Liquid chromatography/tandem mass spectrometry study of forced degradation of azilsartan medoxomil potassium

Article abstract of DOI:10.1002/rcm.7235

Rationale Azilsartan medoxomil potassium (AZM) is a new antihypertensive drug introduced in the year 2011. The presence of degradation products not only affects the quality, but also the safety aspects of the drug. Thus, it is essential to develop an efficient analytical method which could be useful to selectively separate and identify the degradation products of azilsartan medoxomil potassium. Methods AZM was subjected to forced degradation under hydrolytic (acid, base and neutral), oxidative, photolytic and thermal stress conditions. Separation of the drug and degradation products was achieved by a liquid chromatography (LC) method using an Acquity UPLC C18 CSH column with mobile phase consisting of 0.02% trifluoroacetic acid and acetonitrile using a gradient method. Identification and characterization of the degradation products was carried out using LC/electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOFMS). Results A total of five degradation products (DP 1 to DP 5) were formed under various stress conditions and their structures were proposed with the help of tandem mass spectrometry (MS/MS) experiments and accurate mass data. A common degradation product (DP 4) was observed under all the degradation conditions. DP 1, DP 2 and DP 5 were observed under acid hydrolytic conditions whereas DP 3 was observed under alkaline conditions. Conclusions AZM was found to degrade under hydrolytic, oxidative and photolytic stress conditions. The structures of all the degradation products were proposed. The degradation pathway for the formation of degradation products was also hypothesized. A selective method was developed to quantify the drug in the presence of degradation products which is useful to monitor the quality of AZM.

Full text of DOI:10.1002/rcm.7235

Research Article
Received: 9 October 2014
Revised: 21 May 2015
Accepted: 21 May 2015
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2015, 29, 1437–1447
(wileyonlinelibrary.com) DOI: 10.1002/rcm.7235
Liquid chromatography/tandem mass spectrometry study of
forced degradation of azilsartan medoxomil potassium
2**
Debasish Swain1, Prinesh N. Patel1, Ilayaraja Palaniappan2, Gayatri Sahu and
Gananadhamu Samanthula1
*
1National Institute of Pharmaceutical Education and Research, Hyderabad-500037, Telangana, India
2United States Pharmacopeia (USP) India Pvt. Ltd., Hyderabad-500078, Telangana, India
RATIONALE: Azilsartan medoxomil potassium (AZM) is a new antihypertensive drug introduced in the year 2011. The
presence of degradation products not only affects the quality, but also the safety aspects of the drug. Thus, it is essential
to develop an efficient analytical method which could be useful to selectively separate and identify the degradation
products of azilsartan medoxomil potassium.
METHODS: AZM was subjected to forced degradation under hydrolytic (acid, base and neutral), oxidative, photolytic
and thermal stress conditions. Separation of the drug and degradation products was achieved by a liquid
chromatography (LC) method using an Acquity UPLC® C18 CSH column with mobile phase consisting of 0.02%
trifluoroacetic acid and acetonitrile using a gradient method. Identification and characterization of the degradation
products was carried out using LC/electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOFMS).
RESULTS: A total of five degradation products (DP 1 to DP 5) were formed under various stress conditions and their
structures were proposed with the help of tandem mass spectrometry (MS/MS) experiments and accurate mass data.
A common degradation product (DP 4) was observed under all the degradation conditions. DP 1, DP 2 and DP 5 were
observed under acid hydrolytic conditions whereas DP 3 was observed under alkaline conditions.
CONCLUSIONS: AZM was found to degrade under hydrolytic, oxidative and photolytic stress conditions. The
structures of all the degradation products were proposed. The degradation pathway for the formation of degradation
products was also hypothesized. A selective method was developed to quantify the drug in the presence of degradation
products which is useful to monitor the quality of AZM. Copyright © 2015 John Wiley & Sons, Ltd.
High blood pressure, also known as hypertension, is a
prevalent and widespread disorder. As per the WHO health
statistics 2012 report, one in every three adults has high blood
pressure.[1] Hypertension has been reported to be the leading
cause of heart disease and stroke thereby leading to
morbidity and mortality.[2]
treatment of hypertension, either individually or in
combination with other drugs like chlorthalidone and
amlodipine.[4–6] This drug is more effective in lowering the
24-hour blood pressure than the recommended doses of the
previously marketed ARBs.[7–9] It also has a better therapeutic
profile in lowering blood pressure when compared to other
drugs like ramipril.[10] This drug was approved by the Food
and Drug Administration (FDA) in February 2011.[11]
Azilsartan medoxomil potassium (AZM) is a prodrug,
chemically
known
as
1-[{2’-(2,5-dihydro-5-oxo-1,2,4-
oxodiazol-3-yl) [1,1’-biphenyl]-4-yl] methyl]-2-ethoxy-1H-
benzimidazole-7-carboxylic acid (5-methyl-2-oxo-1,3-dioxol-
4-yl) methyl ester potassium salt. The administered prodrug
is converted into azilsartan, the therapeutically active moiety
inside the gut and plasma through ester hydrolysis during the
absorption phase of oral administration.[3] AZM is an
angiotensin receptor blocker (ARB) which is used in the
Forced degradation study of drugs facilitates the
identification of various degradation products of the drug
and also provides information about the behaviour of the
drug under the specified stress conditions. The presence of
degradation products (as impurities) in dosed drugs has
resulted in alarming side effects and also formulation defects
in the past. The formation of epianhydrotetracycline (a
degradation product of tetracycline) resulted in Fanconi’s
syndrome that led to renal failure.[12] Another example was
the formation of polymeric aminopenicillins which led to
allergic reactions attributed to the degradation products and
not the active ingredient.[13] In the recent past the presence of
oversulfated chondroitin sulfate (as an impurity) in heparin
samples caused allergenic reactions and led to fatal
consequences.[14] Hence preliminary information regarding the
degradation products is helpful in evaluating any such effects.
ICH guidelines suggest conducting forced decomposition
studies in order to establish the stability and identify the
* Correspondence to:
S Gananadhamu, Department of
Pharmaceutical Analysis, National Institute of Pharmaceutical
Education and Research [NIPER], Hyderabad-500037,
Telangana, India.
E-mail: gana@niperhyd.ac.in
** Correspondence to: G. Sahu, Analytical Research and
Development, United States Pharmacopeia (USP) India
Pvt. Ltd. Hyderabad-500078, Telangana, India.
E-mail: gayatri_sahu@yahoo.com
Rapid Commun. Mass Spectrom. 2015, 29, 1437–1447
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D. Swain et al.
degradation products of a drug in development.[15] The stress
conditions, in general, include the effect of hydrolysis (acid,
base and neutral), oxidation, temperature and photolysis.
Analytical methods have been reported in the literature for
the determination of AZM alone or in combination with
chlorthalidone. Vekariya et al. developed a reversed-phase
high-performance liquid chromatography (RP-HPLC)
method for quantification of AZM in human plasma.[16] Gorla
et al. developed an ultraviolet (UV) spectrophotometric
method to determine the presence of AZM in bulk drug and
formulations.[17] Ebeid et al. carried out spectrophotometric
and spectrofluorimetric studies to estimate AZM and
chlorthalidone in combination pharmaceutical dosage
(Osworld OPSH-G-16 GMP series, Osworld Scientific
Equipments Pvt. Ltd. India) was set at 40 5°C and relative
humidity (RH) of 75
5.0%. This photostability chamber
has ultraviolet lamps and fluorescent lamps as recommended
by the ICH Q1B guideline.[21]
Forced degradation studies
Forced degradation studies of AZM were carried out under
hydrolytic (acid, base and neutral), oxidative, photolytic and
thermal stress conditions as per ICH guidelines. AZM at a
concentration of 1 mg mL–1 was used to study all the solution
state forced degradation experiments. Acidic hydrolysis was
carried out in 0.1 N HCl for 2 h at 60°C. The basic hydrolysis
was performed in 1 N NaOH at 60°C for 24 h and neutral
hydrolysis was carried out by dilution of the drug sample
with water (previously the drug was solubilised with diluent)
and exposing it to a temperature of 60°C for 1 h. Oxidative
studies were conducted in 6% H2O2 at room temperature
for 3 h. The AZM powder was spread as a layer of about
1 mm thickness in a Petri dish and kept in dry air oven at
105°C for 48 h for thermal degradation studies. For photolytic
degradation studies, the solid samples were exposed to UV
light of intensity 200 Whrm–2 and fluorescent light of intensity
1.2 million lux hours in separate Petri dishes. Control samples
for photolytic degradation were also prepared by wrapping
the Petri dishes with aluminium foil prior to holding them
under similar conditions to those of the stressed samples.
The solution state stability under UV and fluorescent light
was studied by dissolving the drug in the diluent and then
placing it in the photolytic chamber.
forms.[18] Naazneen and Sridevi proposed
a stability-
indicating HPLC assay method for fixed dose combination
of AZM and chlorthalidone.[19] Recently, Ebeid et al. have
developed a stability-indicating method for the combination
of azilsartan medoxomil and chlorthalidone with its
subsequent application to degradation kinetics.[20] However,
no method has been reported for characterization of the
degradation products formed under various stress conditions.
Identification and characterization of degradation products is
very important in the field of drug development. This
prompted the authors to study the forced degradation and
identify the degradation products of AZM using LC/MS/
MS and accurate mass measurements. The degradation
pathway followed by AZM leading to the formation of
various degradation products was also proposed.
EXPERIMENTAL
Sample preparation
Chemicals and reagents
The degradation samples tested with acid and base
hydrolysis were neutralised and diluted to a concentration
of 100 μg/mL of AZM. The samples of oxidative stress
hydrolysis, thermal and photolysis were also prepared in
the same concentration of AZM. All the samples were filtered
using 0.22 μ membrane filter before being analysed and also
stored under refrigerated conditions of 10°C.
Azilsartan medoxomil potassium (AZM) was obtained as a
gift sample from Mylan Labs. Pvt. Ltd., Hyderabad. HPLC
grade acetonitrile (ACN), trifluoroacetic acid (TFA), sodium
hydroxide, hydrochloric acid (35%), and hydrogen peroxide
(30%) were purchased from Merck (Darmstadt, Germany).
High-purity water was obtained from a Milli-Q plus system
(Millipore, Milford, MA, USA). ACN was used as diluent.
All the final sample dilutions were made with the diluent.
All the reagent solutions (0.1 N HCl, 1 N NaOH and 6%
H2O2) used in the forced degradation study were prepared
in a mixture solvent system of water and ACN (50:50 v/v).
RESULTS AND DISCUSSION
Optimisation of the chromatographic conditions
Chromatographic conditions were optimised to achieve a
good separation of the drug and its degradation products
based on peak shape and the resolution. The Aquity UPLC®
CSH C18 column (100 mm × 2.1 mm × 1.7 μ) showed the best
results in terms of retention, peak shape and resolution when
compared to other columns. Various mobile phase
compositions were tried using buffers with pH 3.0, 4.0, 5.5,
0.02% TFA and 0.1% formic acid (FA) with ACN as organic
modifier to obtain a good separation. ACN produced better
retention, peak shapes and low column pressures due to its
inherent property of possessing less viscosity and also higher
organic strength. Best separation was achieved with the
mobile phase of 0.02% TFA (A) and ACN (B) in gradient
method as: Time (min)/% B – 0/40, 0.3/40, 4.0/70, 5.0/70,
5.5/40, 8.0/40. The flow rate was 0.3 mL min–1. An injection
volume of 1.0 μL and column temperature of 25°C were
Instrumentation
A Waters UPLC H-Class chromatographic system with
quaternary solvent manager and photo-diode array (PDA)
detector was used for the initial method development.
For MS analysis
a quadrupole time-of-flight (QTOF)
mass spectrometer (QTOF LC/MS 6540 series, Agilent
Technologies, USA) equipped with an electrospray ionization
(ESI) source was used. The data acquisitions were under the
control of Mass Hunter software. The operating conditions
for AZM in the MS scan in positive mode were optimized
as: fragmentor voltage set at 144 V, capillary voltage set at
3500 V, skimmer at 65 V. Nitrogen gas was used as the drying
(325°C, 10 L/min) and nebulizing gas (40 psi). The MS/MS
studies were carried out using collision-induced dissociation
with nitrogen as the collision gas. A photostability chamber
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LC/MS/MS study of forced degradation of AZM
optimised for the study. The wavelength for the detection of
AZM was chosen to be 254 nm where the drug and other
degradation products showed optimum detection sensitivity.
The degradation products were well separated from the main
peak as well as from each other which indicates the method is
stability indicating. For LC/MS studies, the same method
was successfully transferred and MS conditions were
optimised to obtain good signal and sensitivity.
Photolytic and thermal degradation
AZM was degraded under UV light exposure in the solution
state to yield the degradation product DP 4 (Scheme 1). It was
found to be stable to UV light in the solid state. It was also
stable to fluorescent light under both solution and solid state
stress conditions. There was no degradation observed under
thermal stress conditions.
LC/MS/MS studies of AZM and its degradation products
Degradation of AZM
The developed method was transferred to the LC/MS
method for the characterisation of degradation products.
The structures of the various degradation products formed
were elucidated using MS/MS and also correlating with the
accurate mass data. The fragmentation assignments were
made on the basis of the accurate mass of product ion data
with mass accuracy better than 10 ppm. The elemental
compositions of the proposed structures of degradation
products are given in Table 1.
A total of five degradation products (DP 1 to DP 5) were
identified and characterised by liquid chromatography/tandem
mass spectrometry (LC/MS/MS) analysis. The proposed
structures are shown in Fig. 1. The chromatograms depicting
the degradation of the drug under various stress conditions are
as shown in Fig. 2.
Hydrolysis
The drug degraded under all the hydrolytic conditions
(acidic, basic and neutral). Under acidic conditions four
degradation products were formed (DP 1, DP 2, DP 4 and
DP 5), as shown in Scheme 1. AZM was extensively degraded
to DP 4 (azilsartan free acid) even under mild alkaline
conditions (0.01 N NaOH at room temperature). AZM raw
material normally contains a trace amount of azilsartan free
acid as an impurity. To study the degradation of azilsartan
free acid, the AZM was further subjected to 1 N NaOH at
60°C where initially AZM completely degraded to DP 4
(azilsartan free acid) and later DP 4 was degraded to yield
DP 3, as shown in Scheme 2. In neutral hydrolysis only DP
4 was formed. Since water can act as a weak acid or base,
the degradation pathway for formation of DP 4 in neutral
hydrolysis may proceed as shown in Scheme 1 or 2.
MS/MS of AZM
The ESI-MS (+ ion mode) spectrum of AZM showed a highly
abundant protonated molecule [M+H]+ at m/z 569 ion. The
CID spectrum (Fig. 3) of the protonated molecule [M+H]+
obtained from AZM afforded the following series of product
ions. Consequently, the product ion (Scheme 3) at m/z 541
was obtained from the loss of C2H4 from the precursor and
the second-generation product ion at m/z 523 was formed
by loss of a molecule of H2O from the product ion at m/z
541. The third-generation product ion at m/z 497 was obtained
by loss of CO2 from the ion at m/z 541and the fourth-
generation product ion at m/z 479 was obtained by loss of
H2O from the product ion at m/z 497. The second-generation
product ion at m/z 457 was created by loss of C5H4O3 from
the precursor ion at m/z 569. This was followed by the
product ion at m/z 439, which was formed by loss of H2O
from the product ion at m/z 457. In addition the product ion
at m/z 429 was created by loss of a molecule of C2H4 from
the ion at m/z 457. Furthermore, the product ion at m/z
411was formed by loss of H2O from the ion at m/z 429 and
the product ion at m/z 393 by loss of a molecule of C2H6O
Oxidative stress study
The AZM degraded under oxidative stress conditions to yield
only one degradation product, identified as DP 4, which was
observed under acidic, basic and neutral hydrolytic conditions
(Scheme 1).
Figure 1. Proposed chemical structures of degradation products.
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D. Swain et al.
Figure 2. Overlaid chromatograms of (A) standard (unstressed) of AZM, (B) acid degradation of
AZM, (C) base degradation of AZM, (D) neutral degradation of AZM, (E) oxidative degradation of
AZM, (F) photolysis – UV solution, and (G) photolysis – UV solid.
from m/z 439. Moreover, the product ion at m/z 367was
formed by loss of CO2 from the ion at m/z 411. The product
ion at m/z 279 was produced by loss of the C8H4N2O2 portion
(160 Da) from the product ion at m/z 439.The product ion at
m/z 251 was created by loss of the C10H10N2O3 portion
(206 Da) from the product ion at m/z 457. The product ion at
m/z 233 was formed by loss of the C8H8N2O3 portion
(178 Da) from the ion at m/z 411). The product ion at m/z
207was formed by loss of the C8H4N2O2 portion (160 Da)
from the product ion at m/z 367, the product ion at m/z 180
was obtained by loss of C2HNO2 from the product ion at
m/z 251 and, finally, the product ion at m/z 161 was obtained
by loss of C17H14N2O2 from the product ion at m/z 439 and
the product ion at m/z 113 was obtained by loss of
C25H20N4O5 from the precursor ion at m/z 569.
cleavage of the enol ether bond leading to formation of a
secondary alcohol which tautomerises to ketone and also
hydrolysis of the 1,2,4-oxadiazol-5(2H)-one moiety of AZM to
yield a diazirine group (Scheme 1). The CID spectrum of this
protonated molecule [M+H]+ obtained from DP 1 resulted in
the series of product ions as shown in Fig. 3. The product ion
at m/z 453 was obtained by loss of CO2 from the precursor ion
at m/z 497 and the second-generation product ion at m/z 385
was formed by loss of the C4H4O portion (68 Da) from the
product ion at m/z 453. The third-generation product ion at
m/z 367 was obtained by loss of a molecule of H2O from m/z
385 whereas the fourth-generation product ion at m/z 339 was
obtained by loss of CO from the product ion at m/z 367. The
fourth-generation product ion at m/z 349 was formed by loss
of a molecule of 18 Da from the product ion at m/z 367.
Furthermore, the product ions at m/z 207 and 195 were obtained
with subsequent losses of the C8H4N2O2 and C6H2N2O3
portions from the product ions at m/z 367 and 385, respectively.
The MS/MS experiment and accurate mass of DP 1 indicated
the structure to be (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl-
3-((2’-(1H-diazirin-3-yl)-[1,1’-biphenyl]-4-yl)methyl)-2-oxo-2,3-
dihydro-1H-benzo[d]imidazole-4-carboxylate (Scheme 4).
MS/MS of degradation products
The LC/ESI-MS/MS (+ ion mode) spectrum of DP 1 showed
the protonated molecule [M+H]+ at m/z 497. DP 1 (Fig. 2(B),
retention time (Rt) 2.0 min) is formed only under acidic
hydrolytic conditions. DP 1 is formed by the hydrolytic
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LC/MS/MS study of forced degradation of AZM
Scheme 1. Proposed degradation pathway followed by AZM under acidic hydrolysis, oxidation and photolysis.
Scheme 2. Proposed degradation pathway followed by AZM under alkaline hydrolysis.
Table 1. High-resolution mass spectrometry (HRMS) data of product ions of protonated AZM and its degradation products
Molecular
formula
Theoretical
Experimental
Mass error
(ppm)
Mass shift
from AZM
Stress condition
m/z
m/z
+
AZM
DP 1
DP 2
DP 3
DP 4
–
C30 H25N4O8
569.1667
497.1456
429.1193
416.1605
457.1506
569.1663
497.1458
429.1181
416.1586
457.1503
0.70
–0.40
2.80
–
+
Acid
C27H21N4O6+
C23H17N4O5+
C24H22N3O4+
C25H21N4O5
72.0211
140.0474
153.0062
112.0161
Acid
Base
4.57
Acid, Base,
–0.65
Oxidative, Photolytic
Acid
+
DP 5
C28H21N4O8
541.1354
541.1344
–1.84
28.0313
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D. Swain et al.
Figure 3. MS/MS spectra of AZM, DP 1 and DP 2.
DP 2 ([M+H]+, m/z 429). The LC/ESI-MS/MS spectrum of
DP 2 (Fig. 2(B), Rt 2.3 min) formed under acidic degradation
conditions is shown in Fig. 3. DP 2 is formed as a result of
hydrolytic reactions under acidic conditions. This process
involves ester hydrolysis to liberate the medoxomil
component and gives the free acid form. Also the enol ether
hydrolysis takes place as in DP 1 (Scheme 1). The CID
spectrum of this protonated molecule [M+H]+ obtained from
DP 2 produced the following series of product ions. The
product ion at m/z 385 was formed by loss of a molecule of
CO2 from the precursor ion at m/z 429. The second-generation
product ion at m/z 367 was formed by loss of a molecule of
H2O from the product ion at m/z 385. This was followed by
the third-generation product ion at m/z 349 resulting due to
loss of 18 Da from the product ion at m/z 367. The product ion
at m/z 411 was obtained by loss of H2O from the precursor ion
at m/z 429. The second-generation product ion at m/z 251 was
formed by loss of the C8H4N2O2 portion (160 Da) from the
product ion at m/z 411. Further, the third-generation product
ion at m/z 180 resulted from the product ion at m/z 251 by loss
of the C2HNO2 portion (71 Da). The product ion at m/z 207
was formed by loss of C8H4N2O2 (160 Da) from the product
ion at m/z 367. Finally, the ion at m/z 153 was obtained by loss
of HCN from the product ion at m/z 180. The structure of DP
2 was confirmed by accurate mass measurements and
MS/MS fragmentation data to be 2-oxo-3-((2’-(5-oxo-4,5-
dihydro-1,2,4-oxadiazol-3-yl)-[1,1’-biphenyl]-4-yl)methyl)-2,3-
dihydro-1H-benzo[d] imidazole-4-carboxylic acid (Scheme 5).
DP 3 ([M+H+, m/z 416): The LC/.ESI-MS/MS spectrum of
DP 3 (Fig. 2(C), Rt 3.7 min) is shown in Fig. 4. DP 3 is formed
as a result of the hydrolytic cleavage of the ester group (as in
the case of the formation of DP 1 and DP 2 as shown in
Scheme 1) along with the alkali-catalysed decarboxylation
through successive formation of intermediary oximocar-
boxamic acid (i) and oximo amine (ii), as shown in Scheme 2.
The CID spectrum of this protonated molecule [M+H]+
obtained from DP 3 afforded the following series of product
ions. Consequently, the product ion at m/z 381 was obtained
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LC/MS/MS study of forced degradation of AZM
Scheme 3. Proposed fragmentation pathway for protonated AZM.
Scheme 4. Proposed fragmentation pathway for degradation product DP 1.
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D. Swain et al.
Scheme 5. Proposed fragmentation pathway for degradation product DP 2.
Figure 4. MS/MS spectra of DP 3, DP 4 and DP 5.
by a concurrent loss of H2O and NH3 from the precursor ion
at m/z 416. The second-generation product ion at m/z 353
resulted from loss of a molecule of C2H4 from the product
ion at m/z 381. The product ion at m/z 221 was formed by loss
of the C13H9NO portion (195 Da) from the precursor ion at
m/z 416. Also the product ion at m/z 210 was formed due to
loss of the C10H10N2O3 portion (206 Da) from the precursor
ion at m/z 416.The third-generation product ion resulted from
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LC/MS/MS study of forced degradation of AZM
loss of a molecule of NH3 from the product ion at m/z 210.
Moreover, the product ion at m/z 167 resulted as a loss of
the CONH2 portion from the product ion at m/z 210. The
MS/MS spectra and the accurate mass data confirmed DP 3
to be 1-((2’-carbamoyl-[1,1’-biphenyl]-4-yl)methyl)-2-ethoxy-
1H-benzo[d]imidazole-7-carboxylic acid (Scheme 6).
The LC/ESI-MS/MS spectrum of [M+H]+ at m/z 457
obtained from DP 4 formed under hydrolytic, photolytic
and oxidative conditions is shown in Fig. 4. Esters are
susceptible to alkaline hydrolysis. AZM, being an ester was
extensively hydrolysed to DP 4 under alkaline conditions.
DP 4 was also formed as a major degradant under the
Scheme 6. Proposed fragmentation pathway for degradation product DP 3.
Scheme 7. Proposed fragmentation pathway for degradation product DP 4.
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D. Swain et al.
Scheme 8. Proposed fragmentation pathway for degradation product DP 5.
oxidative and photolytic conditions, as shown in Scheme 1. It
was formed as a minor degradation product under acidic and
neutral hydrolysis. The product ion at m/z 385 is formed with
loss of CO2 and C2H4 from the precursor ion at m/z 457. The
second-generation product ion at m/z 367 was obtained by loss
of H2O from the product ion at m/z 385. The third-generation
product ions, at m/z 349, were formed through loss of a portion
of 18 Da from the product ion at m/z 367. The ion at m/z 207 was
obtained by loss of the C8H4N2O2 portion from the product ion
at m/z 367. The product ion at m/z 439 was obtained by a loss of
H2O from the precursor ion at m/z 457. The second-generation
product ion at m/z 411 resulted as a loss of C2H4 from the
product ion at m/z 439. Consequently, the product ion at m/z
251 formed with loss of the C8H4N2O2 portion from the product
ion at m/z 411. The second-generation product ion at m/z 233
was formed due to loss of the C8H6N2O3 portion (178 Da) from
the product ion at m/z 411. Furthermore, the product ion at m/z
180 was formed by loss of C2HNO2 (71 Da) from the product
ion at m/z 251. A loss of the C9H10 portion (118 Da) from the
product ion at m/z 279 resulted in the third-generation product
ion at m/z 161. The product ion at m/z 439 fragmented to produce
the product ion at m/z 279 with loss of C8H4N2O2. With support
from the accurate mass data and MS/MS studies, DP 4 was
identified as 2-ethoxy-1-((2’-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
3-yl)-[1,1’-biphenyl]-4-yl)methyl)-1H-benzo[d]imidazole-7-
carboxylic acid (Scheme 7).
of protonated AZM and that of DP 5, the data pertaining to the
product ions formed at m/z 429, 411, 367, 349, 279, 251, 207, 180,
153 and 113 has already been discussed above. The MS/MS
data and also the accurate mass data confirmed DP 5 to be
(5-methyl-2-oxo-1,3-dioxol-4-yl)methyl 2-oxo-3-((2’-(5-oxo-4,5-
dihydro-1,2,4-oxadiazol-3-yl)-[1,1’-biphenyl]-4-yl)methyl)-2,3-
dihydro-1H-benzo[d]imidazole-4-carboxylate (Scheme 8).
CONCLUSIONS
The behavior of AZM under forced degradation was studied
under various stress conditions mentioned as per ICH guidelines.
AZM was found to be labile under hydrolytic and oxidative
conditions. A total of five degradation products (DP1 to DP5)
were identified and characterized by liquid chromatography/
tandem mass spectrometric (LC/MS/MS) analysis. The
proposed method can be effectively used to carry out the
estimation and detection of AZM and its degradation products.
Acknowledgements
The authors wish to thank Dr Ahmed Kamal, Project Director,
National Institute of Pharmaceutical Education & Research
[NIPER-H], Hyderabad, for his constant encouragement. The
authors are thankful to Prof. P K Dubey, Department of
Medicinal Chemistry [NIPER-H], for his valuable suggestions.
One of the authors, Mr. Debasish Swain, is thankful to USP
India Pvt. Ltd. for providing the facilities to carry out the
research work and also to the Department of Pharmaceuticals,
Ministry of Chemicals and Fertilizers, New Delhi, India, for
providing a research fellowship.
DP 5 ([M+H]+ , m/z 541). The LC/ESI-MS/MS spectrum of
[M+H]+ at m/z 541 corresponding to DP 5 (Fig. 2B, Rt 4.0 min)
is shown in Fig. 4. Enol ethers are easily hydrolysed by aqueous
acids. The enol ether bond of AZM undergoes acid hydrolysis
to yield the major degradation product DP5 (Scheme 1). Due
to the strong similarities that exist between the MS/MS spectra
wileyonlinelibrary.com/journal/rcm
Copyright © 2015 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2015, 29, 1437–1447

LC/MS/MS study of forced degradation of AZM
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