The Pharma Innovation Journal 2018; 7(4): 217-234
ISSN (E): 2277- 7695
ISSN (P): 2349-8242
NAAS Rating: 5.03
TPI 2018; 7(4): 217-234
© 2018 TPI
www.thepharmajournal.com
Received: 23-02-2018
Accepted: 26-03-2018
Nidhi S Patel
Smt. B. N. B Swaminarayan
Pharmacy College, Salvav, Vapi,
Gujarat, India
Dr. Dulendra P Damahe
Smt. B. N. B Swaminarayan
Pharmacy College, Salvav, Vapi,
Gujarat, India
Dr. Shailesh V Luhar
Smt. B. N. B Swaminarayan
Pharmacy College, Salvav, Vapi,
Gujarat, India
Dr. Sachin B Narkhede
Smt. B. N. B Swaminarayan
Pharmacy College, Salvav, Vapi,
Gujarat, India
Correspondence
Nidhi S Patel
Smt. B. N. B Swaminarayan
Pharmacy College, Salvav, Vapi,
Gujarat, India
Stability indicating RP-HPLC method development
and validation for simultaneous estimation of aspirin
and isosorbide mononitrate in pharmaceutical dosage
form
Nidhi S Patel, Dr. Dulendra P Damahe, Dr. Shailesh V Luhar and Dr.
Sachin B Narkhede
Abstract
A simple, rapid, precise, economic, and accurate Stability-Indicating RP-HPLC method were developed
and validated for the estimation of Aspirin and Isosorbide Mononitrate in pharmaceutical dosage form.
Method include Shiseido C18 (250 mm × 4.6 mm, 5μm) column and Phosphate buffer (pH: 4): Methanol
(30:75 v/v) as mobile phase at 1.0ml/min flow rate. The detection was carried out at 215nm. Rt was
found to be 3.767 min for Aspirin and 2.540 min for Isosorbide Mononitrate. For stability study drugs
were subjected to acid hydrolysis, alkaline hydrolysis, oxidative degradation and thermal degradation.
Pharmaceutical dosage form was more stable than Active pharmaceutical ingredient. The linearity range
was found in range of 15-52.5 μg/mL for Aspirin and 6-21μg/mL Isosorbide Mononitrate. Limit of
Detection for Aspirin and Isosorbide Mononitrate was found to be 1.01μg/mL and 0.77μg/mL
respectively. Limit of Quantitation for Aspirin and Isosorbide Mononitrate was found to be 3.07μg/mL
and 2.34μg/mL respectively.
Keywords: aspirin, isosorbide mononitrate, stability indicating rp-hplc method, forced degradation
studies, validation
Introduction
Materials and Methods
Aspirin is chemically 2-(acetyloxy) benzoic acid. It is use in temporary relief of various forms
of pain, inflammation associated with various conditions, and is also used to reduce the risk of
death and/or nonfatal myocardial infarction in patient with previous infarction or unstable
angina pectoris. It is official in Indian pharmacopeia 2018. It is freely soluble in ethanol and
methanol. Molecular weight of Aspirin is 180.1574 gm/mol and formula is C 9H8O4.
Isosorbide Mononitrate is chemically 8-nitrooxy-2, 6-dioxabicyclo [3. 3. 0] 0ctan-4-ol. It is the
prevention of angina pectoris due to coronary artery disease and the treatment of acute and
chronic angina pectoris, hypertension, and myocardial infarction. It is official in Indian
pharmacopeia 2018. It is freely soluble in ethanol and methanol. Molecular weight of Aspirin
is 191.139 gm/mol and formula is C6H9NO6.
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Aspirin and Isosorbide Mononitrate is obtained from Sidmak
laboratories (India) pvt. Ltd. Valsad.
Instrumentation and Chromatographic method
The analysis of the drug was carried out on a Peak HPLC
system equipped with a reverse phase Shiseido C18 column,
peak pump with auto sampler and a detector running on LC
solution Software. The mobile phase consists of Phosphate
Buffer (pH: 4): Methanol (30:70% V/V) and the flow rate
were maintained at 1.0 ml/min. The mobile phase was freshly
prepared and passed through nylon membrane filter of pore
size of 0.45μm and it was degassed by sonicate for 10min.
before it was used. The elution was monitored at wavelength
of 215nm with UV detector and the injection volume was
10μl.
Determination of maximum absorbance
The standard solutions of Aspirin and Isosorbide Mononitrate
were scanned in the range of 200-400 nm against mobile
phase as blank. Isobestic Point of Aspirin and Isosorbide
Mononitrate at 215 nm. Thus the wavelength selected for the
determination of Aspirin and Isosorbide Mononitrate was
215nm.
Preparation of Standard Stock Solutions:
Aspirin Standard stock-I solution (1000μg/ml): 100mg of
Aspirin was weighed and transferred to a 100 ml volumetric
flask and dissolved in Methanol and sonicated for about 10
min. Volume was make up to the mark with methanol to give
a solution containing 1000μg/ml Aspirin.
Aspirin Standard stock-II solution (300μg/ml): Withdraw
30ml of stock-I solution was diluted to 100 ml with Methanol
to prepare 300μg/ml.
Isosorbide Mononitrate Standard stock-I solution
(1000μg/ml):100mg of Isosorbide Mononitrate was
accurately weighed and transferred to a 100ml volumetric
flask and dissolved in Methanol and sonicated for about 10
min. Volume was make up to the mark with methanol to give
a solution containing 1000μg/ml Isosorbide Mononitrate.
Isosorbide Mononitrate standard stock-II solution
(120μg/ml): Withdraw 12ml of stock-I solution was diluted to
100 ml with Methanol to prepare 120μg/ml.
Assay of marketed formulation (Brand name of Tablet)
20 Tablet were weighed and powdered. Powder equivalent to
30mg of ASP and 12mg of ISMN was weighed and
transferred to 100mL volumetric flask. And 60mL of diluents
was added to it and was sonicated for 10minutes and then
volume was made up to the mark with diluent to give a
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solution containing 300μg/mL Aspirin and 120μg/mL
Isosorbide Mononitrate Solution.
Method validation
The Proposed method was validated according to ICH
guidelines. The parameters assessed were linearity, precision,
accuracy, LOD and LOQ.
System Suitability
System suitability tests are an integral part of liquid
chromatography. They are used to verify that resolution and
reproducibility of chromatography system are adequate for
the analysis to be done. System Suitability was performed on
standard solution and system suitability parameters were
calculated at the start of study for each parameter.
Linearity and Range
The linearity was determined at three levels over the range of
15-52.5μg/ml Aspirin and 6-21μg/ml Isosorbide Mononitrate.
Peak area of above linearity solution preparations were taken
at each concentration three times.
Accuracy
Recovery studies were carried out by addition of standard
drug to the sample at 3 different concentration levels (80%,
100% and 120%) taking into consideration percentage purity
of added bulk drug samples. These solutions were subjected
to re-analysis by the proposed method and Results are
calculated.
Precision Repeatability Study
Standard solutions of 12μg/ml Aspirin and 30μg/ml
Isosorbide Mononitrate were prepared and chromatograms
were recorded. Area was measured of the same concentration
solution three times and % RSD was calculated.
Intra-day precision
Mixed solutions containing 15, 30, 52.5μg/ml Aspirin and 6,
12, 21μg/ml Isosorbide Mononitrate were analysed three
times on the same day %R.S.D was calculated.
Inter-day precision
Mixed solutions containing 15, 30, 52.5μg/ml Aspirin and 6,
12, 21μg/ml Isosorbide Mononitrate were analysed on three
different days and % R. S. D was calculated.
Limit of Detection and Limits of Quantitation
Limit of Detection (LOD)
From the linearity curve equation, the standard deviation (SD)
of the intercepts (response) was calculated. The limit of
detection (LOD) of the drug was calculated by using the
following equation designated by International Conference on
Harmonization (ICH) guideline:
LOD=3.3× Intercept/Slope
Limit of Quantitation (LOQ)
The limit of Quantitation (LOQ) of the drug was calculated by
using the following equation designated by International
Conference on Harmonization (ICH) guideline:
LOQ=10 × Intercept/Slope
Robustness
The robustness of the method was established by making
deliberate minor variations in the following method
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parameters
a) pH of mobile phase: ±0.2
b) Flow rate: ±0.2 ml/min
c) Change in the ratio of component in the mobile phase:
±2%.
Stability studies
Stability Studies was carried out on the drug in order to check
the stability of the drug by providing various stress conditions
like acid, base, oxidation and thermal degradation compared
with normal conditions. The purpose of force degradation
method is to provide evidence that the analytical method is
efficient in determination of drug substances in commercial
drug product in the presence of its degradation products.
Acidic hydrolysis
Take 2 ml solution of Aspirin 1000μg/ml and Isosorbide
Mononitrate 1000μg/ml, 2 ml of 0.1M HCl was added. The
solution was heated for 4hr at 40 °C and transferred to a 10ml
volumetric flask, cooled, neutralized by 0.1M NaOH and
diluted up to mark with methanol to get final concentration
100μg/ml of Aspirin and 100μg/ml of Isosorbide Mononitrate.
Alkaline hydrolysis
Take 2 ml solution of Aspirin 1000μg/ml and Isosorbide
Mononitrate 1000μg/ml, 2 ml of 0.1M NaOH was added. The
solution was heated for 4hr at 40°C and transferred to a 10ml
volumetric flask, cooled, neutralized by 0.1M HCl and diluted
up to mark with methanol to get final concentration 100μg/ml
of Aspirin and 100μg/ml of Isosorbide Mononitrate.
Oxidative degradation
Take 2 ml solution of Aspirin 1000μg/ml and Isosorbide
Mononitrate 1000μg/ml, 2 ml 3% H2O2 was added at room
temperature for 6hours at 60 °C and transferred to a 10ml
volumetric flask, cooled diluted up to mark with methanol to
get final concentration 100μg/ml of Aspirin and 100μg/ml of
Isosorbide Mononitrate.
Thermal degradation
Take 2 ml solution of Aspirin 1000μg/ml and Isosorbide
Mononitrate 1000μg/ml, heat the solution for 24hr at 80 °C
and transferred to a 10ml volumetric flask, cooled diluted up
to mark with methanol to get final concentration 100μg/ml of
Aspirin and 100μg/ml of Isosorbide Mononitrate.
Forced Degradation Study
Acid Hydrolysis
Aspirin, Isosorbide Mononitrate and Formulation Acid
Degradation
1mL of Standard Stock-II Solution of Aspirin, Isosorbide
Mononitrate and Sample Stock Solution were transferred in
three different 10mL volumetric flask; to it 2mL of 0.1 NHCl
at 40 °C was added and kept for 4hrs and then 2mL of 0.1N
NaOH was added for neutralization and diluted up to the mark
with Mobile Phase.
Alkaline Hydrolysis
Aspirin, Isosorbide Mononitrate and Formulation Acid
Degradation
1mL of Standard Stock-II Solution of Aspirin, Isosorbide
Mononitrate and Sample Stock Solution were transferred in
three different 10mL volumetric flask; to it 2mL of 0.1N
NaOH at 40 °C was added and kept for 4hrs and then 2mL of
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0.1N HCl was added for neutralization and diluted up to the
mark with Mobile Phase.
Oxidative Degradation
Aspirin, Isosorbide Mononitrate and Formulation
Oxidative Degradation
1mL of Standard Stock-II Solution of Aspirin, Isosorbide
Mononitrate and Sample Stock Solution were transferred in
three different 10mL volumetric flask; to it 2mL of 3% H 2O2
at 60°C was added and kept for 6hrs and then diluted up to the
mark with Mobile Phase.
Thermal degradation
Aspirin, Isosorbide Mononitrate and Formulation
Oxidative Degradation
Weighed 50mg of both drugs and 50mg equivalent weight of
tablet powder was taken in a clean and dry Petri dish and
covered with Aluminium foil. Petri dish was kept in oven at
80 ºC for 24hr. An Accurately weighed 10mg of dry heated
drug was transferd to 100mL volumetric flask. It was
dissolved using mobile phase and final volume was made up
to 100mL. Solution of 30μg/mL of Aspirin and 12μg/mL of
Isosorbide Mononitrate was prepared from above solution.
Photo degradation
Aspirin, Isosorbide Mononitrate and Formulation Photo
degradation
1mL of Standard Stock-II Solution of Aspirin, Isosorbide
Mononitrate and Sample Solution were transferred in three
different 10mL volumetric flask was kept in Photo stability
chamber for 24hrs and then diluted up to the mark with
Mobile Phase.
Method Validation
Linearity and Range
The linearity is expressed in terms of correlation co-efficient
of linear regression analysis. The linearity of Aspirin and
Isosorbide Mononitrate was found to be in between 15 to
52.5μg/mL and 6 to 21μg/mL.
Limit of Detection and Limit of Quantitation
Limit of Detection (L. O. D.)
The L. O. D. was estimated from calibration curves.
LOD=3.3 × (S. D. /Slope)
Where, S. D. =Standard deviation of the Y-intercept of the
calibration curve.
Slope=Mean slope of calibration curve.
Limit of Quantitation (L.O.Q.)
The L. O. Q. was estimated from calibration curve.
LOQ=10 × (S.D./Slope)
Where, S. D=Standard deviation of the Y-intercept of
calibration curve.
Slope=Mean slope of calibration curve.
Accuracy
Preparation of Recovery Solution
For 80% level 0.5mL of Sample solution, 0.4mL of Stock-II
Solution of ASP and 0.4mL of Stock-II Solution of ISMN
were transferred in 10mL volumetric flask and volume was
made up to the mark with Mobile Phase.
For 100% level: 0.5mL of Sample solution, 0.5mL of Stock-
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II Solution of ASP and 0.5mL of Stock-II Solution of ISMN given below.

were transferred in 10mL volumetric flask and volume was
made up to the mark with Mobile Phase.
For 120% level: 0.5mL of Sample solution, 0.6mL of Stock-
II solution of ASP and 0.6mL of Stock-II Solution of ISMN
were transferred in 10mL volumetric flask and volume was
made up to the mark with Mobile Phase.
Precision
A. Interday Precision
Interday Precision was performed at three different
concentrations of binary mixture of ASP and ISMN on three
different days. The concentrations of the binary mixtures are
given below.
Lower concentration (50%)-ASP-15µg/mL + ISMN-6µg/mL
Middle concentration (100%)-ASP-30µg/mL + ISMN-
12µg/mL
Higher concentration (150%)-ASP-45µg/mL + ISMN-
18µg/mL
Lower concentration (50%)-ASP-15µg/mL + ISMN-6µg/mL
Middle concentration (100%)-ASP-30µg/mL + ISMN-
12µg/mL
Higher concentration (150%)-ASP-45µg/mL + ISMN-
18µg/mL
C. Repeatability
Repeatability study was performed on only one binary
mixture (Middle concentration) of ASP and ISMN repeated
for 6 times.
Middle concentration (100%)-ASP-30µg/mL + ISMN-
12µg/mL
Areas were recorded and % RSD was calculated are shown in
Table 7.17.
Robustness
The solution containing concentration of ASP and ISMN was
analysed in different flow rate, mobile phase and pH.

B. Intraday Precision Results and Discussion

Intraday Precision was performed at three different Development of Rp-Hplc Method
concentrations of binary mixture of ASP and ISMN thrice on Selection of Detection Wavelength
the same day. The concentrations of the binary mixtures are

Fig 1: Selection of Detection Wavelength

Selection of mobile Phase

Trial 1

Fig 2: Mobile Phase Water: Methanol (30:70% V/V)

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Trial 2

Fig 3: Mobile Phase Water: Methanol (20:80 % V/V)

Trial-3

Fig 4: Mobile Phase Phosphate Buffer (pH: 4): Methanol (40:60% V/V)

Trial 4

Fig 5: Mobile Phase Phosphate Buffer (pH: 4): Methanol (30:70% V/V)
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Trial 5

Fig 6: Mobile Phase Phosphate Buffer (pH: 5): Methanol (30:70% V/V)

Chromatogram of Both the drugs and capsule

Chromatogram of Aspirin

Fig 7: Chromatogram of Aspirin

Chromatogram of Isosorbide Mononitrate

Fig 8: Chromatogram of Isosorbide Mononitrate

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Chromatogram of Standard

Fig 9: Chromatogram of Standard

Chromatogram of Formulation

Fig 10: Chromatogram of Formulation

System Suitability Parameter

Table 1: System Suitability Result

Retention Time (min) Tailing Factor Resolution Theoretical Plate
Parameters
(n=6) (T) (n=6) (R) (N)±SD (n=6)
Specifications - NMT 2 MT 1.5 NLT 2000
ASP 3.7488±0.0034 1.4083±0.0166 7243±49.0445
Std.
ISMN 2.5271±0.0016 1.3351±0.0235 8.1173 6591±25.2084

Forced Degradation Study

Acid Hydrolysis

Fig 11: Chromatogram of Acid Hydrolysis Blank

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Fig 12: Chromatogram of ASP under Acid Hydrolysis

Fig 13: Chromatogram of ISMN under Acid Hydrolysis

Fig 14: Chromatogram of Formulation under Acid Hydrolysis

Alkali Hydrolysis

Fig 15: Chromatogram of Alkaline Hydrolysis Blank

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Fig 16: Chromatogram of ASP under Alkaline Hydrolysis

Fig 17: Chromatogram of ISMN under Alkaline Hydrolysis

Fig 18: Chromatogram of Formulation under Alkaline Hydrolysis

Oxidative Degradation

Fig 19: Chromatogram of Oxidative Degradation Blank

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Fig 20: Chromatogram of ASP under Oxidative Degradation

Fig 21: Chromatogram of ISMN under Oxidative Degradation

Fig 22: Chromatogram of Formulation under Oxidative Degradation

Thermal Degradation

Fig 23: Chromatogram of Blank Thermal Degradation

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Fig 24: Chromatogram of ASP under Thermal Degradation

Fig 25: Chromatogram of ISMA under Thermal Degradation

Fig 26: Chromatogram of Sample under Thermal Degradation

Photo Degradation

Fig 27: Chromatogram of Blank Photo Degradation

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Fig 28: Chromatogram of ASP under Photo Degradation

Fig 29: Chromatogram of ISMN under Photo Degradation

Fig 30: Chromatogram of Sample under Photo Degradation

Result of Stability Study

Table 2: Result of Stability Study

% Degradation of API % Degradation of pharmaceutical dosage form
Stress Condition
ASP ISMN ASP ISMN
Acid Hydrolysis 17.42 12.07 15.80 13.60
Alkaline Hydrolysis 12.33 17.33 13.54 18.49
Oxidation 19.62 10.93 19.82 8.95
Thermal 10.17 17.95 11.88 17.14
Photo 10.60 13.90 9.66 14.97

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Method Validation
Linearity and Range
Chromatogram of Linearity

Fig 31: Chromatogram of Linearity

Linearity for ASP

Table 3: Linearity for ASP

Sr. No. Conc. (μg/ml) Mean Area± SD % RSD
1 15 1334.6598 ± 18.4363 1.3813
2 22.5 2552.4068 ± 26.9909 1.0574
3 30 3828.0966 ± 13.9837 0.3652
4 37.5 5040.0700 ± 23.3696 0.4636
5 45 6424.3592 ± 15.3749 0.2393
6 52.5 7566.9398 ± 28.2386 0.3731

Calibration Curve of ASP

Fig 32: Linearity for ASP

Linearity for ISMN

Table 4: Linearity for ISMN

Sr. No. Conc. (μg/ml) Mean Area± SD % RSD
1 6 504.0326±4.5315 0.8990
2 9 995.6004±3.2388 0.3253
3 12 1490.4654±6.7690 0.4541
4 15 1958.8554±13.6184 0.6952
5 18 2557.8624±31.3149 1.2242
6 21 2967.7192±19.2658 0.6491
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Calibration curve of ISMN

Fig 33: Linearity for ISMN

Limit of Detection and Limit of Quantitation

Table 5: Result of LOD and LOQ

Drugs LOD (µg/ml) LOQ (µg/ml)
Aspirin 1.01 3.07
Isosorbide Mononitrate 0.77 2.34

Accuracy
Recovery Data for Aspirin

Table 6: Recovery data for Aspirin

Amount of Amount of Total Mean %
Std. Amount %
Level sample taken standard spiked amount Recovery± SD % RSD
recovered (µg/ml) Recovery
(µg/ml) (µg/ml) (µg/ml) (n=3)
15 12 27 11.824 98.53
80 % 15 12 27 12.022 100.18 98.99±1.038 1.049
15 12 27 11.792 98.26
15 15 30 14.728 98.19
100 % 15 15 30 14.735 98.23 98.60±0.679 0.689
15 15 30 14.908 99.39
15 18 33 17.850 99.16
120 % 15 18 33 17.921 99.56 99.26±0.263 0.265
15 18 33 17.830 99.06

Recovery Data for Isosorbide Mononitrate

Table 7: Recovery data for Isosorbide Mononitrate

Amount of Amount of Total Mean %
Std. Amount %
Level sample taken standard spiked amount Recovery± SD % RSD
recovered (µg/ml) Recovery
(µg/ml) (µg/ml) (µg/ml) (n=3)
6 4.8 10.8 4.757 99.11
80 % 6 4.8 10.8 4.799 99.99 99.91±0.768 0.768
6 4.8 10.8 4.831 100.64
6 6 12 5.955 99.25
100 % 6 6 12 5.889 99.15 98.91±0.660 0.667
6 6 12 5.960 99.34
6 7.2 13.2 7.136 99.11
120% 6 7.2 13.2 7.211 100.15 99.63±1.517 0.519
6 7.2 13.2 7.173 99.63

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Precision
A. Inter day Precision

Table 8: Inter day Precision for Aspirin and Isosorbide Mononitrate

Aspirin Isosorbide Mononitrate
Conc. (µg/ml) Area Mean± S.D. (n=3) % RSD Conc. (µg/ml) Area Mean± S.D. (n=3) % RSD
15 1304.212±22.187 1.7012 6 509.917±7.755 1.5210
30 3833.323±11.184 0.2917 12 1493.686±5.606 0.3753
52.5 7655.723±12.634 0.1650 21 2986.414±6.541 0.2190

B. Intraday Precision

Table 9: Intraday Precision for Aspirin and Isosorbide Mononitrate

Aspirin Isosorbide Mononitrate
Conc. (µg/ml) Area Mean± S.D. (n=3) % RSD Conc. (µg/ml) Area Mean± S.D. (n=3) % RSD
15 1278.245±12.471 0.975 6 499.921±5.502 1.1005
30 3856.348±40.962 1.062 12 1508.137±19.229 1.2750
52.5 7781.767±65.969 0.847 21 3042.513±28.949 0.9515

C. Repeatability

Table 10: Repeatability for Aspirin and Isosorbide Mononitrate

Aspirin Isosorbide Mononitrate
Sr. No. Area Area
1 3902.219 1524.386
2 3926.795 1533.850
3 3961.108 1547.338
4 3988.198 1557.940
5 3907.418 1526.242
6 3855.734 1519.852
Avg 3923.579 1534.934
SD 46.722 14.822
% RSD 1.190 0.965

Robustness
Result of Robustness for Aspirin

Table 11: Robustness for Aspirin

Sr. No. Aspirin
pH Flow Rate Mobile Phase
+ 0.2 unite -0.2 unite + 0.2 unite -0.2 unite + 0.2 % -0.2 %
1 3657.691 3890.231 3480.436 4072.704 3733.018 3798.006
2 3650.361 3901.84 3504.832 4081.317 3763.998 3851.49
3 3668.617 3933.055 3494.333 4101.736 3730.099 3828.384
Avg 3658.89 3908.375 3493.2 4085.252 3742.372 3825.96
S. D. 9.1868 22.1473 12.2373 14.9107 18.7857 26.8242
% RSD 0.2510 0.5666 0.3503 0.3649 0.5019 0.7011

Result of Robustness for Isosorbide Mononitrate

Table 12: Robustness for Isosorbide Mononitrate

Sr. No. Isosorbide Mononitrate
pH Flow Rate Mobile Phase
+ 0.2 unite -0.2 unite + 0.2 unite -0.2 unite + 0.2 % -0.2 %
1 1428.95 1519.726 1352.945 1595.972 1463.16 1492.709
2 1426.085 1524.264 1369.094 1594.361 1470.465 1504.723
3 1433.207 1536.452 1364.982 1602.307 1457.225 1495.678
Avg 1429.414 1526.814 1362.34 1597.547 1463.617 1497.703
S.D. 3.5836 8.6496 8.3923 4.2005 6.6318 6.2578
% RSD 0.2507 0.5665 0.6160 0.2629 0.4531 0.4178

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Assay
Chromatogram of Standard and Sample

Fig 34: Chromatogram of Standard

Fig 35: Chromatogram of Sample (1)

Fig 36: Chromatogram of Sample (2)

Fig 37: Chromatogram of Sample (3)

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Assay of Marketed Formulation (Tablet)

Table 13: Assay Result of Marketed Formulation

Actual conc. Of Conc. Of Drug Found % of Drug Avg. of % Drug
Drug SD (n=3) % RSD
Drug (µg/ml) (µg/ml) found found
74.122 98.83
Aspirin 75 75.075 100.10 99.85 0.92 0.93
75.472 100.63
29.643 98.81
Isosorbide
30 29.934 99.78 99.91 1.17 1.17
Mononitrate
30.345 101.15

Summary and Conclusion

Summary of RP-HPLC Method Development and Validation

Table 14: Summary of Method Development and Validation

Parameters Aspirin Isosorbide Mononitrate
Detection wavelength(nm) 215
Mobile Phase Phosphate buffer (pH:4): Methanol 30:70 (v/v)
Retention time of Standard 3.773 min 2.553 min
Concentration range (μg/mL) 15-52.5 6-21
Resolution of Standard 8.020
Slope 167.5 166.4
Intercept 1198 500.8
Correlation coefficient 0.999 0.998
Regression Coefficient Equation Y=167.5x-1198 Y=166.4x-500.8
LOD(μg/mL) 1.01 0.77
LOQ(μg/mL) 3.07 2.34
80% 98.99±1.038 99.91±0.768
% Recovery± SD 100% 98.60±0.679 98.91±0.660
120% 99.26±0.263 99.63±0.517
Interday Precision (n=3) 1.7012-0.1650 1.5210-0.2190
Intraday Precision (n=3) 0.975-0.847 1.1005-0.9515
Repeatability (n=6; 50 μg/ml) 3923.580±46.722 1534.934±14.822
+ 0.2 units 0.2510 + 9.1868 0.2507 + 3.5836
pH
− 0.2 units 0.5666 + 22.1473 0.5665 + 8.6496
Robustness + 0.2 units 0.3503 + 12.2373 0.6160 + 8.3923
F.R
(% R. S. D + SD) − 0.2 units 0.3649 + 14.9107 0.2629 + 4.2005
+ 2% 0.5019 + 18.7857 0.4531 + 6.6318
M.P
− 2% 0.7011 + 26.8242 0.4178 + 6.2578
Assay 99.85±0.92 99.91±1.17

Conclusion of RP-HPLC Method Development: test parameters were also performed and were found to be
 Developed HPLC method can resolve all degradant peak within acceptable criteria. The method can be
of both drug. No chromatographic interference from successfully employed for the simultaneous
capsule excipients was found. determination of Aspirin and Isosorbide Mononitrate in
 It is concluded that the developed method is specific. The pharmaceutical formulation.

Summary of Forced Degradation Study

Table 15: Summary of Forced Degradation Study

% Degradation % Degradation
Degradation Volume of Stock Final Dilution
Condition Time (hrs) of API of Sample
Type Solution (mL) Up to (mL)
ASP ISMN ASP ISMN
Acidic N HCl 40 ⁰C 1 5 10 17.42 12.07 15.80 13.60
Alkaline 0.1 N NaOH, 40 ⁰C 1 4 10 12.33 17.33 13.54 18.49
Oxidative 3% H2O2 60 ⁰C 1 3 10 19.62 10.93 19.82 8.95
Thermal 80 ⁰C in Oven 1 10 10 10.17 17.95 11.88 17.14
Photo UV Chamber 1 18 10 10.60 13.90 9.66 14.97

Conclusion of Forced Degradation Study Degradation.

The Forced Degradation Study was carried out and it was
found that Aspirin is most stable in Thermal Degradation. Acknowledgements
Whereas, Isosorbide Mononitrate is most stable in Oxidative I am grateful to the Principal Dr. Sachin B. Narkhede and to
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The Pharma Innovation Journal

associate professors in the Quality Assurance Department of International Journal of Pharmaceutical Sciences and
the Dr. Shailesh V. Luhar, Dr. Dulendra P. Damahe and Dr. Research. 2014; 5(11):4858-4864.
Chirag K. Desai. We also thank the management team for 19. M Sambasiva Rao A, Sunil Kumar Reddy A. Ashok
support and for providing chemicals and equipment for this Kumar. New RP-HPLC method for the simultaneous
work. We also thank Sidmak laboratories (India) pvt. Ltd. estimation of Rosuvastatin and Aspirin in pharmaceutical
Valsad, Gujarat, India for providing Aspirin and Isosorbide dosage form. International Journal of Pharmaceutical
Mononitrate give free of charge. Investigations and Research. 2016; 3(1):42-63.
20. Shital M, Patel CN. Patel, VB Patel. Stability indicating
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