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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.
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
Aspirin and Isosorbide Mononitrate is obtained from Sidmak standard solution and system suitability parameters were
laboratories (India) pvt. Ltd. Valsad. calculated at the start of study for each parameter.
parameters 0.1N HCl was added for neutralization and diluted up to the
a) pH of mobile phase: ±0.2 mark with Mobile Phase.
b) Flow rate: ±0.2 ml/min
c) Change in the ratio of component in the mobile phase: Oxidative Degradation
±2%. Aspirin, Isosorbide Mononitrate and Formulation
Oxidative Degradation
Stability studies 1mL of Standard Stock-II Solution of Aspirin, Isosorbide
Stability Studies was carried out on the drug in order to check Mononitrate and Sample Stock Solution were transferred in
the stability of the drug by providing various stress conditions three different 10mL volumetric flask; to it 2mL of 3% H 2O2
like acid, base, oxidation and thermal degradation compared at 60°C was added and kept for 6hrs and then diluted up to the
with normal conditions. The purpose of force degradation mark with Mobile Phase.
method is to provide evidence that the analytical method is
efficient in determination of drug substances in commercial Thermal degradation
drug product in the presence of its degradation products. Aspirin, Isosorbide Mononitrate and Formulation
Oxidative Degradation
Acidic hydrolysis Weighed 50mg of both drugs and 50mg equivalent weight of
Take 2 ml solution of Aspirin 1000μg/ml and Isosorbide tablet powder was taken in a clean and dry Petri dish and
Mononitrate 1000μg/ml, 2 ml of 0.1M HCl was added. The covered with Aluminium foil. Petri dish was kept in oven at
solution was heated for 4hr at 40 °C and transferred to a 10ml 80 ºC for 24hr. An Accurately weighed 10mg of dry heated
volumetric flask, cooled, neutralized by 0.1M NaOH and drug was transferd to 100mL volumetric flask. It was
diluted up to mark with methanol to get final concentration dissolved using mobile phase and final volume was made up
100μg/ml of Aspirin and 100μg/ml of Isosorbide Mononitrate. to 100mL. Solution of 30μg/mL of Aspirin and 12μg/mL of
Isosorbide Mononitrate was prepared from above solution.
Alkaline hydrolysis
Take 2 ml solution of Aspirin 1000μg/ml and Isosorbide Photo degradation
Mononitrate 1000μg/ml, 2 ml of 0.1M NaOH was added. The Aspirin, Isosorbide Mononitrate and Formulation Photo
solution was heated for 4hr at 40°C and transferred to a 10ml degradation
volumetric flask, cooled, neutralized by 0.1M HCl and diluted 1mL of Standard Stock-II Solution of Aspirin, Isosorbide
up to mark with methanol to get final concentration 100μg/ml Mononitrate and Sample Solution were transferred in three
of Aspirin and 100μg/ml of Isosorbide Mononitrate. different 10mL volumetric flask was kept in Photo stability
chamber for 24hrs and then diluted up to the mark with
Oxidative degradation Mobile Phase.
Take 2 ml solution of Aspirin 1000μg/ml and Isosorbide
Mononitrate 1000μg/ml, 2 ml 3% H2O2 was added at room Method Validation
temperature for 6hours at 60 °C and transferred to a 10ml Linearity and Range
volumetric flask, cooled diluted up to mark with methanol to The linearity is expressed in terms of correlation co-efficient
get final concentration 100μg/ml of Aspirin and 100μg/ml of of linear regression analysis. The linearity of Aspirin and
Isosorbide Mononitrate. Isosorbide Mononitrate was found to be in between 15 to
52.5μg/mL and 6 to 21μg/mL.
Thermal degradation
Take 2 ml solution of Aspirin 1000μg/ml and Isosorbide Limit of Detection and Limit of Quantitation
Mononitrate 1000μg/ml, heat the solution for 24hr at 80 °C Limit of Detection (L. O. D.)
and transferred to a 10ml volumetric flask, cooled diluted up The L. O. D. was estimated from calibration curves.
to mark with methanol to get final concentration 100μg/ml of LOD=3.3 × (S. D. /Slope)
Aspirin and 100μg/ml of Isosorbide Mononitrate.
Where, S. D. =Standard deviation of the Y-intercept of the
Forced Degradation Study calibration curve.
Acid Hydrolysis Slope=Mean slope of calibration curve.
Aspirin, Isosorbide Mononitrate and Formulation Acid
Degradation Limit of Quantitation (L.O.Q.)
1mL of Standard Stock-II Solution of Aspirin, Isosorbide The L. O. Q. was estimated from calibration curve.
Mononitrate and Sample Stock Solution were transferred in
three different 10mL volumetric flask; to it 2mL of 0.1 NHCl LOQ=10 × (S.D./Slope)
at 40 °C was added and kept for 4hrs and then 2mL of 0.1N Where, S. D=Standard deviation of the Y-intercept of
NaOH was added for neutralization and diluted up to the mark calibration curve.
with Mobile Phase. Slope=Mean slope of calibration curve.
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Trial 2
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 Standard
Chromatogram of Formulation
Alkali Hydrolysis
Oxidative Degradation
Thermal Degradation
Photo Degradation
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Method Validation
Linearity and Range
Chromatogram of Linearity
Accuracy
Recovery Data for Aspirin
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Precision
A. Inter day Precision
B. Intraday Precision
C. Repeatability
Robustness
Result of Robustness for Aspirin
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Assay
Chromatogram of Standard and Sample
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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.
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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