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RESEARCH ARTICLE Am. J. PharmTech Res. 2012; 2(6) ISSN: 2249-3387
Please cite this article in press as Hotha K et al., Extensive Study of Aspirin and Its Related Impurities
Under Various Stressed Conditions in Low Dose Aspirin and Esomeprazole Magnesium Capsules.
American Journal of PharmTech Research 2012.
Extensive Study of Aspirin and Its Related Impurities Under
Various Stressed Conditions in Low Dose Aspirin and Esomeprazole
Magnesium Capsules
Palavai Sripal Reddy
1,2,
Shakil Sait
1
, Kishore Kumar Hotha
1*
1.Analytical Research and Development, IPDO, Dr. Reddy’s. Ltd. Hyderabad, India-500 072,
2.JNT University, Kukatpally, Hyderabad-500085, A.P, India
ABSTRACT
The objective of the present research work is to develop a isocratic reversed-phase liquid
chromatographic (RP-HPLC) method for the determination of Aspirin in pharmaceutical
pharmaceutical dosage forms for its related impurities in presence of esomeprazole. The
chromatographic separation was achieved on a RP 18 column (100mm×4.6mm, 5 µm). The
isocratic LC method employs mixture of buffer methanol and isopropyl alcohol in the ratio of
(84:13:3 v/v) solutions as mobile phase. The buffer solution contains 6.8g of Potassium
dihydrogen orthophosphate adjusted to pH 2.5 with orthophosphoric acid .The flow rate was 1.5
ml/min and the detection wavelength was 275 nm. In the developed HPLC method, the
resolution between Aspirin and its potential impurity salicylic acid was found to be greater than
4.0. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal
degradation in presence of esomeprazole. Considerable degradation was found to occur in basic
medium and mild degradation observed in acid hydrolysis stress conditions. Degradation product
formed during acidic hydrolysis was salicylic acid. The stress samples were assayed against a
qualified reference standard and the mass balance was found close to 99.5%. The developed RP-
HPLC method was validated with respect to linearity, accuracy, precision and robustness.
Keywords: RP-HPLC; Forced degradation; Validation; Aspirin, Salicylic acid Method
development
*Corresponding Author Email: drhotha@gmail.com
Received 15 November 2012, Accepted 26 November 2012
Journal home page: http://www.ajptr.com/
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INTRODUCTION
Aspirin (ASP), also known as acetylsalicylic acid, is a salicylate drug, often used (Figure. 1) as
an analgesic to relieve minor aches and pains, as an antipyretic to reduce fever and as an anti-
inflammatory Medication. Esomeprazole is a proton pump inhibitor which reduces acid secretion
through inhibition of ATPase in gastric parietal cells
1
. By inhibiting the functioning of this
enzyme, the drug prevents formation of gastric acid.Aspirin, by irreversibly acetylating cyclo-
oxygenase (COX), reduces the production of thromboxane A2 (TXA2) in platelets and prevents
platelet aggregation
2
. Aspirin can also reduce prostacyclin (PGI2) production in endothelial cells
and cause vasoconstriction. One of the side-effects associated with this use of aspirin is
gastrointestinal ulcers. Aspirin has a long history of therapeutic use, not only for its analgesic,
antipyretic and anti-inflammatory properties but also for its anti-thrombotic properties, which are
of value in states of platelet hyperaggregability. Aspirin binds irreversibly to the enzyme cyclo-
oxygenase-1 (COX-1) in platelets, leading to its antiplatelet effect
3
. Side effects of aspirin
treatment are mainly dyspeptic symptoms, gastrointestinal (GI) lesions and increased
gastrointestinal and overall bleeding, which are consequences of the blockage of prostaglandin
synthesis through inhibition of various COX enzymes. This leads to a decrease in mucosal
protection, which in turn predisposes the patient to mucosal lesions such as peptic ulcers and
peptic ulcer bleeding. Esomeprazole is a proton pump inhibitor (PPI) which is indicated,
amongst other indications, for the prevention of gastric and duodenal ulcers associated with
NSAID therapy (including aspirin therapy). There are very many drug products containing
aspirin 100 mg strength enteric coated tablets. The latter are the only low-dose aspirin
monotherapy drug products apart from breaking a 300 mg tablet in half which is probably done
by a small proportion of patients taking low dose aspirin for cardiovascular protection
4-6
.
Aspirin Salicylic acid Esomeprazole
Figure: 1: Chemical Structures of Aspirin, Salicylic acid and Esomeprazole
Combination of esomeprazole and aspirin assay/related impurities method was traditionally
difficult due to their stability in aqueous solutions, dosage variations and their absorption
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differences in the UV region. Literature reveals that aspirin is stable in acidic form where as
esomeprazole stability was found in basic solutions. The objective of the present research work
is to establish specificity and stability of aspirin in presence of esomeprazole and its related
impurities which gives precise and accurate quantitation of aspirin in the pharmaceutical dosage
forms of aspirin and esomeprazole. There were significant number of analytical methods for the
determination of aspirin using HPLC
7-18
, column switching chromatography
19
LC-MS/MS
20
and electrophoresis
21
have been reported for ASP in single form and in combination with other
drugs. There were several reported methods in human plasma also reported including its stability
in biological matrices. Recently Vijaya Bharathi D et al 2012, reported a new collection
procedure in the research article Low dose aspirin estimation: an application to human
pharmacokinetic study states that aspirin stability in acidic diluents rather than basic solutions
due to its sensitivity toward hydrolysis in aqueous and also in biological matrices(Figure-2).
Figure: 2:Degradation Pathway of Aspirin
MATERIALS AND METHODS
Chemicals
Samples of aspirin, esomeprazole and its related impurities were obtained from Drreddys
laboratories limited (Hyderabad, India) HPLC grade acetonitrile, analytical reagent grade
potassium dihydrogen orthophosphate and ortho phosphoric acid, Isopropyl alcohol was
purchased from Merck, Darmstadt, Germany. High purity water was prepared by using Millipore
Milli-Q plus water purification system. All samples and impurities used in this study were of
greater than 95.0% purity.
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Equipment
The HPLC system, used for method development, forced degradation studies and method
validation was waters HPLC system equipped with a diode array detector, from Waters Corp.
(Milford, MA, USA). The output signal was monitored and processes using Empower software
(Waters) Water bath equipped with temperature controller was used to carry out degradation
studies for all solution. Photo stability studies were carried out in a photo stability chamber and
thermal stability studies were performed in a dry air oven (Mack Phar-matech, Hyderabad,
India).
Chromatographic Conditions
The chromatographic column used was water XTerra RP C18 Column 100mm×4.6 mm, 5µm, all
obtained from Waters Corp. (Milford, MA, USA). The isocratic LC method consists buffer:
methanol: isopropyl alcohol in the ratio of (84:13:3 v/v) as mobile phase. The buffer solution
contains 6.8gms of potassium dihydrogen orthophosphate pH adjusted to 2.5 with ortho
phosphoric acid (Buffer).The flow rate of the mobile phase was 1.5 ml/min. The column
temperature was maintained 50°C and the detection was monitored at a wavelength of 275nm.
The injection volume was 20µl. [Buffer pH 2.5: Acetonitrile: Ethanol in the ratio of (50:30:20)]
was used as a diluent.
Preparation of Solutions
A stock solution of aspirin and its related impurity salicylic acid (1 mg·mL–1) was pre-pared by
dissolving appropriate amount in the diluent [Buffer pH 2.5: Acetonitrile: Ethanol in the ratio of
(50:30:20)].
Specificity
Specificity is the ability of the method to measure the analyte response in the presence of its
potential impurities
23
. Stress testing of the drug impurities can help identify the likely
degradation products, which can in turn help establish the degradation pathways and the intrinsic
stability of the molecule and validate the stability indicating power of the analytical procedures
used.
The specificity of the developed LC method for aspirin was determined in the presence of
esomeprazole and its related impurities. Forced degradation studies were also performed on
aspirin to provide an indication of the stability indicating property and specificity of the proposed
method. The stress conditions employed for degradation study includes light (carried out as per
ICH Q1B), Dry heating done at 105° C for about 2 hrs., acid hydrolysis (Refluxed with 1N HCl
solution for about 30 minutes at 60ºC), base hydrolysis (Refluxed with 1N NaOH solution for
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about 30 minutes at 60ºC), water hydrolysis and oxidation (Treated with 30% Hydrogen
peroxide (H
2
O
2
) for about 30 minutes at 60ºC). Sunlight, thermal and UV degradation was also
performed and purity of stressed samples was checked by using Photo diode array detector
(PDA). The purity factor is within the threshold limit obtained in all stressed samples
demonstrates the analyte peak homogeneity. Specificity of the aspirin was shown by spiking all
the esomeprazole and its related impurities the specification level (i.e. 0.15% of analyte
concentration which is 1 mg/mL).
ANALYTICAL METHOD VALIDATION
The developed chromatographic method was validated for linearity, precision, accuracy,
sensitivity, robustness and system suitability
22-24
.
Precision
The precision of test method was evaluated by analyzing six test samples of Low dose Aspirin
and Esomeprazole magnesium capsules 325/40,325/20, 81/40 and 81/20 mg by spiking test
preparation with Salicylic acid impurity at 3.0% and analyzed.
Limit of detection (LOD) and limit of quantification (LOQ)
Limit of detection and limit of quantification were established based on signal to noise ratio. A
series of solutions having aspirin impurity were injected. Limit of detection for impurity was
established by identifying the concentration which gives signal to noise ratio about 3. Limit of
quantification was established by identifying the concentration which gives signal to noise ratio
about 10.
Precision of aspirin impurity at about Limit of Quantification level was conducted. Six test
preparations having impurities at the level of about Limit of quantification were prepared and
injected into the HPLC system.
Linearity and Range
The linearity for aspirin and its impurity salicylic acid from Limit of quantification level to 200%
of the target concentration (3.0 %). Linearity shall be established for Salicylic acid from Limit of
quantification level to 200% of the target concentration (0. 2 %) target concentration of aspirin
unknown salicylic acid impurity and injected into the HPLC system.
Accuracy
Accuracy of aspirin and aspirin impurities at about Limit of Quantification and at 150% of target
concentration level was conducted. Test solutions spiked with Aspirin impurities at about Limit
of Quantification and at 150% of target concentration were prepared in triplicate and injected
into HPLC system
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Robustness
To determine the robustness of the developed method, experimental conditions were deliberately
changed and the resolution (Rs) between aspirin and its impurity salicylic acid was evaluated.
The flow rate of the mobile phase was 1.5 mL·min–1. To study the effect of flow rate on the
developed method, 0.2 units of flow was changed (i.e. 1.3 and 1.7 mL·min–1). The effect of
column temperature on the developed method was studied at 45°C and 55°C instead of 50°C. In
the all above varied conditions, the components of the mobile phase were held constant.
Solution Stability and Mobile Phase Stability
The solution stability of aspirin and its related impurity salicylic acid were carried out by leaving
both spiked sample and unspiked sample solution in tightly capped volumetric flask at room
temperature for 48 h. Content impurity was determined at every 6 h interval, up to the study
period. Mobile phase stability was also carried out for 48 h by injecting the freshly prepared
sample solutions, for every 6 h interval. Salicylic acid impurity was checked in the test solutions.
Mobile phase prepared was kept constant during the study period.
RESULTS AND DISCUSSION
Method Development and Optimization
The main complexity of the present research work is to develop a stability indicating method for
the estimation of aspirin in esomeprazole and aspirin pharmaceutical dosage forms in presence of
esomeprazole. The main target of the chromatographic method is to get the separation of critical
closely eluting degradable peaks of esomeprazole that can interfere with aspirin and its related
impurities. Impurities were co-eluted by using different stationary phases like C18, Phenyl and
cyano and different mobile phases containing buffers like phosphate, sulphate and acetate with
different pH (2–8) and using organic modifiers like acetonitrile, methanol and ethanol in the
mobile phase. After several logical trails and optimization of stationary phase, column
temperature and flow rate and mobile phase pH the chromatographic separation was achieved on
a Waters X terra RP 18 Column 100mm×4.6 mm, 5 µm column, The isocratic LC method
consists buffer: methanol and isopropyl alcohol in the ratio of (84:13:3 v/v) as mobile phase. The
buffer solution contains 6.8 gms potassium dihydrogen orthophosphate in 1000 mL pH adjusted
to 2.5 with Potassium hydroxide solution (Buffer).The flow rate of the mobile phase was 1.5
ml/min. The column temperature was maintained 50°C and the detection was monitored at a
wavelength of 275 nm. The injection volume was 20µl. Mixture of [Buffer pH 2.5: Acetonitrile:
Ethanol in the ratio of (50:30:20)] was used as a diluent. The concentration is 1.0 mg·mL–1 for
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related impurities method. The peak shape of aspirin was found symmetrical. In the optimized
conditions aspirin and salicylic acid impurity was well separated with a resolution of greater than
4.0 and the typical retention times of aspirin and its related impurity was3.72 and 6.02 min
respectively. The system suitability results are given in Table 1 and the developed HPLC method
was found to be specific for aspirin and its three impurity salicylic acid.Figure-3, Figure-4 and
Figure-5 shows the chromatograms of Diluent, Impurity blend solution and test sample solution.
Table-1: System suitability Report
System suitability parameters
Observed value
Acceptance limit
From System suitability solution
The resolution between Aspirin and Salicylic
Acid peaks.
3.7
NLT 2.5
From Standard preparation
% RSD for Aspirin and salicylic Acid Peak
areas for six replicate injections.
2.6/0.5
NMT 10.0 %
The Tailing factor for Aspirin and salicylic
Acid peaks.
1.1/1.0
NMT 2.0
Figure:-3 Typical chromatogram of Diluent
Figure:-4 Typical chromatogram of Impurities blend solution
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Figure:-5 Typical chromatogram of Sample
Results of Forced Degradation Studies
The drug was exposed to 1N HCl at 60°C for 30 min. aspirin has shown mild sensitivity towards
the treatment of 1N HCl. The drug gradually undergone degradation with time in 1N HCl and
prominent degradation was observed (~22%). The representative chromatogram present in
Figure 6.
Figure 6: Typical chromatogram of Acid degradation
Degradation in Basic Solution
The drug was exposed to 1N NaOH at 60°C for 30 h. Aspirin has shown significant sensitivity
towards the treatment of 1N NaOH. The drug gradually undergone degradation with time in 1N
NaOH and degradation was observed (~8%). The representative chromatogram present in
Figure7.
Oxidative Conditions
The drug was exposed to 30% hydrogen peroxide at room temperature for 60 min.Aspirin has
shown no significant sensitivity towards the treatment of 30% hydrogen peroxide and the drug
shown mild sensitivity in oxidative conditions.
Aspirin was stable under forced photo and thermal degradation. From the degradation studies,
Peak purity test results derived from PDA detector, confirmed that the Aspirin peak was
homogeneous and pure in all the analyzed stress samples. The mass balance of stressed samples
was close to 99.5%. After exposing aspirin in sunlight(1.2 mn lux hours) and UV light(200 wt
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hours per sq meter) 1.33 and 0.14 % degradation was observed. After dry heating at 105°C for
2hours and in the presence exposed humidit at 25°C /90%RH about 7 days 0.33 and 0.68 %
degradation was observed. The forced degradation study results are given in Table 2. The
representative chromatogram presents in Figure 7 to Figure 13.
Table-2: Summary of Forced degradation study report
Stress Condition
Drug Product
%
Degradation
Purity
angle
Purity
threshold
Purit
y flag
Refluxed with 1N HCl solution for about 30 mins at
60ºC and neutralized with 1N NaOH
22.15
2.373
90.0
No
Refluxed with 1N NaOH solution for about 30 mins
at 60ºC and neutralized with 1N HCl
8.79
2.983
90.0
No
Treated with 30% Hydrogen peroxide (H
2
O
2
) for
about 30 minutes at 60ºC
4.07
3.161
90.0
No
Refluxed with purified water for about 5 hrs at 60ºC
2.73
0.277
0.661
No
Exposed to Sunlight for about 1.2 Million Lux
hours.
1.33
0.380
0.746
No
Exposed to UV light both at shorter and longer
wavelengths for about 200 watt hours / square
meter.
0.14
0.295
0.640
No
Dry heating done at 105° C for about 2hrs.
0.68
0.280
0.58
No
Exposed to humidity at 25°C, 90% RH for about 7
days
0.33
3.524
90.0
No
Figure 7: Typical chromatogram of Base degradation
Figure 8: Typical chromatogram of oxidation degradation
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Figure 9: Typical chromatogram aqueous stressed preparation
Figure 10: Typical chromatogram of Thermal degradation
Figure 11: Typical chromatogram of sunlight stressed degradation
Figure 12: Typical chromatogram of dry heat stressed degradation
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Figure 13: Typical chromatogram of UV Light stressed degradation
Figure 14: Typical chromatogram of humidity stressed degradation
Table 3: Precision and Accuracy Data of Salicylic acid
Sample
No.
Spike
level
‘ppm’
added
‘ppm’ found
(recovered)
% Recovery
Mean %
recovery
1.
20%
39.7256
38.5450
97.0
97.7
2.
20%
39.7256
39.2600
98.8
3.
20%
39.7256
38.6750
97.4
1.
50%
99.3141
104.2600
105.0
105.5
2.
50%
99.3141
105.0400
105.8
3.
50%
99.3141
104.9100
105.6
1.
100%
198.6281
204.7500
103.1
103.3
2.
100%
198.6281
205.5300
103.5
3.
100%
198.6281
205.1400
103.3
1.
120%
238.3537
245.8950
103.2
104.8
2.
120%
238.3537
253.6950
106.4
3.
120%
238.3587
249.6650
104.7
1.
150%
297.9422
327.4050
109.9
105.3
2.
150%
297.9422
311.6100
104.6
3.
150%
297.9422
311.7400
104.6
4.
150%
297.9422
310.4400
104.2
5.
150%
297.9422
311.1550
104.4
6.
150%
297.9422
309.8550
104.0
Method validation
Precision and Accuracy
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The %RSD of area of a method precision study were 1.5. This aspirin and its related impurity the
good precision of the developed analytical method for related impurities. Experimental data
given in Table 3 and Table 4.
The percentage recovery of aspirin and salicylic acid in pharmaceutical dosage forms ranged
from 103% - 112%. The percentage recovery of aspirin and its impurity ranged from 97% to
105.5% (Table 4).
Table 4: Precision and Accuracy Data of Aspirin
Sample
No.
Spike
level
‘ppm’
added
‘ppm’ found
(recovered)
%
Recovery
Mean %
recovery
1.
20%
3.2144
3.6400
113.2
112.5
2.
20%
3.2144
3.5750
111.2
3.
20%
3.2144
3.6400
113.2
1.
50%
6.4288
6.9550
108.2
106.2
2.
50%
6.4288
6.7600
105.2
3.
50%
6.4288
6.7600
105.2
1.
100%
12.8575
13.5200
105.2
105.4
2.
100%
12.8575
13.5200
105.2
3.
100%
12.8575
13.5850
105.7
1.
120%
16.0719
16.5100
102.7
103.1
2.
120%
16.0719
16.1200
100.3
3.
120%
16.0719
17.0950
106.3
1.
150%
19.2863
21.6450
112.2
107.1
2.
150%
19.2863
20.3450
105.5
3.
150%
19.2863
20.2150
104.8
4.
150%
19.2863
20.2800
105.2
5.
150%
19.2863
20.2150
104.9
6.
150%
19.2863
21.1900
109.9
Table 5: Limit Of Quantification - Precision
Name
Sample No.
% Impurity
Mean
%RSD
Salicylic acid
1
0.015
0.014
4.5
2
0.014
3
0.014
4
0.013
5
0.014
6
0.014
Aspirin
1
0.009
0.009
8.2
2
0.009
3
0.010
4
0.008
5
0.010
6
0.009
Sensitivity
The limit of detection of aspirin and salicylic acid was 0.003% (of analyte concentration, i.e. 1
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mg/ml) for 20µl injection volume. The limit of quantification of salicylic acid was 0.015 % and
0.010 (of analyte concentration, i.e. 1.0 mg/mL) for 20µl injection volume. The precision at
LOQ concentration were below 10 %.Experimental data shown in Table 5 and Table 6.
Table 6: Limit of Quantification – Accuracy
Sample
No.
Spike
level
(ppm)
added
(ppm) found
(recovered)
% Recovery
Mean %
recovery
1
LOQ
0.7960
0.8450
106.2
107.6
2
LOQ
0.7960
0.8450
106.2
3
LOQ
0.7960
0.9100
114.3
4
LOQ
0.7960
0.8450
106.2
5
LOQ
0.7960
0.8450
106.2
6
LOQ
0.7960
0.8450
106.2
Aspirin
1
LOQ
0.5994
0.5850
97.6
99.4
2
LOQ
0.5994
0.5850
97.6
3
LOQ
0.5994
0.6500
108.4
4
LOQ
0.5994
0.5200
86.8
5
LOQ
0.5994
0.6500
108.4
6
LOQ
0.5994
0.5850
97.6
Linearity and Range
Linear calibration plot for related impurities method was obtained over the calibration ranges
tested, i.e. 0.8 to 397 ppm for salicylic acid. The correlation coefficient obtained was greater than
0.999 for salicylic acid impurity. The result shows an excellent cor-relation existed between the
peak area and concentration of impurity. Experimental data presented in Table 7
Table 7: Linearity
Sample Name
Sample ID
Concentration ‘ppm’
Peak Area
Salicylic acid
01
0.8
4633
02
39.7
223647
03
99.3
530509
04
194.7
938616
05
238.4
1305587
06
297.9
1581388
07
397.3
2146851
Aspirin
01
0.6
3482
02
2.5
12309
03
6.5
30841
04
13.0
55836
05
16.0
74782
06
20.0
92427
Robustness
Close observation of analysis results for deliberately changed chromatographic conditions (flow
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860
rate, pH and column temperature) revealed that the resolution between closely eluting peaks,
namely aspirin and its related impurity was always greater than 2.0, illustrating the robustness of
the method.
Solution Stability and Mobile Phase Stability
No significant changes were observed in the content of salicylic acid impuruty during solution
stability and mobile phase stability experiments for related impurities. The %RSD of assay of
aspirin during solution stability and mobile phase stability experiments was within 1.0.The
solution stability and mobile phase stability experiments data confirms that sample solutions and
mobile phase used during assay and related impurities determination were stable up to the study
period of 48 h.
Assay Analysis
Analysis was performed for different batches of Esomeprazole and low dose Aspirin dosage
forms in (n = 3) ranged from 99.95% - 99.96%.
CONCLUSION
In this present research article complete degradation stress studies were reported for aspirin and
its related impurity salicylic acid in presence of esomeprazole. The RP-HPLC method developed
for related impurities was linear, precise, accurate and specific. The method was completely
validated showing satisfactory data for all the method validation parameters tested as per ICH
guidelines. The developed method is stability indicating and can be used for the routine analysis
of production samples and also to check the stability of aspirin samples same procedures on a
different day (inter day precision). To the best of our knowledge the specified method presented
in the article successfully measures aspirin and its related impurities by HPLC in presence of
esomeprazole.
ACKNOWLEDGEMENT
We wish to express our sincere thanks to the Managements of Dr.Reddys Laboratories,
Hyderabad, India for their support and encouragement. Cooperation from colleagues and of
Research & Development and Analytical Research & Development of Dr.Reddy’s Laboratories
Ltd. is appreciated.
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