An unprecedented rearrangement in collision-induced mass spectrometric fragmentation of protonated benzylamines

Article abstract of DOI:10.1002/jms.1089

The collision-induced dissociation (CID) mass spectra of several protonated benzylamines are described and mechanistically rationalized. Under collision-induced decomposition conditions, protonated dibenzylamine, for example, loses ammonia, thereby forming an ion of m/z 181. Deuterium labeling experiments confirmed that the additional proton transferred to the nitrogen atom during this loss of ammonia comes from the ortho positions of the phenyl rings and not from the benzylic methylene groups. A mechanism based on an initial elongation of a C-N bond at the charge center that eventually cleaves the C-N bond to form an ion/neutral complex of benzyl cation and benzylamine is proposed to rationalize the results. The complex then proceeds to dissociate in several different ways: (1) a direct dissociation to yield a benzyl cation observed at m/z 91; (2) an electrophilic attack by the benzyl cation within the complex on the phenyl ring of the benzylamine to remove a pair of electrons from the aromatic sextet to form an arenium ion, which either donates a ring proton (or deuteron when present) to the amino group forming a protonated amine, which undergoes a charge-driven heterolytic cleavage to eliminate ammonia (or benzylamine) forming a benzylbenzyl cation observed at m/z 181, or undergoes a charge-driven heterolytic cleavage to eliminate diphenylmethane and an immonium ion; and (3) a hydride abstraction from a methylene group of the neutral benzylamine to the benzylic cation to eliminate toluene and form a substituted immonium ion. Corresponding benzylamine and dibenzylamine losses observed in the spectra of protonated tribenzylamine and tetrabenzyl ammonium ion, respectively, indicate that the postulated mechanism can be widely applied. The postulated mechanisms enabled proper prediction of mass spectral fragments expected from protonated butenafine, an antifungal drug. Copyright

Full text of DOI:10.1002/jms.1089

JOURNAL OF MASS SPECTROMETRY
J. Mass Spectrom. 2006; 41: 1195–1204
Published online 21 August 2006 in Wiley InterScience
(
www.interscience.wiley.com) DOI: 10.1002/jms.1089
An unprecedented rearrangement in collision-induced
mass spectrometric fragmentation of protonated
benzylamines
∗
Jason Bialecki, Josef Ruzicka and Athula B. Attygalle
Center for Mass Spectrometry, Department of Chemistry and Chemical Biology, Stevens Institute of Technology, Hoboken, New Jersey 07030, USA
Received 31 March 2006; Accepted 20 June 2006
The collision-induced dissociation (CID) mass spectra of several protonated benzylamines are described
and mechanistically rationalized. Under collision-induced decomposition conditions, protonated dibenzy-
lamine, for example, loses ammonia, thereby forming an ion of m/z 181. Deuterium labeling experiments
confirmed that the additional proton transferred to the nitrogen atom during this loss of ammonia comes
from the ortho positions of the phenyl rings and not from the benzylic methylene groups. A mechanism
based on an initial elongation of a C–N bond at the charge center that eventually cleaves the C–N bond to
form an ion/neutral complex of benzyl cation and benzylamine is proposed to rationalize the results. The
complex then proceeds to dissociate in several different ways: (1) a direct dissociation to yield a benzyl
cation observed at m/z 91; (2) an electrophilic attack by the benzyl cation within the complex on the phenyl
ring of the benzylamine to remove a pair of electrons from the aromatic sextet to form an arenium ion,
which either donates a ring proton (or deuteron when present) to the amino group forming a protonated
amine, which undergoes a charge-driven heterolytic cleavage to eliminate ammonia (or benzylamine)
forming a benzylbenzyl cation observed at m/z 181, or undergoes a charge-driven heterolytic cleavage to
eliminate diphenylmethane and an immonium ion; and (3) a hydride abstraction from a methylene group
of the neutral benzylamine to the benzylic cation to eliminate toluene and form a substituted immo-
nium ion. Corresponding benzylamine and dibenzylamine losses observed in the spectra of protonated
tribenzylamine and tetrabenzyl ammonium ion, respectively, indicate that the postulated mechanism can
be widely applied. The postulated mechanisms enabled proper prediction of mass spectral fragments
expected from protonated butenafine, an antifungal drug. Copyright  2006 John Wiley & Sons, Ltd.
KEYWORDS: hydrogen transfer; fragmentation; CID; benzylamines; deuterium-labeled compounds; butenafine; ion/neutral
complex
INTRODUCTION
Although odd-electron ion fragmentations (electron-
ionization mass spectrometry) have been widely examined
and rules established, we know less about even-electron
fragmentation and rearrangement mechanisms. In our quest
for understanding the intricate rearrangement mechanisms
involved in the fragmentation processes of even-electron
ions, we investigated the spectra of several benzylamines
Many benzylamines are biologically important compounds.
For example, several neurotransmitters such as catechol-
1
–3
amines
and drugs such as butenafine (N-4-tert-butyl-
4
4
benzyl-N-methyl-1-naphthalenemethylamine), terbinafine,
5
and ambenonium chloride are benzylamine derivatives.
Mass spectrometry in conjunction with electrospray ioniza-
tion and collision-induced dissociation (CID) shows promise
in the prognosis of various diseases and in the identifi-
cation of metabolic by-products. Recently, selected reac-
tion monitoring (SRM) techniques have been employed for
the identification and quantification of norepinephrine and
(
Fig. 1), and we present our results here.
EXPERIMENTAL
Mass spectrometry
The CID mass spectra were recorded on a Micromass
Quattro 1 spectrometer equipped with an electrospray
source. Samples were introduced to the source as acetoni-
1
catecholamine. Success of all these analytical procedures
depends on conceptual understanding of fragmentation
mechanisms.
ꢀ
2
trile–water–formic acid (9 : 1 : 10 ) solutions for positive-ion
ꢀ
2
MS and as acetonitrile–water–ammonia (9 : 1 : 10 ) solu-
ꢀ
1
tions for negative-ion MS at a flow rate of 320 µl h . The
source temperature was held constant at 80 °C. The argon gas
Ł
Correspondence to: Athula B. Attygalle, Center for Mass
Spectrometry, Department of Chemistry and Chemical Biology,
Stevens Institute of Technology, Hoboken, NJ 07030, USA.
E-mail: athula.attygalle@stevens.edu
pressure in the collision cell was set to attenuate precursor
ion transmission by 30–50%. For experiments performed in
Copyright  2006 John Wiley & Sons, Ltd.

1196
J. Bialecki, J. Ruzicka and A. B. Attygalle
D
reference TMS. All coupling constants (J values) are reported
1
3
D
D
D
D
in Hertz (Hz). All C NMR spectra were decoupled and all
chemical shifts are reported relative to that of CDCl . EI Mass
3
spectra were recorded on an HP 5970 series Mass Selective
Detector coupled to a Hewlett Packard 5890 Series II gas
chromatograph. Accurate mass determinations were carried
out on a Waters Micromass Q-Tof API-US mass spectrometer
equipped with a nanospray source, at a resolution of about
D
D
NH
D
R2C
R2C
CR2
D
D
NH
N
CR2
CR2
1
5 000.
Chemical synthesis
Benzyl alcohol
1
2
, R = H
, R = D
3
4, R = H
5, R = D
Benzoic acid (0.150 g, 1.23 mmol) in THF (0.5 ml) was added
to LiAlH (0.072 g, 1.90 mmol) in diethyl ether (0.5 ml). The
4
D
D
D
D
D
reaction mixture was stirred at ambient temperature for 2 h
and quenched with water (0.08 ml). The inorganic solids
were filtered and washed with diethyl ether. The combined
ether filtrate was dried over Na SO and evaporated to give
D
D
D
D
D
N
D
NH
D
2
4
1
a colorless oil (0.097 g, 72%). H NMR: (300 MHz, CDCl
7
3
) υ
D
D
NH
D
D
D
D
D
13
.33 (5H, m), 4.61 (2H, s), 1.94 (1H, s); C NMR: (300 MHz,
D
CDCl
3
) υ 140.81, 128.39, 127.44, 126.87, 64.99; EI-MS: m/z (%),
D
Cž
1
08 (M , 80), 107 (65), 91 (20), 79 (100), 77 (70), 65 (10), 51
D
D
D
(
30), 39 (20).
6
7
8
Benzyl bromide
Benzyl alcohol (0.097 g, 0.90 mmol) in acetonitrile (5 ml) was
CH3
brominated with triphenylphosphine (0.378 g, 1.45 mmol)
H
6
N
and bromine (0.24 g, 1.5 mmol). The reaction mixture
N
was heated for 2 h at 55 °C and worked up with 30%
3
hydrogen peroxide (0.5 ml) in 5% aqueous NaHCO (10 ml).
The aqueous mixture was extracted with pentane–diethyl
ether (2 : 1). The organic layer was separated, dried over
Cl
Br
9
10
2 4
Na SO , concentrated, and chromatographed on silica gel
Figure 1. Compounds used for fragmentation studies.
(pentane–ether 2 : 1). The eluate was evaporated to give
0
0 2
1
Dibenzylamine (1), N,N-bis([1 ,1 - H2]benzyl)amine (2),
benzyl bromide (0.12 g, 80% yield). H NMR: (300 MHz,
2
13
N,N-bis([2,3,4,5,6- H5]benzyl)amine (3), tribenzylamine (4),
CDCl
CDCl
3
) υ 7.36 (5H, m), 4.48 (2H, s); C NMR: (300 MHz,
0
0 2
N,N,N-tris([1 ,1 - H2]benzyl)amine (5),
3
) υ 144.03, 128.99, 128.78, 128.40, 46.43; EI-MS: m/z (%),
170/172 (M , 10/10), 91 (100), 65 (20), 39 (15).
Cž
2
N,N,N-tris([2,3,4,5,6- H5]benzyl)amine (6),
2
N,N-bis([2,6- H2]benzyl)amine (7),
2
N,N-([2,3,4,5,6- H2]benzyl)benzylamine (8),
Dibenzylamine (1)
tetrabenzylammonium bromide (9), butenafine hydrochloride
10).
Benzyl bromide (0.010 g, 0.058 mmol) was added to a stirred
solution of acetonitrile (1 ml) and tetrahydrofuran (0.5 ml)
(
and 30% aqueous ammonia (0.125 ml, 2.20 mmol). The
reaction mixture was stirred for 1 h at 60 °C, diluted with
deuteriated solvents D
Co., St Louis, MO), and CD
Isotope Lab., MA, USA) were used.
2
O (99.8 atom% D; Aldrich Chemical
COOD (99 atom% D; Cambridge
water and extracted with diethyl ether. The organic layer
was separated, concentrated, applied on a preparative thin-
layer chromatography plate and eluted with hexanes–ethyl
3
General procedures
acetate (4 : 1). The product was isolated as a colorless oil from
1
All solvents and reagents were obtained from commercial
suppliers (Aldrich Chemical Co., St Louis, MO) and used
as purchased. Butenafine HCl was isolated from the anti-
the second major band (R
(400 MHz, CDCl
1.76 (1H, s); C NMR: (400 MHz, CDCl
f
D 0.45)(0.003 g, 25%). H NMR:
3
) υ 7.34 (8H, m), 7.27 (2H, m), 3.82 (4H, s),
1
3
3
) υ 140.28, 128.39,
Cž

fungal cream Lotrimin Ultra (Schering-Plough HealthCare
128.15, 126.95, 53.13; EI-MS: m/z (%), 197 (M , 10), 196 (15),
Products, Berkeley Heights, NJ). All synthetic products were
detected as one spot on thin-layer chromatography analysis
120 (10), 106 (60), 91 (100), 65 (20), 39 (10); Accurate mass
C
ESI-MS [M C H] for C14
H16N calc. 198.1286, found 198.1283.
(
precoated 0.25 mm silica gel plates purchased from Fisher
Scientific, Hampton, NH) unless specified. All NMR spectra
were recorded from a Varian INOVA 400, a GE 300 or a Var-
Tribenzylamine (4)
Benzyl bromide (0.010 g, 0.058 mmol) was added to a stirred
solution of acetonitrile (1 ml) and tetrahydrofuran (0.5 ml)
and 30% aqueous ammonia (0.025 ml, 0.44 mmol). The
ian VXR-200 spectrometer using CDCl
3
as solvent. Chemical
1
shifts for H NMR are reported in ppm relative to the internal
Copyright  2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 1195–1204
DOI: 10.1002/jms

CID of protonated benzylamines
1197
reaction mixture was stirred for 1 h at 60 °C, diluted with
a white solid from the major band (R
D 0.85) (0.003 g, 25%).
f
1
water, and extracted with diethyl ether. The organic layer
was separated, concentrated and applied to a preparative
thin-layer chromatography plate. The plate was eluted with
hexanes–ethyl acetate (10 : 1). The product was isolated as a
H NMR: (400 MHz, CDCl
3
) υ 7.40 (6H, td, J D 8.4, 1.2 Hz),
7.31 (6H, tt, J D 7.6, 1.6 Hz), 7.22 (3H, tt, J D 7.2, 1.6 Hz);
1
3
C NMR: (400 MHz, CDCl
3
) υ 139.54, 128.76, 128.20, 126.84,
Cž
57.92; EI-MS: m/z (%), 293 (M , 20), 216 (25), 200 (20), 93
C
white solid from the major band (R
f
D 0.85) (0.003 g, 25%).
(100), 67 (10), 40 (5); Accurate mass ESI-MS [M C H] for
1
2
H NMR: (400 MHz, CDCl
3
) υ 7.41 (6H, d, J D 7.2 Hz), 7.31
C
21
H
16
H
6
N calc. 294.2129, found 294.2133.
(
6H, t, J D 7.6 Hz), 7.24 (3H, t, J D 6.8 Hz), 3.56 (6H, s);
1
3
2
C NMR: (400 MHz, CDCl
3
) υ 139.64, 128.73, 128.20, 126.83,
[2,3,4,5,6- H ]Benzyl alcohol
5
Cž
2
5
57.91; EI-MS: m/z (%), 287 (M , 20), 210 (20), 196 (20), 91
[2,3,4,5,6- H ]Benzoic acid (0.150 g, 1.18 mmol) in THF
C
(
C
100), 65 (20), 39 (10); Accurate mass ESI-MS [M C H] for
(0.5 ml) was reduced with LiAlD4 (0.072 g, 1.71 mmol) in
diethyl ether (0.5 ml) by a procedure similar to that described
for benzyl alcohol to give a colorless oil (0.097 g, 72%). H
21
H
22N calc. 288.1752, found 288.1757.
1
0
0 2
13
[
1 ,1 - H ]Benzyl alcohol
NMR: (300 MHz, CDCl
3
) υ 4.62 (2H, s), 1.92 (1H, s); C NMR:
2
Benzoic acid (0.150 g, 1.21 mmol) in THF (0.5 ml) was
reduced by LiAlD (0.072 g, 1.71 mmol) in diethyl ether
0.5 ml) by a procedure similar to that described for benzyl
(300 MHz, CDCl
MS: m/z (%), 113 (M , 100), 112 (40), 96 (20), 83 (90), 81 (40),
68 (10), 54 (40), 42 (15).
3
) υ 140.83, 128.41, 127.47, 126.86, 64.97; EI-
Cž
4
(
alcohol. The product was obtained as a colorless oil (0.097 g,
1
2
7
2%). H NMR: (300 MHz, CDCl
3
) υ 7.34 (5H, m), 1.93 (1H, s);
[2,3,4,5,6- H ]Benzyl bromide
5
1
3
2
5
C NMR: (300 MHz, CDCl
3
) υ 140.82, 128.39, 127.44, 126.85,
[2,3,4,5,6- H ]Benzyl alcohol (0.097 g, 0.86 mmol) in ace-
Cž
65.01; EI-MS: m/z (%), 110 (M , 80), 109 (60), 92 (20), 81 (80),
tonitrile (5 ml) was brominated with triphenylphosphine
(0.378 g, 1.45 mmol) and bromine (0.24 g 1.5 mmol) by a pro-
cedure similar to that described for benzyl bromide to give
the desired product (0.12 g, 80% yield). H NMR: (300 MHz,
) υ 4.49 (2H, s); C NMR: (300 MHz, CDCl
128.98, 128.78, 128.40, 46.45; EI-MS: m/z (%), 175/177 (M
7
9 (60), 67 (10), 52 (20), 40 (10).
0
0
0 2
2
1
[
[
(
1
1 ,1 - H ]Benzyl bromide
2
0
13
1 ,1 - H
5 ml) was brominated with triphenylphosphine (0.378 g,
.45 mmol) and bromine (0.24 g, 1.5 mmol) by a procedure
2
]Benzyl alcohol (0.097 g, 0.88 mmol) in acetonitrile
CDCl
3
3
) υ 144.01,
Cž
,
10/10), 96 (100), 69 (15), 42 (10).
0
0
similar to that described for benzyl bromide to give [1 ,1 -
H
CDCl
29.00, 128.77, 128.42, 46.44; EI-MS: m/z (%), 172/174 (M
0/10), 93 (100), 66 (15), 40 (10).
2
1
2
2
]benzyl bromide (0.12 g, 80% yield). H NMR: (300 MHz,
N,N-Bis[2,3,4,5,6- H ]benzylamine (3)
[2,3,4,5,6- H ]Benzyl bromide (0.010 g, 0.057 mmol) was
added to a stirred solution of acetonitrile (1 ml) and tetrahy-
drofuran (0.5 ml) and 30% aqueous ammonia (0.125 ml,
5
1
3
2
3
) υ 7.37 (5H, m); C NMR: (300 MHz, CDCl
3
) υ 144.09,
5
Cž
1
1
,
2
.20 mmol). The reaction mixture was stirred for 1 h at 60 °C,
diluted with water, extracted with diethyl ether, and the
product was isolated by preparative thin-layer chromatog-
0
0 2
N,N-Bis[1 ,1 - H ]benzylamine (2)
[
2
0
0
2
1 ,1 - H
to a stirred solution of acetonitrile (1 ml) and tetrahydrofuran
0.5 ml) and 30% aqueous ammonia (0.125 ml, 2.20 mmol).
2
]Benzyl bromide (0.010 g, 0.058 mmol) was added
raphy (hexanes-ethyl acetate, 4 : 1) as a colorless oil was from
1
(
the second major band at R
(400 MHz, CDCl
(400 MHz, CDCl
f
D 0.45 (0.003 g, 25%). H NMR:
1
3
The reaction mixture was stirred for 1 h at 60 °C, diluted with
3
) υ 3.82 (4H, s), 1.69 (1H, s); C NMR:
water and extracted with diethyl ether. The organic layer
was separated, concentrated and applied to a preparative
thin-layer chromatography plate. The plate was eluted with
hexanes–ethyl acetate (4 : 1). The product was isolated as a
3
) υ 140.29, 128.38, 128.15, 126.94, 53.14; EI-
MS: m/z (%), 207 (M , 10), 206 (15), 125 (10), 111 (50), 96
(100), 68 (15), 42 (5); Accurate mass ESI-MS [M C H] for
Cž
C
2
C
14
H
6
H10N calc. 208.1910, found 208.1914.
colorless oil from the second major band (R
f
D 0.45)(0.003 g,
1
2
2
5%). H NMR: (400 MHz, CDCl
3
) υ 7.34 (8H, m), 7.27 (2H,
N,N,N-Tris[2,3,4,5,6- H ]benzylamine (6)
5
1
3
2
5
m), 1.70 (1H, s); C NMR: (200 MHz, CDCl
1
3
) υ 140.28, 128.40,
); EI-MS: m/z
[2,3,4,5,6- H ]Benzyl bromide (0.010 g, 0.057 mmol) was
1
28.18, 126.98, 53.14; H NMR: (400 MHz, CDCl
3
added to a stirred solution of acetonitrile (1 ml) and tetrahy-
drofuran (0.5 ml) and 30% aqueous ammonia (0.025 ml,
0.44 mmol). By a procedure similar to that used for N,N,N-
Cž
(
4
%), 201 (M , 10), 200 (5), 124 (10), 108 (40), 93 (100), 67 (10),
0 (10); Accurate mass ESI-MS [M C H]C for C14
H
12
2
H
4
N
0
0
2
calc. 202.1534, found 202.1534.
tris[1 ,1 - H ]benzylamine (5), a white solid was obtained
2
1
from the major TLC band (R
NMR: (400 MHz, CDCl
f
D 0.85) (0.003 g, 25%).
H
0
0 2
13
N,N,N-Tris[1 ,1 - H ]benzylamine (5)
3
) υ 3.58 (6H, s); C NMR: (400 MHz,
2
0
0
2
[
1 ,1 - H
to a stirred solution of acetonitrile (1 ml) and tetrahydrofuran
0.5 ml) and 30% aqueous ammonia (0.025 ml, 0.44 mmol).
2
]Benzyl bromide (0.010 g, 0.058 mmol) was added
CDCl
3
) υ 139.66, 128.74, 128.20, 126.84, 57.90; EI-MS: m/z (%),
302 (M , 20), 220 (20), 107 (10), 96 (100), 68 (15), 42 (5); Accu-
rate mass ESI-MS [M C H] for C21
Cž
C
2
(
7
H H15N calc. 303.2694,
The reaction mixture was stirred for 1 h at 60 °C, diluted with
found 303.2700.
water and extracted with diethyl ether. The organic layer
was separated, concentrated and applied to a preparative
thin-layer chromatography plate. The plate was eluted with
hexanes–ethyl acetate (10 : 1). The product was obtained as
2
[2,6- H ]Toluene
2
2,6-Dibromotoluene (0.040 g, 0.16 mmol) and LiAlD
(0.030 g) were mixed with diethyl ether (0.30 ml) and heated
4
Copyright  2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 1195–1204
DOI: 10.1002/jms

1198
J. Bialecki, J. Ruzicka and A. B. Attygalle
overnight in a sealed tube at 80 °C. The tube was cooled to
RESULTS AND DISCUSSION
RT and several drops of water were added to quench excess
reducing agent. The product was extracted with pentane and
The CID spectrum of protonated dibenzylamine (1, Fig. 1)
shows a significant peak at m/z 181 for a loss of NH
Fig. 2(A)). Although such a loss appears trivial, the mech-
3
dried over MgSO . The solvent was removed under reduced
4
(
1
pressure at 0 °C to afford a clear liquid (0.010 g, 67%). H
anism is not straightforward since the protonated molecule
should undergo a complicated skeletal rearrangement to
eliminate a molecule of ammonia.
NMR: (400 MHz, CDCl
3
) υ 7.25 (2H, d, J D 7.6 Hz), 7.16 (1H,
Cž
t, J D 7.2 Hz), 2.35 (3H, s); EI-MS: m/z (%), 94 (M , 75), 93
(
100), 66 (10), 52 (10), 40 (15).
For example, to eliminate a molecule of ammonia
from protonated dibenzylamine, two carbon–nitrogen bonds
must be broken, the charge on the nitrogen should be
neutralized, the two benzyl groups must be connected to
form the m/z 181 ion, and a hydrogen must be transferred
to the nitrogen atom (Scheme 1). Interestingly, the CID
spectrum of protonated tribenzylamine (4) (Fig. 3(A)) also
shows a peak at m/z 181 for a corresponding neutral loss
2
[
2,6- H ]Benzyl bromide
2
N-Bromosuccinimide (0.017 g, 0.096 mmol) was added to
a solution of [2,6- H
tetrachloride (0.5 ml) and benzoyl peroxide (1 mg) The
mixture was heated in a sealed vial at 80 °C for 4 h. Pentane
2
2
]toluene (0.010 g, 0.11 mmol), carbon
(
2 ml) was added and the organic layer was washed with
water (2 ð 2 ml), and dried over MgSO . The solvent was
4
of benzylamine. Accurate mass determinations showed that
removed under reduced pressure at 0 °C to afford crude [2,6-
C
2
the m/z 181 peak represents C14
H
13 (Table 1). We undertook
H
2
]benzyl bromide (0.010 g, 60%). EI-MS: m/z (%), 172/174
Cž
extensive studies of isotope-labeled compounds to ascertain
the mechanisms underlying the formation of the m/z 181
and other product ions of protonated benzylamines. The
compounds employed in the investigation are depicted in
Fig. 1.
(
M
, 10/10) 93 (100), 66 (15), 40 (10).
2
N,N-Bis[2,6- H ]benzylamine (7)
N-Bromosuccinimide (0.017 g, 0.96 mmol) was added to a
solution of [2,6- H
2
2
2
]toluene (0.010 g, 0.11 mmol), chloroform
Interestingly, deuterium exchange experiments of diben-
zylamine (1) and tribenzylamine (4) ascertained that the
exchangeable hydrogens remained bonded to nitrogen dur-
ing CID fragmentation to eliminate ammonia (Figs 2(B) and
3(B)). The seemingly palpable source for the additional
hydrogen required for the elimination of ammonia from
protonated dibenzylamine is the adjacent benzylic hydro-
gens. However, when the benzylic hydrogens were replaced
(
0.5 ml), and benzoyl peroxide (1 mg). The mixture was
heated in a sealed vial at 80 °C for 4 h. Acetonitrile (2 ml) was
added to the mixture followed by 30% aqueous ammonia
(
and the solvents were removed under reduced pressure. Two
drops of 12 M hydrochloric acid was added to the residue to
form a salt. The acid was evaporated under reduced pressure
and the residue was washed with ether to yield a pure white
solid (0.003 g, 35%). A small portion of the hydrochloride
0.250 ml, 4.41 mmol). The mixture was heated at 60 °C for 2 h
1
salt was converted to the free amine for EI-MS analysis. H
NMR: (400 MHz, CDCl
3
) υ 7.45 (2H, t, J D 7.6 Hz), 7.26 (1H,
d, J D 7.2 Hz), 4.15 (6H, s) 1.62 (1H, s); EI-MS: m/z (%), 201
Cž
(
(
2
M
, 10), 200 (15), 122 (10), 108 (50), 93 (100), 66 (15), 40
H
H
-NH3
C
2
CH2
10); Accurate mass ESI-MS [M C H] for C14
12 4
H H N calc.
N
02.1528, found 202.1526.
2
N-[2,3,4,5,6- H ]Benzyl-N-benzylamine (8)
5
m/z 198
m/z 181
2
[
2,3,4,5,6- H
5
]Benzyl bromide (0.010 g, 0.06 mmol) in diethyl
Scheme 1. Elimination of ammonia from protonated
dibenzylamine.
ether (0.10 ml) was added to a stirred solution of benzylamine
0.014 g, 0.13 mmol) in THF (0.20 ml). The reaction mixture
was heated at 60 °C for 2 h, washed with 5% aqueous
(
NaHCO
of the product N-[2,3,4,5,6- H
benzylamine. EI-MS: m/z (%), 202 (M , 25), 201 (20), 125
3
(2 ð 2 ml) and evaporated to give a mixture
Table 1. Accurate masses of product ions derived from
protonated dibenzylamine (1) and tribenzylamine (4)
2
5
]benzyl-N-benzylamine and
Cž
Elemental Accurate
Relative
(
10), 120 (10), 111 (55), 106 (50), 96 (100), 91 (100); Accurate
C
2
Comp- Nominal compo-
mass
(calculated)
Observed error
mass ESI-MS [M C H] for C14
11 5
H H N calc. 203.1591, found
a
ound
m/z
sition
mass
(ppm)
2
03.1593.
C
C
1
181
C14H13
C7H8N
C7H7
181.1017
106.0657
91.0548
196.1126
181.1017
120.0813
91.0548
181.1018
106.0655
91.0547
196.1126
181.1020
120.0815
91.0547
0.4
ꢀ1.5
ꢀ0.7
0.1
1.5
1.6
Tetrabenzyl ammonium bromide (9)
Tribenzylamine (0.010 g, 0.035 mmol) in dry THF (0.2 ml)
was added to a stirred solution of benzyl bromide (0.010 g,
1
06
C
9
1
C
4
196
C14H14N
0
.06 mmol) in dry diethyl ether (0.1 ml). The reaction
C
1
1
81
20
C14H13
mixture was heated at 60 °C for 2 h. The white precipitate
C
C8H10N
formed was filtered and washed with ether to yield a
mixture (0.017 g) of tribenzylamine hydrobromide and the
C
9
1
C7H7
ꢀ1.0
a
product tetrabenzylammonium bromide. Accurate mass ESI-
Accurate masses were determined on the Q-Tof instrument at
C
MS [M ꢀ Br] for C28
H28N calc. 378.2216, found 378.2220.
R ³ 15 000.
Copyright  2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 1195–1204
DOI: 10.1002/jms

CID of protonated benzylamines
1199
C
Figure 2. Product ion spectra of [M C H] ions of m/z 198 of dibenzylamine (1) (A), m/z 200 of dibezylamine recorded in D2O (B),
0
0 2
2
m/z 202 of N, N-bis([1 ,1 - H2]benzyl)amine (2) (C), and m/z 208 of N, N-bis([2,3,4,5,6- H5]benzyl)amine (3) (D), recorded at a
collision energy setting of 15 eV.
by deuterons and the spectrum of the deuteriated benzy-
lamine (2) was recorded, to our surprise no deuterium atom
was eliminated in the process of eliminating an ammonia
molecule (Fig. 2(C)). Similarly, benzylic hydrogens do not
provide the extra hydrogen atom required for the elimination
of a benzylamine molecule from protonated tribenzylamine
from a phenyl ring was exclusively eliminated in the process
of budding an ammonia (Fig. 2(D), peak at m/z 190) or a
benzylamine molecule (Fig. 3(D), peak at m/z 190).
In order to rationalize these observations, we postulate
the mechanism given in Scheme 2. An initial elongation of
a C–N bond at the charge center leads to the consequent
cleavage of the C–N bond forming an ion/neutral complex
of benzyl cation and benzylamine. A simple dissociation of
the complex leads to the benzyl cation that represents the
most intense peak observed at m/z 91, or 93 and 96 for
deuterated species, in Figs 2 and 3. On the other hand, the
(
Fig. 3(C)). It follows that a hydrogen from a phenyl ring
must transfer specifically to the nitrogen atom during the
elimination of ammonia or benzylamine. Support for this
proposition was obtained from the CID spectra of ring-
deuteriated amines 3 and 6. As expected, a deuterium atom
R
H
H
NHR
NH2R
CH2
N
NHR
+
NH2R
CH2
1
4
, R = H
, R = CH2Ph
11
12
Scheme 2. Fragmentation mechanism proposed for elimination of ammonia or benzylamine from protonated benzylamines.
Copyright  2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 1195–1204
DOI: 10.1002/jms

1200
J. Bialecki, J. Ruzicka and A. B. Attygalle
1
1
1
00
A
B
C
CH2 91
Bn
H
2
N
H
88
N
CH2
CH2
N
H
181
196
120
0
00
CH2 91
Bn 289
D
N
D
N
CH2
CH2
N
D
181
197
121
0
00
CD2 93
D2C
CD2⋅Ph
N
H
H
294
CD2
N
CD2
D2C
CD2
D
N
H
CD2
CD2
185
124
199
0
D
D
D
D
100
D
D
D
D
D
D
9
6
D
D
D
CH2
303
D
D
D
D
D
D
D
D
H
D
D
D
2 6 5
CH C D
H
D
D
CH2
CH2
D
N
D
N
D
D
D
D
D
D
D
D
N
H
D
D
1
90
206
D
D
125
0
50
100
150
200
250
300
m/z
C
Figure 3. Product ion spectra of [M C H] of m/z 288 of tribenzylamine (4) (A), m/z 289 of tribenzylamine recorded in D2O (B), m/z
0 0 2
2
2
94 of N,N,N-tris([1 ,1 - H2]benzyl)amine (5) (C), and m/z 303 of N,N,N-tris([2,3,4,5,6- H5]benzyl)amine (6) (D), recorded at a collision
energy setting of 20 eV.
benzyl cation could initiate an electrophilic attack on the ipso,
ortho, meta, or para positions of the benzylamine by removing
a pair of electrons from the aromatic sextet yielding an
to eliminate ammonia (from amine 1; R H), or benzylamine
(from amine 4; R CH Ph), thus forming a benzylbenzyl
that is observed at m/z 181 (Figs 2 and 3). The
2
9
,10
cation
7
arenium ion (11, Scheme 2). Although the arenium ion bears
benzylbenzyl cation could well undergo a ring closure to
yield a protonated dihydroanthracene isomer as described by
less stability than that associated with aromatic systems, the
ion carries some stability of its own by resonance. A more
realistic structure for this highly reactive arenium ion is that
illustrated as structure 13.
1
1
Gr u¨ tzmacher and Dohmeier-Fischer. Moreover, the mech-
anism proposed for the loss of ammonia observed in the
CID spectrum of protonated noradrenaline, a related com-
pound, could well undergo a transition through an arenium
1
2
ion. It is well known that intramolecular proton exchanges
occur in arenium ions at a very rapid phase. For example,
1
1 ring hydrogens in protonated ˛,ω-diphenylalkanes are
NHR
H
1
3
known to randomize within a span of 1 µs (Scheme 3).
Protons are deemed to ‘ring walk’ in isolated arenium ions
1
4
via an edge-protonated species. If this model is valid,
the hydrogen required for the elimination of an ammonia
molecule should not originate specifically from the ortho
position.
13
The highly reactive arenium ion then stabilizes itself by
regenerating the aromatic ring by donating a ring proton
In fact, the CID spectrum of protonated compound
7 (dibenzylamine with ortho-dideuteriated phenyl rings)
supports the ‘proton dance’ model. The spectrum shows
(
or deuteron when present) to the amino group, thereby
generating a protonated amine intermediate 12 (Scheme 2),
which could undergo a charge-driven heterolytic cleavage
3 2
peaks for the loss of both NH and NDH at m/z 185, and 184,
respectively, at an approximate ratio of 3 to 2 (Fig. 4(A)). On
8
Copyright  2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 1195–1204
DOI: 10.1002/jms

CID of protonated benzylamines
1201
C
2
Figure 4. Product ion spectra of [M C H] of m/z 202 of N, N-bis([2,6- H2]benzyl)amine (7) (A), and m/z 203 of
2
N, N-([2,3,4,5,6- H2]benzyl)benzylamine (8) (B), recorded at a collision energy setting of 15 eV. (Insets show expansion of m/z
183–187 region).
The formation of a benzylbenzyl cation (m/z 181) in
gas phase has been well investigated. For example, the
H
H
H
H
(
CH2)n
(CH2)n
14
reaction of gaseous benzyl cations with benzyl methyl ether
leads to m/z 181 as a condensation product. Similarly,
protonated α,ω-diphenylalkanes
1
1
H
H
H
the fragmentation of a protonated dimer formed under
H
H
H
10
chemical ionization of benzyl acetate produces m/z 181.
H
The formation of this m/z 181 ion from the protonated dimer
of benzyl acetate had been rationalized by two consecutive
losses of acetic acid. However, no attempts had been made
to confirm the exact origin of the transferred hydrogen
atoms during the rearrangement. Applying the arenium ion
mechanism discussed here, we propose a rationale for the
consecutive acetic acid losses, in which an aromatic proton
is eliminated during the loss of the first acetic acid molecule
(Scheme 4).
A comparison of CID spectra of protonated dibenzy-
lamine (1) and tribenzylamine (4) revealed another inter-
esting feature. Under identical CID conditions, protonated
compound 4 produces a more intense signal at m/z 181 than
compound 1. We asked ourselves if the tetrabenzylammo-
nium ion (9) would generate an even more intense m/z 181
signal than that of tribenzylammonium ion under identical
conditions. As expected, the CID spectrum of the tetra-
benzylammonium ion showed a significantly more intense
signal at m/z 181 than those of protonated amines 1 and 4
protonated benzene
Scheme 3. Ring Walk mechanism in arenium ions proposed by
Kuck et al. (Refs 13 and 14).
the other hand, the CID spectrum of protonated compound
8
(dibenzylamine with one perdeuterated benzene ring),
which demonstrates a favored loss of NH over that of
NDH even though both rings are statistically equivalent,
provided confirmative evidence for the favored breakage of
a C–H bond over a C–D bond owing to an isotope effect
Fig. 4(B)). In the presence of such a pronounced isotope
effect, it is clear that a loss of NDH is somewhat biased
over that of NH (original ortho protons are eliminated more
rapidly than the others). The most plausible explanation for
the observed ratios is that the complete H/D equilibrium
had not been achieved before the rearomatization took place
by donating a proton to the amine group.
3
2
(
2
3
HO
O
HO
O
HO
OH
O
O
O
O
O
O
O
CH2
O
AcOH
AcOH
H
CH2
m/z 301
m/z 181
Scheme 4. Mechanism proposed for fragmentation of protonated dimer of benzyl acetate.
Copyright  2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 1195–1204
DOI: 10.1002/jms

1202
J. Bialecki, J. Ruzicka and A. B. Attygalle
100
A
198
9
1
1
NH2
30
106
181
0
288
1
00
B
C
9
H
N
181
196
120
0
1
00
378
181
N
9
1
196
0
50
100
150
200
m/z
250
300
350
C
Figure 5. Product ion spectra of [M C H] of m/z 198 dibenzylamine (1) (A), m/z 288 of tribenzylamine (4) (B), and that of m/z 378 of
C
[
M ꢀ Br] of tetrabenzylammonium bromide (9) (C). All spectra were recorded under the same conditions and settings (collision
energy 18 eV) over a period of 1 h.
(
Fig. 5). Moreover, the tetrabenzylammonium ion produced
(168 Da). Since both product ions are of even mass and
a drastically low-intense benzyl-cation signal at m/z 91. Pre-
sumably, the formation of the arenium ion leading to m/z 181
ion relieves the steric congestion of the ion/neutral complex
more effectively than that delivered by a direct formation
of the benzyl cation and tribenzylamine. To a lesser extent,
steric effects must also play a role in the fragmentation of
protonated tribenzylamine (4) as well. However, the elec-
trophilic attack of the benzyl cation on the amine is most
favorable with the ion/neutral complex derived from the
tetrabenzylammonium ion (9). This is due to the fact that the
benzyl groups are more numerous and more closely located
in the ion/neutral complex of tetrabenzylammonium ion
than those derived from protonated tribenzylamine (4) and
dibenzylamine (1). Consequently, the formation of m/z 181
would then be expected to be less favorable in the case of
contain one nitrogen atom, the probable structure for the
C
m/z 30 ion must be H
120 ion should be H
2
C
NH
2
, while that for the m/z
Ph. The proposed formula
C
2
C
NH CH
2
for the benzylimmonium ion is supported by its accurate
mass (Table 1). The proposed loss of diphenylmethane is
interesting because it shows similarities to the formation of
m/z 181. In order to eliminate a diphenylmethane molecule,
a carbon–nitrogen bond and carbon–carbon bond must be
broken and the two aromatic groups must be reconnected.
We asked ourselves if the benzyl groups are connected
through a similar mechanism involving the formation of
the arenium ion. The CID spectra of protonated deuterated
dibenzylamine (2) and tribenzylamine (5) showed a loss of
2
[ H
2
]diphenylmethane (170 Da) to give peaks at m/z 32 and
m/z 124 (Figs 2(C) and 3(C)), respectively, indicating only one
methylene group gets eliminated with the leaving NH , or
4
, and even lesser in 1, which in fact is congruent with the
2
observed data.
NHR, moiety. For example, the intermediate arenium ion
11 originating from an ortho attack undergoes a charge-driven
heterolytic cleavage to restore the aromatic ring, thereby
producing the relevant immonium ion (Scheme 5). A similar
mechanism can be postulated for the fragmentation of the
arenium intermediate that originates from an ipso attack
(Scheme 6). Support for the postulated pathways was
Beside m/z 181, CID spectra of protonated amines 1
and 4 show a pair of low-intensity signals at m/z 30 and
1
06, and 120 and 196, respectively (Figs 2(A) and 3(A)). We
postulate that the ions that correspond to peaks at m/z 30
and m/z 120 in the spectra of protonated amines 1 and
4
originate by eliminating a molecule of diphenylmethane
H
NHR
NHR
H
R
H
R
H
CH2
H2C
N
+
N
NHR
11
1
4
, R = H
, R = CH2Ph
Scheme 5. Proposed mechanism for elimination of diphenylmethane from protonated di- and tri-benzylamine (ortho attack).
Copyright  2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 1195–1204
DOI: 10.1002/jms

CID of protonated benzylamines
1203
H
R
H
R
H
CH2
N
NHR
H2C
N
+
NHR
1
4
, R = H
, R = CH2Ph
Scheme 6. Proposed mechanism for elimination of diphenylmethane from protonated di- and tri-benzylamine (ipso attack).
CH3
R
H
NHR
+
H
N
H
NHR
CH2
1
4
, R = H
, R = CH Ph
2
Scheme 7. Mechanism proposed for elimination of toluene from protonated di- and tri-benzylamine.
obtained by the CID spectra of deuterated compounds 3 and
(or dibenzylamine) to the benzylic cation occurs within the
proposed ion/neutral complex to eliminate toluene as a neu-
tral fragment (Scheme 7). The CID spectra of deuteriated
2
6
. The elimination of [ H10]diphenylmethane (178 Da) to yield
signals at m/z 30 and m/z 125 (Figs 2(D) and 3(D)) indicates
that no deuterium exchange occurs from the phenyl rings to
amines 3 and 6 show peaks at m/z 111 and 206, respectively,
C
2
the H
2
C
NHR immonium ion during this fragmentation.
for a loss of 97 Da for [ H
5
]toluene (Figs 2(D) and 3(D))
Moreover, CID spectra of ions derived from the proto-
nation of amines 1 and 4 show peaks at m/z 106 and m/z
96, respectively, both of which are formed by a significant
loss of elements of toluene (Figs 2(A) and 3(A)). Similar to
the above-mentioned m/z 30 and m/z 120 ions, the m/z 106
and m/z 196 species are even-mass ions that differ by 90 Da,
which formally represents a benzyl group minus one hydro-
corroborating that no deuterium transfer occurs from the
ring in this fragmentation. Since the aromatic groups do not
join, no arenium ion intermediates are expected. Our mech-
anism is not unique since similar hydride abstractions have
been observed in other ion/neutral complexes that eliminate
1
1
5,16
toluene.
The antifungal drug butenafine hydrochloride (10) is a
salt of a tertiary amine, as well as a modified dibenzylamine.
To demonstrate the applicability of our mechanisms, we
predicted that compound 10 will undergo CID fragmen-
tations similar to those observed for dibenzylamine and
tribenzylamine. In fact, the CID spectrum of butenafine (10)
showed four low-intensity signals at m/z 287, 176, 170, and
gen. In contrast, CID spectra derived from deuterium-labeled
2
compounds 3 and 6 show a loss of 95 Da for [ H
3
]toluene
(
Figs 2(C) and 3(C)). The accurate masses of m/z 106 and
m/z 196 ions agree with proposed elemental compositions
Table 1). This observation indicates that a hydride abstrac-
tion from a methylene group of the neutral benzylamine
(
100
141
318
H
N
CH3
H
CH
3
N
147
CH3
H
H2C
N
H
N
1
70 176
3
0
CH3
287
300
0
50
100
150
200
250
m/z
Figure 6. Product ion spectrum of m/z 318 of [M ꢀ Cl]C of butenafine hydrochloride (10).
Copyright  2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 1195–1204
DOI: 10.1002/jms

1204
J. Bialecki, J. Ruzicka and A. B. Attygalle
4
4, all of which could be accurately predicted by our mecha-
6. Nicholas M, Fabre B, Pilard JF, Simonet J. A new efficient route
to 3-vinylthiophene. J. Heterocycl. Chem. 1999; 36: 1105.
nisms (Fig. 6). This indicates that the fragmentation patterns
described are universal, and are useful in predicting CID
fragmentations of protonated benzylamine derivatives, sev-
eral of which are biologically and medicinally important
compounds.
Developing predictive rules for low-energy CID spectra
is very important for the identification of drugs and their
metabolites in biological systems. Using artificial intelligence
for this purpose has been attempted, but has not yet
been widely successful. Predictive rules formulated without
detailed labeling studies could be misleading. Here, we
have accomplished a detailed study on the fragmentation of
benzylamine derivatives, and have demonstrated how our
conclusions can be used for the identification of benzylamine
drugs and related compounds.
7. Olah GA. Stable carbocations. CXVIII. General concept and
structure of carbocations based on differentiation of trivalent
(classical) carbenium ions from three-center bound penta-
of tetracoordinated (nonclassical) carbonium ions. Role of
carbocations in electrophilic reactions. J. Am. Chem. Soc. 1972; 94:
808.
8
. Brouwer DM, Mackor EL, MacLean C. In Carbonium Ions, vol. 2.
Olah GA, Schleyer PvR (eds). Wiley: New York, 1970; 837.
. Nicolaides A, Radom L. Seven-membered ring or phenyl-
substituted cation? Relative stabilities of the tropylium and
benzyl cations and their silicon analogues. J. Am. Chem. Soc.
9
1994; 116: 9769.
10. Cameron D, Cooks RG. Characterization of the chemical
ionization condensation products for the benzyl acetate system
using mass-analyzed ion kinetic energy spectrometry. J. Am.
Chem. Soc. 1979; 101: 316.
1
1. Gr u¨ tzmacher H-F, Dohmeier-Fischer S. Polycondensation of
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Acknowledgements
This investigation was supported by funds from Stevens Institute of
Technology.
1
2. Rogalewicz F, Bourcier S, Hoppilliard Y. Decomposition of
protonated noradrenaline and normetanephrine assisted by
NH2 migration studied by electrospray tandem mass
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Copyright  2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 1195–1204
DOI: 10.1002/jms