bicarbonate (50 mM, 12 mL) in a 100 mL round-bottomed in tandem MS experiments was set to 15 eV. Gas chromatog-
flask followed by the addition of a mixture of acetic anhy- raphy (GC) and MS experiments were done on an Agilent
dride and methanol (1 : 2, v : v, 30 mL). The solution was 6890/5973 GC-MS instrument (Waldbronn, Germany) after
kept at ambient temperature for 14 h, evaporated to dryness derivatization of the analytes to their trimethylsilylated
by means of a rotary evaporator under vacuum, and the dry analogs (vide infra). The GC was equipped with an HP1
residue was stored in a desiccator over phosphorus pentoxide capillary column (Agilent, Waldbronn, Germany), length 17
under vacuum for 5 h. The crude N-acetylated norephedrines m, i.d. 0.25 mm, film thickness 0.11 µm. The carrier gas was
were dissolved in MSTFA (10 mL), and the solution was helium at a constant flow of 1.5 mL min–1 and a temperature
incubated at 80°C for 30 min, giving rise to N-acetyl-N,O- program was employed starting at 100°C, increasing by
trimethylsilylated norephedrines. After cooling to ambient 20°C min–1 up to 300°C. The MS was operated in the full-scan
temperature, 2H3-iodomethane (5 mmol) were added, the mode (50–500 u), and electron ionization (70 eV) was used
flask was sealed properly and the reaction mixture was kept giving rise to characteristic fragment ions upon dissociation
at 80°C. After 24 h, the methylation reaction was completed, of the analytes. Liquid chromatography-mass spectrometry
M. Thevis, G. Opfermann and W. Schänzer, Eur. J. Mass Spectrom. 10, 673–681 (2004) 675

Alternative reaction conditions

(LC-MS) was carried out on an Agilent 1100 Series HPLC
(Waldbronn, Germany) equipped with an Agilent Zorbax In order to study the role of the introduced N-acetyl
XDB-C8 column (4.6 × 150 mm, particle size 5 µm) interfaced residue, the benzylic hydroxyl group and the trimethyl-
by ESI to the QTrap mass spectrometer described above. The silylating reagent during the alkylation reaction, as well
solvents used were (a) 5 mM ammonium acetate in 0.1% as to probe for general methylation mechanisms, various
acetic acid and (b) acetonitrile. A gradient was employed experiments were conducted with phenylethylamine. First,
starting with 95% A for 1 min decreasing to 50% A for 5 min phenylethylamine, which does not have a benzylic hydroxyl
at a flow rate of 800 µL min–1 with a static post-column split function, was acetylated and methylated according to the
of 1 : 10. Full scan mass spectra were recorded from 50 to protocol described above. Second, acetic anhydride was
500 u using unit resolution. substituted by 2H6-acetic anhydride as well as trifluoro-
acetic anhydride, followed by regular alkylation. Third,
Derivatization of compounds for GC-MS analysis regular acetylation was followed by methylation without
In order to increase volatility and gas chromatographic MSTFA but in acetonitrile in the presence of potassium
properties of ephedrines, substances were trimethylsilylated carbonate according to established alkylation procedures.26
using MSTFA. About 500 ng of each analyte were dissolved N-acetylated p-hydroxy-α-(1-aminoethyl)benzyl alcohol
in 100 µL of MSTFA and the solutions were incubated at (50 mg) was dissolved in acetonitrile (19 mL), CD3I (200 µL)
60°C for 20 min. When experiments included deuterium- was added and the solution was incubated at 60°C. Fourth,
labeled TMS residues, MSTFA was substituted by a mixture regular acetylation was followed by methylation without
of 2 H 18 -bis-trimethylsilyltrifluoroacetamide (BSA) and MSTFA but an alternative trimethylsilylation reaction
acetonitrile (1 : 4, v : v). mixture consisting of hexa-methyldisilazane (HMDS, 9 mL)

Table 1. Fragment ions of trimethylsilylated α-(1-aminoethyl)benzyl alcohol (phenylpropanolamine) and p-hydroxy-α-(1-

aminoethyl)benzyl alcohol (p-hydroxyphenylpropanolamine), their N-acetylated and N-acetylated and N-methylated analogs gener-
ated by GC-MS upon electron ionization. Relative abundances are indicated in brackets.

Compound No. mol wt M+– 15 m/z 179 M+ – 106 m/z 147 M+– 179 m/z 73
(Da)

Phenylpropanolamine bis-TMS 7 295 280(8) (1) (5) 116 (100) (48)

N-acetylphenylpropanolamine-bis-TMS 8 337 322(10) (4) (3) 158 (100) (50)

N-acetyl-N-trideuteromethyl-phenyl-
9 282 267(8) (57) 176(38) 103(80) (47)
propanolamine-TMS

N-trideuteromethyl-phenylpropanolamine-
10 312 297(4) (1) (4) 133 (100) (42)
bis-TMS

Compound mol wt M+ – 15 m/z 294 m/z 267 m/z 193 M+ – 194 M+ – 267 m/z 73
(Da)

p-hydroxyphenylpropanolamine tris-TMS 11 383 368(9) (1) (10) (2) 116 (100) (32)

p-hydroxy-N-acetylphenylpropanolamine-
12 425 410(5) (100) (3) 158(52) (58)
tris-TMS
p-hydroxy-N-acetyl-N-trideuteromethyl-
13 370 355 (5) (3) (100) (3) 176(15) 103(28) (28)
phenylpropanolamine-bis-TMS
p-hydroxy-N-trideuteromethyl-
14 400 385(4) (2) (1) 133 (100) (40)
phenylpropanolamine-tris-TMS
p-hydroxy-N-trideuteromethyl-
15 328 313(2) (11) (1) 61 (100) (20)
phenylpropanolamine-bis-O-TMS
p-hydroxy-N-trideuteromethyl- 16 346 M+– 18 m/z 285 m/z 202 M+ – 285 m/z 82
phenylpropanolamine-bis-O-2H9 –TMS 328(2) (12) (1) 61 (100) (30)
676 Synthesis of Deuterated Ephedrines

and trimethylchlorosilane (TMCS, 1 mL). All other reaction (m.p. 119°C), and by means of aqueous hydrochloric acid
conditions were identical to those described above. and incubation at elevated temperatures, these compounds
are converted into each other until an equilibrium is estab-
lished. 1 Under the employed reaction conditions, α-(1-
Results and discussion aminoethyl)benzyl alcohol maintained its stereochemical
orientation yielding 2H3-ephedrine, while p-hydroxy-α-
The employed route of synthesis yielded 75 and 70 mg (1-aminoethyl)benzyl alcohol generated 2H3-p-hydroxy-
of the desired products 5 and 6. In Table 1, main fragment pseudoephedrine by inversion of the benzylic hydroxyl
ions of the trimethylsilylated starting material [i.e. α-(1- function upon alkaline hydrolysis.
aminoethyl)benzyl alcohol and p-hydroxy-α-(1-amino-
ethyl)benzyl alcohol], as well as their trimethylsilylated Characterization and purity of synthesized compounds
reaction intermediates (i.e. N-acetylated and N-acetylated
and N-methylated analogs) are summarized. Ephedrines Elemental analysis

have two stereogenic carbons {assigned 1 and 2 in Scheme Elemental analysis was performed for the products 5 and
1, 1 giving rise to levorotatory ephedrine [1 – (R), 2 – (S); 6. 2H3-ephedrine (5): anal. (C10H12D3NO) C: calcd, 72.69;
[α]D25 = –41] and dextrorotatory pseudoephedrine [1 – (S), found, 71.73; H(/D): calcd, 9.15; found, 9.63; N: calcd, 8.48;
2 – (S); [α]D25 = +52]}. The properties of these compounds found, 8.17. Deuterated 2H3-p-hydroxypseudoephedrine
are significantly different, as demonstrated with the melting (6) provided evidence for the presence of water in the final
points of ephedrine (m.p. 38–40°C) and pseudoephedrine product. The measured composition accounts for the hemi-

Table 2. 1H and C chemical shifts (ppm) of ephedrine, 2H3-ephedrine and 2H3 p-hydroxypseudoephedrine analyzed in CD3OD at
13

200 MHz.

Protons Ephedrinea 2
H3-Ephedrinea 2
H3 p-Hydroxy- Carbons Ephedrinec 2
H3-Ephedrine 2
H3 p-Hydroxy-
pseudoephedrineb pseudoephedrine
1.081 1.079 0.812
H(a) 1 10.3 10.3 15.0
(d, 3JHH = 6.76 Hz) (d, 3JHH = 6.76 Hz) (d, 3JHH = 6.47 Hz)
H(b) 2.777(s) — — 2 31.7 -— —

H(c) 3.425 (m) 3.438 (m) 2.731(m) 3 61.8 61.6 62.0

5.115 5.101 4.208
H(d) 4 72.0 72.0 79.0
(d, 3JHH = 2.94 Hz) (d,3JHH = 3.23 Hz) (d, 3JHH = 8.82 Hz)
7.160
H(e) 7.265 (m) 7.239 (m) 5 141.7 141.7 134.7
(d, 3JHH = 8.53 Hz)
6.771
H(f) 6 127.3 127.1 129.6
(d, 3JHH = 8.53 Hz)
7 130.3 130.2 116.5

8 129.5 129.3 158.9

9 130.3 130.2 116.5

10 127.3 127.1 129.6

a b c
protons assigned as follows protons assiged as follows carbons assigned as follows

— not detected (protons exchanged by deuteration)

M. Thevis, G. Opfermann and W. Schänzer, Eur. J. Mass Spectrom. 10, 673–681 (2004) 677

hydrate 2 H 3 -p-hydroxypseudoephedrine × ½ H 2 O which at carbon C-4 owing to deviant stereochemical orientation

has been described for ephedrines in the literature:1 anal of the attached hydroxyl group (Table 2). In addition, in the
(C10H12D3NO2) C: calcd, 63.16; found, 63.52; H(/D): calcd, case of 6, all shifts of aromatic carbons (C-5–C-10) were
8.42; found, 8.39; N: calcd, 7.37; found, 7.08. strongly influenced by the C-8-linked hydroxyl function.

Nuclear magnetic resonance spectroscopy Mass spectrometry: ESI-MS/MS

Compounds 5 and 6 were dissolved in CD 3 OD and Mass spectrometric characterization of synthesized

proton, as well as 13C NMR measurements, were carried products was performed by means of ESI-MS/MS, LC-MS
out providing detailed information on their composition and GC-MS. The ESI product ion spectra of (M + H)+ of
and stereochemistry. 27,28 The resulting resonances were ephedrine (m/z 166), 2H3-ephedrine (5) (m/z 169), p-hydroxy-
compared with spectra of commercially available ephedrine ephedrine (m/z 182) and 2H3-p-hydroxypseudoephedrine (6)
that was not modified by stable isotope labeling (Table 2). (m/z 185) demonstrated the influence of the trideuteromethyl
In the case of 5, the singlet representing the protons H(b) residue on selected fragment ions (Figure 1). In all spectra,
was absent owing to its exchange by deuteria, while all other the base peak was generated by an initial loss of water (–18
signals demonstrated identical chemical shifts relative to u) from the protonated molecule. With ephedrine (1) and its
non-labeled ephedrine. Compound 6 generated signals with deuterated analog (5), the resulting ions at m/z 148 and 151,
significantly deviant chemical shifts due to the two differ- respectively, subsequently eliminated a methyl radical giving
ences in molecular composition (p-hydroxyl function) and rise to fragments at m/z 133 and 136. Here, the product ion
structure (orientation of benzylic hydroxyl group), which spectrum of 5 [Figure 1(b)] indicated that the expeled methyl
were assigned to distinct hydrogens allowing stucture confir- residue was primarily carbon 3 (Scheme 1), but with MS3
mation (Table 2). 13C-NMR data substantiated the proton experiments inducing dissociation of the product ion at m/z
NMR results. Significant differences in chemical shifts were 151, abundant fragments were observed at m/z 136 and 133
observed between the analyzed ephedrines (1 and 5) and 6 (data not shown). Hence, an elimination of either the methyl

a) 148.2 c) 164.3
100% 100%
CH3
90% 90%
CH3 HO CH3
80% 80% N
HO CH3 H
Relative abundance

Relative abundance

70% N 70%

60%
H 60%

50% 50%
149.1
40% 40%
OH 133.1

30% 30%
117.1
115.1 105.2
20% 20%
133.1
10% 10% 182.1
91.1 107.1
166.2
70 80 90 100 110 120 130 140 150 160 70 80 90 100 110 120 130 140 150 160 170 180

m/z m/z

b) d)
100% 151.2 100% 167.3

90% 90%
CH3
80% 80%
CH3 HO CD3
Relative abundance

Relative abundance

70% 70% N
HO CD3 H
60% N 60%
H
50% 50%

40% 40%
152.3
30% 30% OH
20% 117.1 20%
136.2 149.3
115.1
10% 10% 105.2
133.2
91.1 107.1 185.3
169.2
70 80 90 100 110 120 130 140 150 160 60 70 80 90 100 110 120 130 140 150 160 170 180

m/z m/z

Figure 1. ESI product ion spectra of (a) m/z 166 of ephedrine (1, DP = 10, CE = 30), (b) m/z 169 of 2H3-ephedrine (5, DP = 10, CE = 30),
(c) m/z of p-hydroxyephedrine (2, DP = 20, CE = 20) and (d) m/z 185 of 2H3-p-hydroxypseudoephedrine (6, DP = 20, CE = 20).
678 Synthesis of Deuterated Ephedrines

group, carbon 3 and the N-linked residue, is possible. A ular ion in the EI mass spectra, but fragments resulting from
different fragmentation route from (M + H)+–18 included the the loss of a methyl residue (M+ – 15) or other characteristic
neutral loss of methylamine (–31 u), which produced the ion dissociation processes are listed in Table 1. The elimination
at m/z 117 in both product ion spectra of ephedrine and its of a methyl radical was frequently observed with numerous
labeled counterpart, followed by an additional elimination compounds analyzed by GC-EI/MS after trimethylsilylation,
of hydrogen generating m/z 115. The product ion at m/z 91 but its origin is not necessarily an introduced TMS function as
is proposed to be the benzyl cation (C6H5CH2+) presumably demonstrated in the literature, for example, for steroids.30,31
stabilized as the tropylium cation,29 which was generated Ephedrines comprise two methyl groups and the loss of these
directly from the protonated molecule as it was not observed during mass spectrometric analysis is possible, as observed
in any product ion spectra of (M + H) +–18. Information with ESI-CAD-MS. In GC-MS analyses, the nitrogen-
substantiating the proposed composition of the ion at m/z linked methyl group was retained in the M+ – 15 product ion,
91 is given by spectra of 2H5-ephedrine, which bears five because no ion accounting for the release of –CD3 (M+ – 18)
deuteria at the benzene ring.14 Proposed fragmentation routes was detected in the mass spectra of 5 and 6. To differentiate
of ephedrine are summarized in Scheme 2. The product ion between the methyl residue at C- 3 and those included in
spectra of p-hydroxyephedrine and 6 demonstrated anal- TMS groups, 6 was derivatized with BSA, causing the intro-
ogous dissociation behavior in compliance with an addi- duction of two 2H9 – TMS residues at the hydroxyl functions.
tional hydroxyl function, increasing m/z of the precursor In contrast to 2H3-p-hydroxypseudoephedrine-bis-O-TMS
ion and its product ions by 16 u. Loss of water, methyl resi- (Table 1, 15), the TMS-labeled analog (mol. wt = 346) did
dues including either carbon 3 or the nitrogen-linked methyl not present an M+ – 15 ion but M+ – 18, proving the initial
group and methylamine were also observed. There were elimination of the methyl group from a TMS residue (Table
slight differences in the relative abundance of fragment ions 1, 16). With α-(1-aminoethyl)benzyl alcohol-N,O-bis-TMS
between these two stereoisomers, in particular regarding (mol. wt = 295), M+ – 15 was observed at m/z 280 and the
the stability of the product ion (M + H)+–18. Its intensity, most abundant fragment ion was m/z 116 (Table 1, 7), which
compared with fragment ions of smaller m/z ratios, was is proposed to be generated upon α-cleavage, as depicted
much more predominant in the product ion spectrum of 6 in Scheme 3(a). With the introduction of an acetyl residue
than of p-hydroxyephedrine. at the primary amino function, the respective fragment ion
at m/z 116 was incremented by 42 u to m/z 158 (Table 1, 8)
and upon trideuteromethylation of compound 8, the product
Mass spectrometry: GC-MS ion was decremented by 55 u to m/z 103 (Table 1, 9) owing
Using GC/MS, products 5 and 6, as well as all reaction inter- to the exchange of a TMS residue (73 u) by a trideuterome-
mediates, were confirmed and characterized as their trimeth- thyl group (18 u). In addition, the acetylated and alkylated
ylsilylated derivatives. None of the analytes show a molec- a-(1-aminoethyl)benzyl alcohol (Table 1, 9) gave rise to a
fragment ion at m/z 176, which is suggested to result from a
neutral loss of benzaldehyde (106 u) as proposed in Scheme
3(c). Here, the N-linked acetyl group is essential because
3 3 a TMS residue has to migrate from the benzylic hydroxyl
CH3 CH3
+ CH function to the remaining ion and comparable fragments
HO - H2O + CH3
1
2 N 3
1
2 N were missing in the spectra of analogous compounds lacking
H
H H the acetylation at the nitrogen (Table 1). In agreement with
an increment of 88 u, due to an additional oxygen resulting
m/z 166 in another TMS derivatization site, product ions produced
m/z 148 by analogous dissociation behaviors were observed with the
intermediate products of the synthesis of 6. Starting from
- CH3NH2 p-hydroxy-a-(1-aminoethyl)benzyl alcohol, acetylation and
trimethylsilylation gave rise to compound 12 (Table 1), the
mass spectrum of which contains major fragment ions at m/z
3
3
CH2 410 and 267 (counterparts of m/z 322 and 158 of 8, respec-
CH tively). The subsequent methylation enabled the elimination
1 2
- H2 1 2
+ + of p-hydroxybenzaldehyde (M+ – 194, Table 1, 13) corre-
sponding to the expulsion of benzaldehyde (M+ – 106) from
9. After hydrolysis of TMS and acetyl residues, the isolated
products were trimethylsilylated and analyzed by GC-MS.
m/z 115 m/z 117 The resulting EI mass spectra primarily contained the M+ – 15
Scheme 2. Proposed fragmentation routes of ephedrine after ions, m/z 133 generated by α-cleavage in accordance to m/z
electrospray ionizsation and collisionally-activated dissociation 116 [Scheme 3(a)] and the TMS-specific fragment at m/z 73
(CAD). (Table 1, 10 and 14).
M. Thevis, G. Opfermann and W. Schänzer, Eur. J. Mass Spectrom. 10, 673–681 (2004) 679

a)
CH3
CH3
TMS O + H
N . H
TMS +N

TMS
m/z 116
b)

. CH3
TMS O
+
TMS O H
+ N
TMS

m/z 179
c)

CH3 CH3
+ CD3 . + CD3
. N
CH3
N
CH3
O
Si O TMS O

m/z 176 (M+ - 106 u)

Scheme 3. Postulated fragmentation routes of α-(1-aminoethyl)benzyl alcohol derivatives: a) generation of the common fragment
ions m/z 116 (and corresponding analogs), b) m/z 179, and c) m/z 176 (M+ – 106). Charge-driven fragmentation is proposed with all
pathways starting either from the nitrogen or the benzylic oxygen.

Probing for the methylation mechanism—alternative reaction

utilized to reversibly protect these reactive sites; however,
conditions
attempts at subsequent methylation, following established
Classical alkylation reactions are commonly performed procedures using acetonitrile, iodomethane and potassium
in organic solvents such as acetone or acetonitrile employing carbonate, yielded only 1% of the desired product after 24 h.
alkyl halides in the presence of a base.26 Because multiple As reactions employing MSTFA instead of acetonitrile and
methylation at the primary amino function as well as alkyla- potassium carbonate were completed after 24 h, the influence
tion of the phenolic hydroxyl group of p-hydroxylated of various parameters on the methylation mechanism used
compounds had to be minimized, initial acetylation was for the synthesis of deuterated ephedrines was investigated,

Table 3. Mass spectral data of α-(1-aminoethyl)benzyl alcohol derivatives analyzed by GC-MS using electron ionization. Relative
abundances (%) of fragment ions are shown in brackets.
Compound No. mol. wt M+ M+ – 15 m/z 104 m/z 91 M+ – 91 m/z 77
(Da)
N-Acetyl-phenylethylamine 17 163 (28) 148(2) (100) (31) 72(16) (10)

N,N-bis-acetyl-phenylethylamine 18 205 (2) — (100) (29) 114(2) (8)

N-Acetyl-N-trideuteromethyl-
19 180 (35) 165(3) (32) (28) 89(100) (9)
phenylethylamine
N-Trifluoroacetyl-phenylethylamine 20 217 (4) — (100) (64) 126(10) (5)
N-Trifluoroacetyl-N-trideuteromethyl-
21 234 (7) — (100) (26) 143(92) (5)
phenylethylamine
N-Trideuteroacetyl-phenylethylamine 22 166 (16)- — (100) (25) 75(10) (11)
N-Trideuteroacetyl-N-methyl- 23 180 (18) M+ – 18 (52) (25) 89(100) (20)
phenylethylamine 162(2)
680 Synthesis of Deuterated Ephedrines

using the more simple molecule phenylethylamine (PEA) as and the Manfred-Donike Society, Cologne, for financial
a model compound. All generated substances were measured support.
and identified by means of GC-MS (17–23, Table 3).
Based on these results, a methylation mechanism,
including a trimethylsilyation-promoted enolization of the References
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