tpi 2018; 7(4): 217-234 and validation for simultaneous … · a solution containing 1000μg/ml...

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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



Gujarat, India

Dr. Shailesh V Luhar



Gujarat, India

Dr. Sachin B Narkhede



Gujarat, India

Correspondence

Nidhi S Patel



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 C9H8O4.

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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The Pharma Innovation Journal

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

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



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

0.1N HCl was added for neutralization and diluted up to the

mark with Mobile Phase.

Oxidative Degradation

Aspirin, Isosorbide Mononitrate and Formulation




three different 10mL volumetric flask; to it 2mL of 3% H2O2

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


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


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



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



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

B. Intraday Precision

Intraday Precision was performed at three different

concentrations of binary mixture of ASP and ISMN thrice on

the same day. The concentrations of the binary mixtures are

given below.

Lower concentration (50%)-ASP-15µg/mL + ISMN-6µg/mL


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.


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.

Results and Discussion

Development of Rp-Hplc Method

Selection of Detection Wavelength

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


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Trial 5


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

Parameters Retention Time (min)

(n=6)

Tailing Factor

(T) (n=6)

Resolution

(R)

Theoretical Plate

(N)±SD (n=6)

Specifications - NMT 2 MT 1.5 NLT 2000

Std. ASP 3.7488±0.0034 1.4083±0.0166

8.1173

7243±49.0445

ISMN 2.5271±0.0016 1.3351±0.0235 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


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

Stress Condition % Degradation of API % Degradation of pharmaceutical dosage form

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

Level

Amount of

sample taken

(µg/ml)

Amount of

standard spiked

(µg/ml)

Total

amount

(µg/ml)

Std. Amount

recovered (µg/ml)

%

Recovery

Mean %

Recovery± SD

(n=3)

% RSD

80 %

15 12 27 11.824 98.53

98.99±1.038 1.049 15 12 27 12.022 100.18

15 12 27 11.792 98.26

100 %

15 15 30 14.728 98.19

98.60±0.679 0.689 15 15 30 14.735 98.23

15 15 30 14.908 99.39

120 %

15 18 33 17.850 99.16

99.26±0.263 0.265 15 18 33 17.921 99.56

15 18 33 17.830 99.06

Recovery Data for Isosorbide Mononitrate

Table 7: Recovery data for Isosorbide Mononitrate

Level

Amount of

sample taken

(µg/ml)

Amount of

standard spiked

(µg/ml)

Total

amount

(µg/ml)

Std. Amount

recovered (µg/ml)

%

Recovery

Mean %

Recovery± SD

(n=3)

% RSD

80 %

6 4.8 10.8 4.757 99.11

99.91±0.768 0.768 6 4.8 10.8 4.799 99.99

6 4.8 10.8 4.831 100.64

100 %

6 6 12 5.955 99.25

98.91±0.660 0.667 6 6 12 5.889 99.15

6 6 12 5.960 99.34

120%

6 7.2 13.2 7.136 99.11

99.63±1.517 0.519 6 7.2 13.2 7.211 100.15

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


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


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)



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Assay of Marketed Formulation (Tablet)

Table 13: Assay Result of Marketed Formulation

Drug Actual conc. Of

Drug (µg/ml)

Conc. Of Drug Found

(µg/ml)

% of Drug

found

Avg. of % Drug

found SD (n=3) % RSD

Aspirin 75

74.122 98.83

99.85 0.92 0.93 75.075 100.10

75.472 100.63

Isosorbide

Mononitrate 30

29.643 98.81

99.91 1.17 1.17 29.934 99.78

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

% Recovery± SD

80% 98.99±1.038 99.91±0.768

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

Robustness

(% R. S. D + SD)

pH + 0.2 units 0.2510 + 9.1868 0.2507 + 3.5836

− 0.2 units 0.5666 + 22.1473 0.5665 + 8.6496

F.R + 0.2 units 0.3503 + 12.2373 0.6160 + 8.3923

− 0.2 units 0.3649 + 14.9107 0.2629 + 4.2005

M.P + 2% 0.5019 + 18.7857 0.4531 + 6.6318

− 2% 0.7011 + 26.8242 0.4178 + 6.2578

Assay 99.85±0.92 99.91±1.17

Conclusion of RP-HPLC Method Development:

Developed HPLC method can resolve all degradant peak

of both drug. No chromatographic interference from

capsule excipients was found.

It is concluded that the developed method is specific. The

test parameters were also performed and were found to be

within acceptable criteria. The method can be

successfully employed for the simultaneous

determination of Aspirin and Isosorbide Mononitrate in

pharmaceutical formulation.

Summary of Forced Degradation Study

Table 15: Summary of Forced Degradation Study

Degradation

Type Condition

Volume of Stock

Solution (mL) Time (hrs)

Final Dilution

Up to (mL)

% Degradation

of API

% Degradation

of Sample

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

The Forced Degradation Study was carried out and it was

found that Aspirin is most stable in Thermal Degradation.

Whereas, Isosorbide Mononitrate is most stable in Oxidative

Degradation.

Acknowledgements

I am grateful to the Principal Dr. Sachin B. Narkhede and to

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associate professors in the Quality Assurance Department of

the Dr. Shailesh V. Luhar, Dr. Dulendra P. Damahe and Dr.

Chirag K. Desai. We also thank the management team for

support and for providing chemicals and equipment for this

work. We also thank Sidmak laboratories (India) pvt. Ltd.

Valsad, Gujarat, India for providing Aspirin and Isosorbide

Mononitrate give free of charge.

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tpi 2018; 7(4): 217-234 and validation for simultaneous … · a solution containing 1000μg/ml...

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