Citalopram hydrobromide: Degradation product characterization and a validated stability-indicating LC-UV method

Article abstract of DOI:10.1590/S0103-50532011000500005

Five degradation products (I-V) of citalopram hydrobromide (CTL) were formed under different forced degradation conditions. Products I and II were formed under hydrolytic conditions while product III-V were formed under photolytic conditions. Products II

Full text of DOI:10.1590/S0103-50532011000500005

J. Braz. Chem. Soc., Vol. 22, No. 5, 836-848, 2011.
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Article
A
Citalopram Hydrobromide: Degradation Product Characterization and a Validated
Stability-Indicating LC-UV Method
Manav Sharma, Parikshit R. Jawa, Ravinder S. Gill and Gulshan Bansal*
Department of Pharmaceutical Sciences and Drug Research, Punjabi University,
Patiala, 147 002, India
Cinco produtos de degradação (I-V) do hidrobromidrato de citalopram (CTL) foram formados
sob condições diferentes de degradação forçada. Os produtos I e II foram formados sob condições
hidrolíticas, enquanto que os produtos III-V sob condições fotolíticas. Os produtos II e IV
foram encontrados como impurezas conhecidas, citalopram carboxamida e citolopram-N-óxido,
respectivamente. O produto I foi encontrado como sendo uma nova impureza, que foi caracterizada
como 3-hidroxicitalopram N-óxido. A droga e todos os cinco produtos de degradação foram
resolvidos de uma maneira otimizada em uma coluna C8 com fase móvel composta de acetonitrila e
tampão acetato de amônia (pH* 4,5) na vazão de 0,50 mL min-1. O método foi linear, preciso com
RSD < 3 (desvio padrão relativo) e exato com recuperação de 88-97% no intervalo de concentração
5-500 mg mL-1 de citalopram. Os limites de determinação (LOD) e de quantificação (LOQ) foram
1 mg mL-1 e 5 mg mL-1, respectivamente. A análise por arranjo de fotodiodo de uma solução da
droga degradada contendo a droga e todos os cinco produtos de degradação revelou picos isolados,
puros. Sendo assim, o método pode ser sugerido como estável.
Five degradation products (I-V) of citalopram hydrobromide (CTL) were formed under different
forced degradation conditions. Products I and II were formed under hydrolytic conditions while
product III-V were formed under photolytic conditions. Products II and IV were found known
impurities as citalopram carboxamide and citalopram N-oxide, respectively. Product I was found
to be a new impurity which was characterized as 3-hydroxycitalopram N-oxide. The drug and all
five degradation products were optimally resolved on a C8 column with mobile phase composed
of acetonitrile and ammonium acetate buffer (pH* 4.5) flowing at a rate of 0.50 mL min-1. The
method was linear, precise RSD < 3 (relative standard deviation) and accurate (recovery being
88-97%) in the concentration range of 5-500 mg mL-1 of citalopram. The limits of detection
(LOD) and of quantitation (LOQ) were 1 mg mL-1 and 5 mg mL-1, respectively. The photodiode
array (PDA) analysis of the degraded CTL solution containing the CTL and all five degradation
products revealed each peak to be pure. Hence, the method was suggested to be stability-indicating.
Keywords: citalopram, forced degradation, liquid chromatography-mass spectrometry
(LC-MS), degradation products, mass fragmentation
Introduction
drug makes it susceptible to degradation due to chemical
reactivities of these groups under hydrolytic, oxidative, and
photolytic environments,3,4 which will eventually produce
varied impurities or degradation products. Four process-
related impurities of CTL [citalopram carboxamide (A),
citalopram cyano N-oxide (B), citalopram N-oxide (C) and
citalopram Di N-oxide (D)] reported in literature5 are listed
in Figure 1. Recently, Raman et al.6 have characterized a
new process-related impurity as citalopram butanone (E)
(Figure1).
Citalopram hydrobromide (CTL) (Figure 1) is a
highly selective and potent serotonin reuptake inhibitor
which potentiates the serotonergic activity to produce
antidepressant action.1 It is also reported to reverse
the memory impairment.2 Chemically, it is (RS)-
1-[3-(dimethylamino)propyl]-1-(p-fluorophenyl)-5-
phthalan carbonitrile. Presence of nitrile group, fused
teterahydrofuran ring, and C-N linkage in structure of the
The International Conference on Harmonization (ICH)
guidelines Q3A and Q3B require the characterization
*e-mail: gulshanbansal@rediffmail.com

Vol. 22, No. 5, 2011
Sharma et al.
837
10
11
NC
O
12
9
2
8
5
N
7
3
1
12
6
4
F
Citalopram
O
OH
O
N
C
O
H2N
H2N
O
O
O
N
N
N
F
F
F
3-Hydroxy citalopram
Citalopram carboxamide (A; II)
Citalopram cyano N-oxide (B)
carboxamide (I)
N
C
NC
NC
O
O
O
O
N
O
N
N
O
O
F
F
F
Citalopram N-oxide (C; IV)
Citalopram Di-N-oxide (D)
Citalopram butanone (E)
Figure 1. Citalopram and its known impurities (A-E) and degradation products (I, II and IV).
of all possible impurities and degradation products in a
drug substance and product.7 Further, the ICH guidelines
Q1A(R2) recommend forced degradation of the drug
substance under varied conditions like hydrolysis, oxidation,
heat and photolysis to generate all possible impurities or
degradation products in order to facilitate their structural
characterization and also in development and validation
of a stability-indicating assay method (SIAM).8 However,
there is no systematic forced degradation study on CTL.
Moreover, spectral and mass fragmentation data of only a
few process-related impurities is available in literature.6,9
Further, there are numerous analytical methods available
for quantification of CTL in formulations and biological
matrixes.10 Both the European Pharmacopoeia and United
States Pharmacopeia have mentioned high performance
liquid chromatography (HPLC) methods for separation of
citalopram and some of its related substances,11 but there
is no method suitable to SIAM.
Experimental
Chemicals and reagents
CTL was obtained as a generous gift sample from Ind-
Swift Laboratories Ltd. (Dera Bassi, India). Acetonitrile
and methanol (both HPLC grade) and hydrochloric acid,
sodium hydroxide pellets, ammonium acetate and acetic
acid (all AR grade) were purchased from Ranbaxy Fine
Chemicals (SAS Nagar, India). Milli-Q water obtained
from Milli-Q water purification system (Synergy, Millipore,
USA) in the laboratory was employed to prepare all
reagents and solutions.
Instrumentation
The hydrolytic degradation studies were performed
using a high-precision water bath equipped with a digital
temperature controller (Narang Scientific Works, New
Delhi, India) capable of maintaining temperature within
1 °C. The thermal degradation study was conducted in
a high precision hot air oven (Narang Scientific Works,
New Delhi, India) having a temperature precision of
2 °C. Photodegradation studies were performed in a
photostability chamber (KBF 240;WTB Binder, Tuttlingen,
Hence, the present study is undertaken to conduct ICH
prescribed systematic forced degradation study on CTL to
identify its potential degradation products and to develop
and validate a SIAM for accurate quantification of CTL
in the presence of its impurities and degradation products.
The aim is to provide and substantiate the present data on
impurities and quantification methods.

838
Citalopram Hydrobromide: Degradation Product Characterization
J. Braz. Chem. Soc.
Germany) which was equipped with a light bank consisting
of two UV (OSRAM L73) and four fluorescent (OSRAM
L20) lamps and capable of controlling temperature
and relative humidity in the range of 2 °C and 5%,
respectively. The light system complied with Option 2 of the
ICH guideline Q1B.12 The LC-UV (liquid chromatography-
ultraviolet detection) analyses were carried on an HPLC
system consisted of 515 binary pumps, 2487 dual
wavelength detector, Rheodyne manual injector, and
Millennium 2.01 software (Waters Corp., Milford, USA).
The chromatographic separations were achieved on C8
column (150 mm × 4.6 mm i.d., 5 mm, Spherisorb®) (Waters
Corp., Milford, USA).A Nucleosil C8 (8 mm × 4.6 mm i.d.)
guard column was placed before the analytical column.
The photodiode array (PDA) analysis was performed on
an HPLC system consisting of a 600 E pump, 996 PDA
detector, 717 autoinjector and degasser module (Waters
Corp., Milford, USA). The liquid chromatography-mass
spectrometry (LC-MS) studies and mass spectral analyses
were performed in positive electrospray ionization
(+ESI) mode using a Bruker Daltonics GmbH microTOF
instrument (Bremen, Germany) controlled by microTOF
control software version 2.0. The LC system of LC-MS was
an 1100 series liquid chromatograph (Agilent Technologies
Inc., Wilmington, USA), controlled by Hystar (version 3.1)
software. The column used for LC-MS studies was the same
as that for LC-UV analyses. Fourier transform infrared
(FT-IR) spectra were recorded using 1711 Perkin Elmer IR
spectrophotometer (Shelton, CT, USA) while the nuclear
magnetic resonance of 1H (NMR spectrum) was recorded
on Avance II 400 spectrophotometer (Bruker, Fallanden,
Switzerland).
intervals. Whenever necessary, they were neutralized by
acid or alkali and diluted up to 10 times with mobile phase
before injection.
Chromatographic conditions
The drug and all degradation products formed under
different forced conditions were optimally resolved on C8
column (150 mm × 4.6 mm i.d., 5mm, Spherisorb®) with
a mobile phase composed of acetonitrile and ammonium
acetate buffer (0.025 mol L-1) (35:65 v/v, pH* 4.5) at a flow
rate of 0.50 mL min-1. The injection volume was 20 mL,
and the eluent was monitored at 240 nm. The method was
validated for its stability-indicating nature. Appropriate
blank was injected before each analysis.
Isolation and characterization of degradation product
The major degradation product (II) formed in
0.1 mol L-1 NaOH after refluxing at 85 °C for 35 h was
isolated as fine crystals after neutralizing the solution
with 0.1 mol L-1 HCl and cooling the reaction solution in
refrigerator for 24 h. The crystals were filtered, washed with
cold water, and dried in a vacuum desiccator. Purity of the
product was ascertained by the optimized chromatographic
method. Its structure was ascertained by comparing its
1
FT-IR, H NMR and mass spectral data with that of the
drug. The other degradation products formed in sufficient
amounts were characterised through LC-PDA and LC-MS
studies (in both –ESI and +ESI modes, i.e. negative and
positive ion modes) using a mixed degraded sample
which was prepared by mixing drug solutions subjected to
different forced conditions so that all degradation products
were present in one solution.
Forced decomposition study
The drug was subjected to hydrolytic degradation in
2 mol L-1 HCl, water and 0.2 mol L-1 NaOH at 85 °C over
48 h. The oxidative degradation was performed in 30%
H2O2 solution at room temperature over 48 h. For thermal
degradation, the solid drug was sealed in amber coloured
borosilicate glass vials, which were maintained at 50 °C
for 31 days. The photolytic studies were performed by
exposing solid drug spread as a thin layer in Petri-plates
as well as drug solutions in 2 mol L-1 HCl, water, and
0.01 mol L-1 NaOH in borosilicate vials to the light in
the photostability chamber for 16 days during which the
total light exposure was 1.2 × 106 lux. A parallel set of
solid drug and drug solutions was kept in dark chamber
for the same period to serve as dark control. The drug
concentration in all solution phase studies was 0.1% m/v.
Samples were withdrawn at zero time and at prefixed time
Method validation
The optimized method was validated by evaluating
linearity, precision, accuracy, limit of detection (LOD),
limit of quantitation (LOQ), selectivity and ruggedness as
per ICH guidelines Q2(R1).13
Linearity and range
A stock solution of CTL (1 mg mL-1) in water was
prepared in triplicate and each solution was serially diluted
with mobile phase to prepare standard solutions having
concentrations in the range of 5 to 500 mg mL-1. Each set
of stock and standard solutions was analyzed using the
optimized chromatographic conditions to generate three
calibration plots in order to select the concentration range
providing maximally linear relationship with the response.

Vol. 22, No. 5, 2011
Sharma et al.
839
Precision
chromatographed on a C18 column (150 mm × 4.6 mm i.d.,
Spherisorb®) with mobile phase composed of phosphate
buffer (0.02 mol L-1, pH 3.0) and acetonitrile (60:40 v/v)
flowing at a rate of 0.8 mL min-1 using 240 nm as
detection wavelength. The drug eluted at 4.5 min but
degradation products formed under different conditions
were not resolved. The phosphate buffer was replaced
with ammonium acetate buffer to make the method
LC-MS compatible, and flow rate was decreased to
0.4 mL min-1 to resolve the degradation products. It eluted
the drug at 19.1 min but degradation products formed in
photodegraded drug solution in 2 mol L-1 HCl were not
resolved. To optimize the chromatographic conditions
for separation of drug and all degradation products in
a single isocratic run, the mixed degraded solution was
employed so that all degradation products were present in
one solution. Numerous alterations in flow rate and mobile
phase compositions did not improved resolution of the
degradation products. Even use of ternary mobile phase
composed of buffer (pH 3.0), acetonitrile, and methanol in
varied proportions did not resolve the degradation products.
A critical analysis of structure of the drug followed
by prediction of various degradation products based on
chemical reactivities of the drug molecule under varied
forced degradation conditions and various chromatograms
obtained during the method development process suggested
that the possible degradation products are polar than
the drug itself. Further, differences in polarities of these
degradation products were expected to be very small which
is responsible for very close elution of these products.
Based on this hypothesis, it was predicted that elution of the
degradation products may be delayed by increasing polarity
of stationary phase. Hence, C18 column was replaced by C8
column (150 mm × 4.6 mm i.d., 5 mm, Spherisorb®) which
was run over by mobile phase composed of ammonium
acetate solution (0.02 mol L-1) and acetonitrile (40:60,
pH* 6.0) at a rate of 1 mL min-1. The drug eluted at 19 min
but degradation products were noted as merged peaks
ahead of the drug peak. Lowering of pH* to 5.5 increased
the sharpness of peaks. Increase in proportion of aqueous
phase to 65% and further lowering the pH* to 4.5 improved
the resolution with the drug eluting at 34 min. Further
increase in aqueous component dashaped the peaks and also
delayed drug elution to 80 min. Increase in concentration
of ammonium acetate from 0.02 to 0.025 mol L-1 resulted
in a highly resolved chromatogram with all peaks well
separated from each other. Hence, the optimum isocratic
separation of drug and degradation products was achieved
on C8 column (150 mm × 4.6 mm i.d., 5 mm, Spherisorb®)
with mobile phase composed of ammonium acetate
(0.025 mol L-1) and acetonitrile (65:35, pH* 4.5) at a flow
The intra-day precision was evaluated by analysing
(n = 6) each of the three drug concentrations (40, 80
and 160 mg mL-1) on the same day. The same drug
concentrations were analysed on three different days to
evaluate inter-day precision. The precisions were expressed
as percent relative standard deviation (% RSD) of the
calculated concentrations.
Accuracy
The photodegraded drug solutions in acidic and alkaline
media were mixed in equal proportions to prepare a mixed
degraded solution. The later was mixed further mixed with
equal volume of mobile phase to prepare unfortified sample
solution. It was also mixed separately with an equal volume
of each of the three standard drug solutions (80, 160 and
320 mg mL-1) so that the drug concentrations in the mixed
solution was fortified by 40, 80, 160 mg mL-1, respectively.
The unfortified and each of the fortified solutions was
prepared and analysed in triplicate. The accuracy was
expressed a percent recovery of the drug from the fortified
solutions vis-à-vis unfortified solution.
LOD, LOQ and selectivity
The LOD and LOQ of the drug was determined by
signal-to-noise ratio method by measuring signals from
samples with known low concentrations of drug (0.5,
1.0, 5.0 mg mL-1 for LOQ and 0.25, 0.50, 1.0 mg mL-1 for
LOD). The signals were compared with those of blank to
establish the minimum concentration at which the analyte
was reliably detected or quantified. The overall selectivity
was established through determination of purity and
resolution factor of each degradation product peak using
a PDA detector.
Robustness
Various chromatographic parameters including flow
rate, detection wavelength, and mobile phase composition
were slightly varied deliberately and the mixed degraded
solution was analysed (n = 3) using optimized as well as
each varied chromatographic conditions. The calculated
drug concentration obtained through each varied conditions
was compared with that obtained through the optimized
conditions.
Results and Discussion
Development of SIAM
Initially a standard drug solution and drug solutions
subjected to varied forced degradation conditions were

840
Citalopram Hydrobromide: Degradation Product Characterization
J. Braz. Chem. Soc.
Figure 2. LC chromatogram showing separation of CTL and its all degradation products (I-V) in a single isocratic run.
rate of 0.5 mL min-1 using 240 nm as detection wavelength.
A typical chromatogram of mixture sample is shown in
Figure 2. PDA analysis of the mixed solution established
the purity of drug and each degradation product peak
which indicated the method to be a selective SIAM. The
method was applied to analyse samples generated under
each forced degradation condition to study degradation
behaviour of the drug.
products III (RT 13.75 min, i.e. retention time) and IV
(RT 17.52 min) in addition toI and II were formed in alkaline
medium (Figure 3I). A new product V (RT 26.33 min) was
formed along with products I, II and IV in acidic medium
(Figure 3J). The peak area of product I in all media under
photolytic conditions was greater than under hydrolytic
conditions. It suggested that formation of I was accelerated
by light. Further, formation of products III-V in acidic
and alkaline media upon exposure to light in solution state
indicated that the drug is photosensitive.
Forced degradation behaviour
In total, five peaks due to degradation products (I-V)
formed under different forced degradation conditions were
noted in the chromatograms (Figure 3). Chromatogram
of standard solution of drug showed the drug peak (CTL)
at 36.67 min along with a small impurity peak (II) at
12.41 min (Figure 3A). The latter was also noted as
degradation product in all forced degradation conditions
except in oxidative medium. The drug degraded almost
completely (98.99%) to product II in 0.1 mol L-1 NaOH
at 85 °C after just 0.5 h. The extent of degradation was
significantly decreased in 0.01 mol L-1 NaOH (about
28% after 4 h) and another product I was formed which
eluted at 9.58 min (Figure 3B). The rate of degradation
was comparatively much lower in acidic medium (about
41% in 2 mol L-1 HCl at 85 °C after 48 h) wherein product
II was formed in minute amount along with product I
(Figure 3C). Insignificant degradation (< 3%) was noted
in water at 85 °C (Figure 3D) and in oxidative condition
after 48 h (Figure 3E). Exposure of the drug in dry state
to temperature of 50 °C (Figure 3F) and UV-Vis light
(Figure 3G) resulted in neither any increase in peak area
of II nor formation of any other product which suggested
that the drug in solid state was stable to heat and light.
However, rate and pattern of degradation under photolytic
conditions was significantly different from that under
hydrolytic conditions. The rate of degradation in all media
under photolytic condition was accelerated. No product
other than I and II was formed in water (Figure 3H) while
Characterization of degradation products
The degradation products I, II and IV were characterized
(Figure 1) through spectral analyses and LC-MS studies.
The major product (II), formed in all forced conditions
was isolated from alkali decomposition solution. The
comparative FT-IR spectral data of drug and II (Table 1)
revealed absence of −C≡ N stretching band at 2238 cm-1
in II indicating that nitrile group was absent. Appearance
of bands at 3393 cm-1 for N–H stretch, 1601 cm-1 for C=O
stretch (amide I band) and 1559 cm-1 for N–H bend (amide
II band) suggested an amide functionality in II. The C−F
stretching and aliphatic C−H bending vibrations were noted
in both CTL and II. Comparison of 1H NMR spectra of the
Table 1. Comparative FT-IR data of CTL and II
Vibrational band
Assignment (cm-1)
II
3393
CTL
N–H str. (-CONH-)
C–H str. (aromatic)
C–H str. (aliphatic)
C≡ N str.
absent
3090, 3062
2963, 2853
2238
3067, 3041
2956, 2871
Absent
1601
C=O (-CONH-)
N–H bend
absent
1559
absent
CH2 bend
1482
1487
C–F stretching
1384
1390

Vol. 22, No. 5, 2011
Sharma et al.
841
Figure 3. LC chromatograms of standard solution of the drug (A), drug subjected to alkaline hydrolysis (B), acid hydrolysis (C), neutral hydrolysis (D),
oxidation (E), dry heat (F) and drug subjected to photodegradation in dry state (G), in water (H), in alkali (I) and in acid (J).
drug and II (Table 2) revealed that all signals in spectrum
of II were almost similarly placed as in that of CTL except
an additional 2 proton singlet at d 7.62 which disappeared
upon D2O exchange. It supported the presence of an amide
group as also suggested by FT-IR spectra. The mass spectra
of CTL and II (Figures 4A and 4B) in +ESI mode showed
the parent molecules as M+1 peaks at m/z 324.54 and m/z
343.40 corresponding to molecular masses of 324 and 342,
respectively. The even molecular mass of II indicated even
number of nitrogen atoms in the molecule similar to the
drug. Increase in 18 units in mass of II vis-à-vis drug was
attributed to addition of water which was possible under
all hydrolytic conditions due to hydrolysis of C≡ N group.
Further, the MS2 scan of II (Figure 5B) showed a fragment
at m/z 325.81 due to loss of 17 amu which could be formed
by loss of NH3 from the parent ion (m/z 343.40). It affirmed

842
Citalopram Hydrobromide: Degradation Product Characterization
J. Braz. Chem. Soc.
Table 2. Comparative 1H NMR spectral data of CTL and II
Proton*
CTL
II
d value
3.14
Description
2H, t, J 7.6 Hz
2H, m
d value
2.76
Description
2H, t, J 7.6 Hz
2H, m
1
2
1.78-1.65
2.46-2.30
7.54-7.50
7.06-7.01
7.57
2.18-2.14
2.30-2.23
7.49-7.45
6.97-7.02
7.34
3
2H, m
2H, m
4,5
2H, m
2H, m
6,7
2H, m
2H, m
8
1H, s
1H, d, J 8.0
1H, d, J 8.0
1H, s
9
7.63
1H, s
7.84
10
7.64
1H, d, Jallylic 0.8 Hz
1H, d, Jgem 13.0 Hz
1H, d, Jgem 13.0 Hz
6H, s
7.64
11a
5.27
5.19
1H, d, Jgem 13.0 Hz
1H, d, Jgem 13.0 Hz
6H, s
11b
5.15
5.09
12
2.76
2.47
–CONH2
-
-
7.62
2H, s
* Refer to Figure 1 for proton assignments.
Figure 4. MS scans of CTL (A), product II (B), and LC-MS scans of product I (C) and product IV (D).

Vol. 22, No. 5, 2011
Sharma et al.
843
Figure 5. MS2 scans of CTL (A), product II (B) and LC-MS2 scan of product I (C).
the presence of a primary amino group in II. Based on
these results, the product II was suggested to be formed
through hydrolysis of nitrile group to amide without any
other change in the drug molecule. It was confirmed by
comparative mass fragmentation pattern of CTL and II
(Figure 6) proposed on the basis of their MS and MS2 scans
(Figures 4 and 5). The MS2 scan of CTL (Figure 4A) showed
fragment peaks at m/z 280, 262, 236, 185, 123, 109 and
83. The peaks at m/z 262 and 109 were observed similarly
as in a study by Mullera et al.14 The fragmentation pattern
of CTL was conveniently proposed (Figure 6) revealing
that fragmentation was triggered by protonation on either
nitrogen of dimethylamino or oxygen of benzofuranyl
moieties. The heaviest fragment at m/z 280 was formed by
lossofdimethylamine(45amu)fromtheparention(m/z325)
similar to that reported by Raman et al.6 The fragment of m/z
262 was formed either directly from the parent ion6 or from
m/z 280 by loss of a H2O molecule through rearrangement.
Loss of an acetylene molecule (26 amu) from tropyllium
ion of m/z 262 is proposed to produce fragment of m/z 236,
which finally loose CH≡ C-C≡ N to give fragment of m/z 185.
The base peak (m/z 109) and other peaks were proposed to
form by another fragmentation pathway involving oxygen of
benzofuran. The MS2 scan of product II (Figure 5B) showed
fragment peaks at m/z 298, 280, 254, 235, 185, 123, 109 and
83. The fragment of m/z 298, 280 and 254 were formed from
II (parent ion m/z 343) similarly as fragments of m/z 280,
262 and 236 were formed from the drug. The fragment of
m/z 185 was formed from m/z 236 by loss of CH≡ C-C≡ N
similartothedrug. Itsupportedthatthetertiaryaminomoiety
was intact. Formation of fragments of m/z 109, 123 and 83
in both the drug and II by the same fragmentation pathway
suggested that no change occurred in the p-fluorophenyl
ring of the drug molecule. Fragment of m/z 235 which could
only be formed by loss of ammonia and subsequent loss
of CO (2 amu) to produce fragment of m/z 298 confirmed
the presence of –CONH2 group in II. Hence the product
II was characterized as 1-(3-dimethylaminopropyl)-1-(4-
fluorophenyl)-1,3-dihydro-isobenzofuran-5-carboxamide
(citalopram carboxamide) which is a known impurity of
CTL (Figure 1).
LC-PDA and LC-MS studies
UV absorbance spectra of each peak obtained through
PDA analysis revealed that drug and all five degradation
productshavealmostsamewavelengthmaxima(240 5nm).
It suggested that chromophore in the drug was retained in

844
Citalopram Hydrobromide: Degradation Product Characterization
J. Braz. Chem. Soc.
R
R
R
O
H
O
H
O
N
N
H
NH(CH3)2
F
F
m/z 280; CTL
m/z 298; II
F
m/z 325 (M+1); CTL
m/z 343 (M+1); II
CTL; R = -CN
II; R = -CONH2
R
R
OH2
R
R
O
H
N
H2O
F
F
F
F
m/z 262; CTL
m/z 280; II
m/z 325 (M+1); CTL
m/z 343 (M+1); II
R
OH
HC
C
R
R
F
F
m/z 236; CTL
m/z 254; II
m/z 185
N
F
F
F
F
F
m/z 83
m/z 123
m/z 109
O
O
H2N
O
O
O
N
N
N
CO
NH3
F
F
F
m/z 326
m/z 298
m/z 343 (M+1); II
Figure 6. Proposed common mass fragmentation pattern of CTL and product II.
the degradation products. For characterization of degradation
products I, III, IV and V, the mixed degraded solution was
subjected to LC-MS studies in the +ESI mode. Products
III and V were not detected in the LC-MS chromatogram
probably due to poor ionizability or trace concentrations.
Change in ionization mode from +ESI to –ESI, numerous
variations in ionization conditions including end plate offset,
capillary voltage, collision cell RF, nebulizer pressure, dry
gas flow rate, and dry temperature in both –ESI and +ESI
modes, and even use of concentrated degraded solutions
did not help detection of these analytes, in LC-MS
chromatograms. Hence, these were not characterized in the
present study. The line spectra obtained through LC-MS
analysis of products I and IV (Figures 4C and 4D) showed
the parent ion peaks (M+1) at m/z 358.54 and 340.59,
respectively indicating their molecular masses being 358 and
340, respectively. Their structures were proposed based on
their mass values, mass fragmentation pattern, and known
chemical reactivities of different functional groups present
in CTL in different media.3,4
Product I was formed in acidic medium under photolytic
as well as hydrolytic conditions. Its even molecular mass
(358 amu) indicated even number of nitrogen atoms in
it (The Nitrogen Rule). Its structure was characterized
through mass fragmentation pattern (Figure 7) proposed
through its LC-MS scans. Increase in molecular mass
of I by 34 amu with respect to CTL could be attributed
to addition of a water molecule (18 amu) and an oxygen
atom (16 amu) in the drug molecule. Addition of water
was justified due to hydrolysis of –C≡ N group in the drug
under hydrolytic conditions to form –CONH2. Formation
of I under photolytic conditions also suggested addition
of oxygen atom to form N-oxide in the first instance.
But it is not expected because I is also formed under
hydrolytic conditions. The furan ring is reported to be
under substitution reaction to form hydroxyfuran through
hydroperoxide intermediate in aqueous medium in the
presence of air.4 Hence, the lone oxygen atom is suggested
to be substituted on the furan ring as –OH group. This
proposition is supported by fragment peaks in LC-MS

Vol. 22, No. 5, 2011
Sharma et al.
845
OH
O
OH2
O
O
O
H2N
H2N
H2N
O
O
ESI
+ ve
N
N
N
H2O
F
F
F
Product I
m/z 341
m/z 359 (M+1)
ESI + ve
H2O
OH
O
O
O
F
H2N
H
H2N
N
m/z 109
N
F
m/z 359 (M+1)
F
F
m/z 323
m/z 123
Figure 7. Proposed mass fragmentation pattern of product I.
scan of I at m/z 341 which was proposed to form from the
parent ion (m/z 358.54) by loss of a H2O molecule (18 amu)
triggered by protonation at –OH group introduced in the
furan ring. Further loss of a H2O molecule from m/z 341
through rearrangement was responsible to form another
fragment at m/z 323. Fragment peaks at m/z 109 and 123
similarly as in LC-MS scans of CTL and II indicated
that the p-fluorophenyl moiety is intact. Based on these
results and discussions, the most possible structure for
I was proposed to be 1-(3-dimethylaminopropyl)-1-(4-
fluorophenyl)-3-hydroxy-1,3-dihydroisobenzofuran-5-
carboxylic acid amide (3-hydroxy citalopram carboxamide)
(Figure 1).
and 83. The mass fragmentation pattern of IV is proposed in
Figure 8.The fragment ofm/z 323, 305 were possibly formed
by loss of two water molecule one after the other from m/z
341 triggered by protonation of oxygen of the N-Oxide at
tertiary amine. However, no such fragments can be formed
if photoxidation occurs at C≡ N group. Further, the peaks at
m/z 123, 109 and 83 were noted similarly as in MS2 scan
of the drug, I and II indicating that the p-fluorophenyl ring
was intact. Hence, product IV was proposed to be citalopram
N-oxide, a known impurity (Figure 1) which is formed by
photoxidation of tertiary amino nitrogen of CTL.
Degradation pathways
The product IV was formed in acidic medium under
photolytic conditions. Its even molecular mass (340 amu)
indicated even number of nitrogen atoms in it. Elimination
of both the nitrogen atoms from the drug molecule is not
possible under the photolytic conditions which implied
that both the nitrogen atoms of the drug were intact in IV.
Further, a difference of 16 amu with respect to mass of the
drug indicated that IV was formed by addition of an oxygen
atom to the drug molecule. Under the photolytic conditions,
this addition of an oxygen atom is expected to produce an
N-oxide product through photoxidation at nitrogen of either
the tertiary amino or nitrile group resulting in two possible
structures, i.e., citalopram N-oxide (IVa) and citalopram
cyano N-oxide (IVb) (Figure 8). IVb seemed to be less
probable because nitrogen of C≡ N is more electronegative
(sp hybridized) in comparison to that of tertiary amine (sp3
hybridized). It makes the photoxidation of C≡ N less probable
than tertiary amine. The LC-MS2 scan of IV (Figure 5C)
showed fragment peaks at m/z 323, 305, 279, 228, 123, 109
Based on the above results, the degradation pathways
of CTL under hydrolytic and photolytic conditions are
outlined in Figure 9. The drug was hydrolysed to product
II through the well-known hydrolytic reaction of nitriles
to amides in aqueous media.3 The product I could be
formed from II through substitution reaction at methylene
of dihydrobenzofuran ring under the influence of air
and light in aqueous environment via a tetrahydrofuran
hydroperoxide intermediate.4 Alternatively, the product
I could be formed directly from the drug through some
unknown route. The drug underwent photoxidation at
nitrogen of the tertiary amine to form the product IV.
Method validation
The response for the drug was strictly linear in
the concentration range of 5-500 mg mL-1. The mean
( SD) of slope, intercept and correlation coefficient are

846
Citalopram Hydrobromide: Degradation Product Characterization
J. Braz. Chem. Soc.
N
N
C
C
O
O
O
O
H
ESI
+ ve
N
m/z 323
N
F
F
m/z 341 (M+1)
Product IVa
NC
NC
NC
O
O
O
ESI
+ ve
N
N
N
OH
H2O
O
F
F
F
m/z 323
m/z 341 (M+1)
Product IVb
NC
NC
N
N
H2O
F
m/z
305
F
F
m/z 123
F
NC
m/z 109
N
N
HC
C
CN
F
F
F
m/z 228
m/z 83
m/z 279
Figure 8. Proposed mass fragmentation pattern of product IV.
O
NC
NC
H2N
O
O
O
N
N
H+/OH-
H2O
N
O
hν
Photooxidation
F
F
F
Citalopram
Product IV
Product II
unknown route
hν/air H2O
O
O
OH
O.OH
O
H2N
H2N
O
N
N
F
F
Product I
Hydroperoxide Intermediate
Figure 9. Degradation pathways of CTL.
Table 3. Intra-day and inter-day precision studies
98333 ( 1464), 84370 ( 13776) and 0.9998 ( 0.0002),
respectively. The method was sufficiently precise for the
drug over the selected concentration range. The RSD values
of intra-day and inter-day precision studies were < 1 and
< 3%, respectively (Table 3). Excellent recoveries (88-97%)
were achieved at each added concentration of the drug
(Table 4) indicating the method to be sufficiently accurate
for quantification of CTL. The LOD and LOQ for the drug
Actual conc.
Measured conc. (mg mL-1); %RSD
(mg mL-1)
Intra-day (n = 6)
40; 0.58
Inter-day (n = 3)
41; 2.59
40
80
80; 0.24
80; 1.23
160
159; 0.32
160; 1.12

Vol. 22, No. 5, 2011
Sharma et al.
847
Table 4. Recovery studies
of Pharmaceutical Education and Research, SAS Nagar,
India of for facilitating photodegradation and LC-MS
studies and to Ind-Swift Laboratories, Panchkula (India)
for providing citalopram hydrobromide as a generous gift
to conduct the study.
Fortified conc.
(mg mL-1)
Calculated conc.
Recovery
(%)
(mg mL-1); %RSD (n = 6)
40
35; 2.34
73; 0.77
155; 0.95
88
91
97
80
160
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resolution of the drug peak from the nearest resolving peak
was always > 5 indicating the method specific to the drug.
The method was also selective to degradation products as
all the peaks were pure, which was proved through PDA
purity studies. Good separations and recoveries were
always achieved during robustness studies, indicating that
minor deviations in chromatographic conditions do not
affect quantification of CTL.
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Acknowledgement
The authors are thankful to Prof. Saranjit Singh, Head of
Department of PharmaceuticalAnalysis, National Institute

848
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Submitted: July 1, 2010
Published online: January 27, 2011