Isolation and structural characterization of degradation products of aceclofenac by HPLC, HRMS and 2D NMR

Article abstract of DOI:10.14233/ajchem.2019.21798

The stability of aceclofenac under stress conditions was assessed to identify the degradation products. So, it was subjected to stress conditions like acid, base and oxidation, according to ICH guideline Q1A (R2). One degradation product formed when the drug was subjected to acid stress. Three degradation products were formed during the basic stress condition. The drug substance was found to be stable to oxidative stress. The degradants formed during the stress were separated on a C-18 column using gradient preparative HPLC elution. The only product (DP-2) formed during the acid stress and this one is same as of one of the three degradation products (DP-1, DP-2, DP-3) were formed during base stress. 1D and 2D NMR spectra and mass spectral analysis supported the proposed structures for the products. The products DP-2 and DP-3 have been reported earlier but this is the first report of product DP-1 as a degradation product of aceclofenac.

Full text of DOI:10.14233/ajchem.2019.21798

Asian Journal of Chemistry; Vol. 31, No. 4 (2019), 851-854
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2019.21798
Isolation and Structural Characterization of Degradation
Products of Aceclofenac by HPLC, HRMS and 2D NMR
1,2
1,2
1
1
SANTHOSH GUDURU , V.V.S.R.N. ANJI KARUN MUTHA , B. VIJAYABHASKAR , MURALIDHARAN KALIYAPERUMAL ,
2
2
1,*
RAGHU BABU KORUPOLU , KISHORE BABU BONIGE and CHIDANANDA SWAMY RUMALLA
1Department of Medicinal Chemistry, GVK Biosciences Pvt. Ltd, IDA Mallapur, Hyderabad-500076, India
2Department of Engineering Chemistry, Andhra University, Visakhapatnam-530003, India
*Corresponding author: E-mail: chidanand_swamy@yahoo.co.in
Received: 5 November 2018;
Accepted: 19 December 2018;
Published online: 27 February 2019;
AJC-19297
The stability of aceclofenac under stress conditions was assessed to identify the degradation products. So, it was subjected to stress
conditions like acid, base and oxidation, according to ICH guideline Q1A (R2). One degradation product formed when the drug was
subjected to acid stress. Three degradation products were formed during the basic stress condition. The drug substance was found to be
stable to oxidative stress. The degradants formed during the stress were separated on a C-18 column using gradient preparative HPLC
elution. The only product (DP-2) formed during the acid stress and this one is same as of one of the three degradation products (DP-1,
DP-2, DP-3) were formed during base stress. 1D and 2D NMR spectra and mass spectral analysis supported the proposed structures for
the products. The products DP-2 and DP-3 have been reported earlier but this is the first report of product DP-1 as a degradation product
of aceclofenac.
Keywords: Aceclofenac, Degradation products.
To the best of our knowledge, there is only one report on
the stress stability study of aceclofenac in tablet dosage form
by HPLC. The study showed that the degradant formed is diclo-
fenac by comparison of its standard chromatogram in both
acidic and basic stress conditions [9]. Other reports [10-15] were
found on simultaneous estimation of aceclofenac along with
other NSAIDs by HPLC .All the above cited literature revealed
that the drug is unstable under various stress conditions tested
and few other degradants were formed. However, they were
not characterized. This study was performed to know about the
degradation pattern and to characterize the degradants by
isolating each of them using various spectroscopic techniques.
INTRODUCTION
Aceclofenac belongs to a class of non-steroidal antiinflam-
matory drugs (NSAIDs). It is prescribed for the relief of pain
and inflammation in muscle-related inflammatory conditions,
rheumatoid arthritis, osteoarthritis and ankylosing spondylitis
[1,2]. Aceclofenac acts by inhibiting cyclooxygenase during
prostaglandin synthesis and thereby exhibiting its antiinflam-
matory effect [3,4].
Storage of drugs may cause physico-chemical degradation.
Stability testing of an active pharmaceutical ingredient is there-
fore performed under various conditions and is required to
document these results in drug development process. Identi-
fication, characterization of degradation products is required
according to guidelines issued by various regulatory authorities
and International Conference on Harmonization (ICH) and
other regulatory authorities [5-7]. Even small amounts of impu-
rities may affect the quality of pharmaceutical products and could
produce unwanted evil effects on the health of patients taking
such drugs. So methods indicating the stability are important to
assess the impurities formed during the storage [8].
EXPERIMENTAL
Aceclofenac was obtained as a kind gift sample from an
industry (Hyderabad, India). HPLC grade solvents and buffers
were used. Water used was Milli-Q grade.
All the analysis was performed on anAgilent 1290 Infinity
LC System (Agilent, Santa Clara, CA,USA) with ACQUITY
BEH C18 ( 2.1 × 50 mm, 1.7 µm particle size) column; column
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852 Guduru et al.
Asian J. Chem.
mAU
400
300
200
100
0
temperature at 45 ºC; Binary pump with mobile phaseA: 0.1 %
formic acid in water; mobile phase B: 100 % acetonitrile; T/%
of B: 0.0/2.0, 0.5/2.0, 3/30, 8/60, 11/98, 13/98, 14/2, 15/2;
Diluent: mobile phase; flow rate 0.6 mL/min; Detection: 215
nm.
Preparative HPLC equipped withWaters Quaternary gradient
module 2545, Waters Photo DiodeArray detector module 2998;
WatersAuto sampler 2707;Waters Fraction collector III (Waters
Corp., Milford, MA, USA); column: Kromasil C-18 (150 ×
25 mm) 10µ, mobile phase A: 0.1 % formic acid (aq.); mobile
phase B:acetonitrile: T % of B: 0.0/30, 8.0/70, 11/70, 11.1/98,
13/98, 13.1/30, 15/30; flow rate : 20 mL/min.
Samples were analyzed with Waters micro mass Q-TOF
equipped with an electrospray ionization (ESI) source. Samples
were operated in positive and negative mode to enable to dete-
ction of degradants. Leucine encephalin (m/z: 555.62268 Da)
was used as reference lock mass calibrant to achieve the typical
mass accuracies with Mass Lynx software. The optimal conditions
of analysis were as follows: capillary voltage of 3.5 kV, sample
cone at 25 V and the scan range from 100 to 2200 Daltons.
The source temperature was 120 ºC and the desolvation temper-
ature set at 350 ºC. Nitrogen was used as nebulization gas at
flow rate of 750 L/ h.
NMR spectra of the degradation impurities were recorded
in DMSO-d6 (Cambride Isotope Limited) on 400 MHz Bruker
Avance-III HD NMR spectrometer equipped with broad band
observe (BBO) probe. The 1H and 13C chemical shifts are reported
on δscale in ppm, relative to tetramethyl silane (TMS) as internal
standard. The spectra were set to δ 0.00 ppm in 1H NMR (TMS)
and δ 39.50 ppm in 13C NMR (DMSO-d6).
0.5 N NaOH
2
2
4
4
6
6
8
8
10
10
12
14 min
mAU
400
300
200
100
0
0.5 N HCl
12
14
min
mAU
600
400
200
0
5 % H2O2
2
4
6
8
10
12
14 min
Fig. 1. Acid, base and hydrogen peroxide degradation chromatograms of
aceclophenac drug substance
products were labeled as DP-1, DP-2 and DP-3. The extensive
analysis of HRMS and 1D, 2D NMR data confirmed the struc-
tures of the degradation products. Literature survey revealed
that DP-1, DP-2 and DP-3 were already reported. However,
this is the first report of DP-1 as a degradation product of aceclo-
fenac. Structures of aceclofenac and its degradation products
in various stress conditions are shown in Fig. 2.
22
O
21
20
OH 23
18 O
O 19
19
O
17
16
Stress methods: The drug was subjected to stress as per
ICH guideline Q1A (R2) [5] to identify the potential degradants
those could be generated during the storage. The details of the
stress methods are shown in Table-1.
11
18 O
10
9
13
Cl
17
12
OH 20
16
15
H
N
7
5
12
NH 15
4
3
7
6
1
11
8
TABLE-1
13 Cl
Cl 14
4
5
STRESS CONDITIONS FOR OPTIMUM DEGRADATION
8
10
3
2
Cl 14
2
9
Stress
condition
Concentration of
stressor
Exposure
condition
Duration
6
DP-1
1
Acid-0.5 N HCl
Base-0.5 N NaOH
5 % H2O2
Reflux-50 °C
Reflux-50 °C
Reflux-50 °C
12 h
12 h
12 h
2-(2-((2,6-dichlorophenyl)amino)-
phenyl)-2-oxoacetic acid
Hydrolysis
Oxidation
Aceclofenac
2-(2-(2-((2,6-dichlorophenyl)amino)-
phenyl)acetoxy)acetic acid
Chemical Formula: C14H9Cl2NO3
Exact Mass: 309.00
Chemical Formula: C16H13Cl2NO4
Exact Mass: 353.02
RESULTS AND DISCUSSION
18
O
After 12 h of reflux, degradations were observed. 1 mL
of the resultant acid, base and peroxide mediated degradation
solution were dissolved in mobile phase and a sample of 10 µL
was subjected to LC-MS analysis. Drug solution treated with
acid showed one degradant and base showed three degradants
(Fig. 1). In that one degradant product (DP-2) is same for both
acid and base degradation study. No degradation of the drug
was observed in peroxide treated drug solution. Acid and base
treated solutions were taken up for isolation of all the three
detected degradants.
1
2
6
13
Cl
17
3
16
12
OH 19
14 Cl
15
H
N
5
5
4
15
4
3
7
8
Cl13
18
6
1
11
N
7
9
17
O
8
10
Cl 14
10
2
9
12
16
11
DP-3
DP-2
2-(2-((2,6-dichlorophenyl)amino)-
phenyl)acetic acid
Chemical Formula: C14H11Cl2NO2
Exact Mass: 295.02
1-(2,6-dichlorophenyl)indolin-2-one
Chemical Formula: C14H9Cl2NO
Exact Mass: 277.01
Isolation of acid and base degradation products: As
describes above the methods used in preparative HPLC for
isolation of degradants. The fractions corresponding to the peaks
in each study were collected and dried by lyophilization. The
Fig. 2. Structures of aceclofenac drug substance and its degradation products

Vol. 31, No. 4 (2019)
Isolation of Degradation Products of Aceclofenac by HPLC, HRMS and 2D NMR 853
Structure elucidation of degradation product (DP-1):
The HRMS showed a deprotonated molecular ion peak at m/z
307.9887 [M-H]− corresponding to m.f. C14H9NO3Cl2. The 1H
NMR spectrum revealed that the DP-1 had seven aromatic protons
and one NH exchangeable proton. Carboxylic acid proton was
not observed as degradant having moisture. H-16 and H-20 protons
(Fig. 2) of aceclofenac drug substance was not observed in
DP-1. The 13C NMR spectrum showed 14 aromatic carbons.
One of carbon observed at 192.6 ppm indicating that DP-1
having keto-carbonyl group which was not present in drug
(aceclofenac). The HSQC analysis revealed that there are 7
methines in the compound. In HMBC experiment, H-11 (7.65
ppm) correlated with 16th position carbonyl group at 192.6
ppm (Fig. 3) indicating that 16th position CH2 group of drug
(aceclofenac) was converted to carbonyl group.All these analysis
supporting to structure of DP-1 as shown in Fig. 2. 1H and 13C
chemical shift values was assigned as shown in Table-2 by
using 1H, 13C, COSY, edited HSQC and HMBC experiments.
Structure elucidation of degradation product (DP-2):
The HRMS showed a protonated molecular ion peak at m/z
278.0140 [M+H]+ corresponding to m.f. C14H9NOCl2. The 1H
NMR spectrum revealed that DP-2 had 7 aromatic protons
and one aliphatic CH2 protons. DP-2 degradant don't have exch-
angeable protons of NH and acid protons of drug substance
(aceclofenac).The13C NMR spectrum showed 13 aromatic carbons
and one aliphatic carbon. One of carbon observed at 173.2 ppm
belong to amide carbonyl group HSQC analysis revealed that
there are 7 methines and one methylene in the compound. In
HMBC experiment, H-16 (3.89ppm) correlated with C-17 (173.2
ppm), C-7 (142.8 ppm), C-11 (124.9ppm) and C-12 (124.6 ppm)
as shown in Fig. 4. All these analysis supporting to structure
of DP-2 as shown in Fig. 2. Proton and carbon chemical shift
values are assigned as shown in Table-2 by using 1D and 2D
NMR data. The compound was already reported earlier as a
degradant. However, its spectra data is not published [14].
H16
H1
9
H 2,6
H11
H8
10
DP-2
gHMBC, 400 MHz
15
1,9
2,6,11
8
10
1
DP-1
HMBC, 400 MHz
2
6
3
8
14 Cl
5
4
11,12
8
Cl
15
13
7
N
9
7
17
18
O
19
O
10
12
17
16
18
O
11
17
8
13
17
10
Cl
OH
16
1,2,6
20
15
3,5,11,12
H
12
5
4,9
N
8
6
4
2
F2 (ppm)
4
3
7
6
7
11
Fig. 4. 13C HMBC spectrum of DP-2
17
16
10
8
1
Cl
9
2
14
H-11,C-16
Structure elucidation of degradation product (DP-3):
10
8
6
4
2
F2 (ppm)
The HRMS showed a deprotonated molecular ion peak at m/z
Fig. 3. 13C HMBC spectrum of DP-1
294.0088 [M-H]− corresponding to m.f. C14H11NO2Cl2. The
TABLE-2
1H AND 13C NMR DATA OF ACECLOFENAC AND ITS DEGRADATION PRODUCTS
Aceclofenac
1H(PPM)
7.19
7.51
–
DP-1
DP-2
DP-3
Assignment
13C (PPM)
125.9
129.1
130.9
137.2
142.9
116.2
127.8
120.8
130.7
123.1
–
1H(PPM)
7.43
7.65
–
13C (PPM)
129.1
129.2
133.7
136.4
148.3
113.5
136.4
117.7
133.8
133.74
–
1H(PPM)
7.62
7.76
–
13C (PPM)
132
129.3
134.4
129.9
142.8
108.5
127.8
122.9
124.9
124.6
–
1H(PPM)
7.17
7.51
–
13C (PPM)
125.5
129.1
129.9
137.1
142.7
116
127.5
120.8
130.9
124.1
–
1
2,6
3,5
4
7
8
–
–
–
–
–
–
–
–
6.3
7.07
6.87
7.26
–
6.29
7.46
6.88
7.65
–
–
9.88
–
–
–
–
–
6.39
7.21
7.09
7.39
–
–
–
3.89
–
–
6.29
7.05
6.86
7.21
–
9
10
11
12
13,14
15
16
17
18
19
20
21
22
23
–
–
6.95
3.91
–
–
–
–
7.3
3.7
–
–
12
–
–
–
–
–
36.8
171
192.6
166.7
–
–
–
–
–
–
35.1
173.2
–
–
–
–
–
–
37.9
173.4
–
–
–
–
–
–
–
–
–
–
–
–
–
–
4.65
–
61.1
169
–
–
–
–
13.1
–
–
–

854 Guduru et al.
Asian J. Chem.
1H NMR spectrum revealed that DP-3 had 7 aromatic protons,
1 aliphatic CH2 group, two exchangeable protons of one NH
and one carboxylic acid.All protons of drug substance (aceclo-
fenac) were observed in DP-3 except H-20 proton (Fig. 2). The
13C NMR spectrum showed 13 aromatic carbons and one aliphatic
carbon. One of carbon observed at 173.4 ppm indicated that
DP-3 having acid carbonyl group. HSQC analysis revealed
that there are 7 methines and 1 methylene in the compound.
In HMBC experiment, H-16 (3.7 ppm) correlated with 17th
position carbonyl group at 173.4 ppm, C-7 (142.7 ppm), C-11
(130.9 ppm) and C-12 (124.1 ppm) as shown in Fig. 5 indicated
that 16th position CH2 group is present in between of benzene
group and carboxylic acid. This was found to be diclofenac
and has already been reported as a degradant in the stability
studies earlier [9]. All these analysis supporting to structure
of DP-3 as shown in Fig. 2. 1H and 13C chemical shift values
was assigned as show in Table-2 by using 1H, 13C, COSY, edited
HSQC and HMBC experiments.
[9-14]. However, to the best of our knowledge, this is the first
report on the structure characterization of degradation product
(DP-2).
ACKNOWLEDGEMENTS
The authors thank the Management of GVK Biosciences
Pvt. Ltd., Hyderabad, India for supporting this work.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interests
regarding the publication of this article.
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O
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H16,C-11
12
11
7
H16,C-7
H16,C-17
17
8
6
4
2
F2 (ppm)
Fig. 5. 13C HMBC spectrum of DP-3
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Forced degradation of aceclofenac was performed as per
the ICH guidelines. The drug was subjected to acid, base and
oxidative stressed conditions and three degradation products
were found to be formed.All the products were selectively sepa-
rated and fully characterized by various 2D NMR and mass
spectroscopic methods experiments. The degradation products
DP-2 and DP-3 were reported as the impurities of aceclofenac
https://doi.org/10.1365/s10337-008-0829-6.