2806 X. Wang, M. Li and A. M. Rustum
LC-PDA (photo-diode array)-MSn analyses
OH
14
12
O
N
N
OH
11
10
17
N
N
A Thermo Fisher Scientific LTQ-Orbitrap mass spectrometer
was used in positive ion mode for all the LC-PDA-MS and
LC-PDA-MS/MS experiments. An ACE3 C8 column
(150 ꢂ 4.6 mm, 3 mm; Mac Mod, Chadds Ford, PA, USA)
was used with the temperature controlled at 408C. Mobile
phase A consisted of 10 mM ammonium acetate and mobile
phase B consisted of acetonitrile. A linear gradient was
1
9
N
Cl
N
Cl
S
6
4
5
S
B
MW 419
A
MW 403
O
N
OH
OH
N
N
N
O
started from 25% to 60%
B in 20 min, followed by
O
N
Cl
N
Cl
equilibration at 25% B for 5 min at a flow rate of 1.5 mL/
min. For all the LC/ESI-MS experiments, the LC flow was
split 10:1 prior to the mass spectrometer, and ꢁ150 mL/min
of the flow was directed into the ESI source of the mass
spectrometer. The operating conditions for the ESI ion source
were as follows: spray voltage 4.5 kV, capillary voltage 36 V,
tube lens voltage 85 V, capillary temperature 2758C, sheath
gas (N2) pressure 45 (arbitrary units), and auxiliary gas (N2)
pressure 5 (arbitrary units). For all the LC/APCI-MS
experiments, the entire LC flow was directed into the APCI
source of the mass detector. The operating conditions for the
APCI source were as follows: vaporizer temperature 4508C,
capillary temperature 3008C, capillary voltage 26 V, tube lens
85 V, sheath gas (N2) 45 (arbitrary units of pressure), and
auxiliary gas (N2) 5 (arbitrary units of pressure). The mass
scale was calibrated at the beginning of a working day using
a solution of a polytyrosine mixture. Full scan mass spectra
were acquired in the range m/z 100–800 with a resolving
power of 60 000 at m/z 400. CID experiments were conducted
using helium as the collision gas. Low-resolution MS/MS
experiments were performed in the LTQ mass spectrometer,
with a precursor ion isolation width of 2.0 m/z units and
normalized collision energy of 28%.
S
S
C
MW 435
D
MW 419
Figure 1. Structures of perphenazine (A) with the numbering
of key positions, perphenazine 14-N-oxide (B), perphenazine
14,17-N,N-dioxide (C), and perphenazine 17-N-oxide (D).
molecule.3,4 These unique fragmentation pathways occur-
ring in the thermal degradation processes are due to thermal
energy activation in the vaporizer of the APCI source and not
to collision-induced dissociation (CID).3,4,7
Perphenazine (Fig. 1) is one of the first generation of
antipsychotic drugs; it has three tertiary amine groups.
During a recent oxidative stress study of perphenazine
performed in our laboratory, six N-oxide degradants were
generated. In this paper we report the observation of several
unique fragment ions observed in the APCI mass spectra of
two N-oxide degradants; the occurrence of these unique
fragments has not been previously reported. These novel
fragmentation pathways were observed only in APCI mode
but not in ESI and CID, suggesting they are thermally
induced degradation products in the APCI process. The
structures of the two N-oxide compounds (perphenazine 14-
N-oxide and perphenazine 14,17-N,N-dioxide) are shown in
Fig. 1.
RESULTS AND DISCUSSION
Deoxygenation and dehydration of N-oxides
The UV chromatogram at 254 nm of the hydrogen peroxide-
stressed perphenazine sample is shown in Fig. 2. Multiple
oxidative degradants were observed and they can be divided
into two categories: three mono-oxidized ones (peaks 3, 5 and
6) and three di-oxidized ones (peaks 1, 2 and 4). The mono-
oxidized 17-N-oxide of perphenazine is the most abundant
degradant under these liquid-phase stressing conditions. It is
interesting to note that this degradant is also the most
abundant oxidative degradant in a solid formulation of
perphenazine under stability testing (data not shown). The
identities of these degradants were confirmed by various 1D
and 2D 1H, 13C and 15N NMR analyses (The 1H NMR spectra
of perphenazine and perphenazine 17-N-oxide are shown in
the Supporting Information). The full details of the structural
characterization will be published elsewhere. In this paper,
we focus on the unique fragment ions observed during the
LC/APCI-MS analysis of two of the N-oxide degradants:
perphenazine 14-N-oxide and perphenazine 14,17-N,N-
dioxide (Fig. 1). The ESI and APCI mass spectra of these
two degradants are shown in Figs. 3 and 4, respectively. The
ESI mass spectra displayed only the protonated molecules,
while the APCI mass spectra displayed a number of fragment
ions in addition to the protonated molecules. The fragment
ion at m/z 404 in the APCI mass spectrum of perphenazine 14-
EXPERIMENTAL
Chemicals
Perphenazine is an in-house reference standard. Acetonitrile,
methanol and ammonium acetate (all HPLC grade) were
purchased from Fisher Scientific (Pittsburgh, PA, USA).
Trifluoroacetic acid (TFA), 30% (wt%) H2O2 solution and ꢁ2–
3% H128O2 solution were purchased from Sigma (St. Louis,
MO, USA).
Sample preparations
An aliquot of 20 mL of 30% (wt%) H2O2 solution was added to
1 mL of 1 mg/mL perphenazine methanol solution. The
mixture was heated in a heating block (Fisher Scientific) at
508C for up to 2 h and then analyzed by LC/ESI-MS and LC/
APCI-MS.
To prepare the 18O-isotope-labeled perphenazine N-oxide
compounds, 40 mL of a ꢁ2–3% (wt%) H128O2 solution was
added to 1 mL of 1 mg/mL perphenazine methanol solution.
The resulting mixture was heated in a heating block (Fisher
Scientific) at 608C for up to 3 h and then analyzed by LC/
APCI-MS.
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 2805–2811
DOI: 10.1002/rcm