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PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES

DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR

DETERMINATION OF MESALAMINE IN TABLET DOSAGE FORMS

Venugopal Darak1*, Arvind B Karadi1, S Appal raju1, MD Arshad1 and Ashok L Ganure2

1Department of Pharmaceutical Analysis, H.K.E.S’s College of Pharmacy, Sedam Road, Gulbarga-

585104.(Karnataka) 2K. J. College of Pharmacy, Kalol (Gujrat)

ABSTRACT A simple, Rapid and Reproducible HPLC method has been developed for the estimation of Mesalamine in bulk drug and its Pharmaceutical dosage forms using RP C18 column. The mobile phase consists of Acetonitrile and water in the ratio of 60:40v/v and was pumped at a flow rate of 0.6ml/min at 25±1oc. The detection was carried out at 330nm and the calibration curve was linear in the range of 20-100μg/ml, Retention time was found to be 3.09min for run time of 5min. The method was statistically validated for its linearity, Precision and accuracy. Intra and Inter-day variation study was carried out and found to be less than 3% showing reasonable precision of the assay method. Parameters of validation obtained prove the accuracy of the method and its applicability for the determination of Mesalamine in tablet dosage formulations. Keywords: Mesalamine, HPLC, Mobile phase. INTRODUCTION

Chemically Mesalamine is 5-amino salicylicacid[1-4], belongs to a group of anti-

inflammatory drug. It is structurally related to salicylates and active in inflammatory

bowel disease[5-6].Tablet formulations containing 250, 400 and 500 mg are available in

the market. It is official in USP. Literature survey reveales that few visible

spectrophotometric methods[7-9] , HPLC methods have been reported for estimation of

Mesalamine in single component formulations[10-12]. Hence, an attempt has been made to

develop simple HPLC method for its determination in pharmaceutical formulations with

good accuracy, precision and economy.

Experimental

Instrumentation

A Shimadzu HPLC instrument, equipped with a Luna C18 reverse phase column

(250mm x 4.6mm.,5μ), an LC-20AT pump and variable wavelength programmable

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UV/visible detector SPD-20A, was employed in this study. Chromatographic analysis

and data acquisition was monitored by using spinchrome software. A 20 μl Hamilton

injection syringe was employed for sample injection.

Degassing of mobile phase was done by using a ultrasonic bath sonicator,

electronic balance (Shimadzu) was used for weighing the materials. Class ‘A’ Broil glass

ware (Borosil, India) was employed for volumetric and general study in entire study.

Drug

The reference sample of Mesalamine was supplied by Cosmo Pharma Pvt Ltd,

Goa. The Branded tablets formulation of Mesalamine (Walasa, Mesacol) were purchased

from local market.

Chromatographic Condition:

The contents of the mobile phase were Acetonitrile and Water in the ratio of

60:40v/v. The contents of the mobile phase were filtered before use through 0.45μ

membrane filter. The flow rate of the mobile phase was maintained at 0.6ml/min. The

column temperature was maintained at 25±1oc and detection was carried out at 330nm by

UV detector. The run time was set at 5min and the volume of the injection loop was 20μl.

Prior to injection of the drug solution the column was equilibriated for at least 30min

with mobile phase flowing through the system. The data acquired, stored and analyzed

with software spinchrome (Shimadzu).

Procedure:

About 50mg of Mesalamine was accurately weighed and dissolved in HPLC

grade water in a 50ml volumetric flask so as to give 1mg/ml. subsequent dilutions of

solution was made with mobile phase to get concentration of 20-100μg/ml of

Mesalamine. The standard solution prepared above are injected 5 times in to the column

at a flow rate of 0.6ml/min. The peak areas of drug concentration were calculated. The

regression equation was used to estimate the amount of Mesalamine in pharmaceutical

dosage forms (Tablets).

Mesalamine solution containing 20, 40 and 60μg/ml were subjected to the

proposed HPLC analysis for finding out intra and inter-day variations. The recovery

studies were carried out by adding known amount of Mesalamine to the pre analyzed

samples and subjecting them to the proposed HPLC method.

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Assay of Mesalamine in Tablets:

Twenty tablets each containing 400mg were taken weighed and powdered. An

tablet powdered equivalent to 100mg was accurately weighed and transferred to 100ml

volumetric flask containing 50ml HPLC grade water. The contain of flask was sonicated

for 20min to dissolve Mesalamine and the volume was made up to the 100ml with water

and resulting solution was filtered through 0.45μ membrane filter. 2ml of this solution

was taken diluted to 100ml with mobile phase. This (20μl) was injected 5times into the

column. The mean peak area of 5 such determinations were calculated and the drug

contents in the tablets was quantified using regression equation.

Figure 1 A Typical Chromatogram of Mesalamine Standard 20µg/ml

Figure 2 A Typical Chromatogram of Mesalamine Formulation 20µg/ml

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RESULTS AND DISCUSSION The present study was carried out to develop simple sensitive, precise and

accurate reverse phase HPLC method for the analysis of Mesalamine in pharmaceutical

dosage forms. The column pressure varied from 96kg/cm2. The retention time for

Mesalamine was 3.09min for a runtime of 5min. Each sample was injected 5 times in to

the column and same retention time were obtained in all cases. The peak areas of

different concentration set up as above were calculated and presented in Table 1. A good

linear relationship (r=0.9996) was obtained between concentration of Mesalamine and the

respective peak areas. Calibration curve was found to be in the concentration range of 20-

100μg/ml (Table-2). when solution containing 20, 40 and 60μg/ml were analyzed by the

proposed HPLC method for finding out intra and inter-day variations. A low % RSD

variation was observed 0.24 (Table-3). This shows the present HPLC method was highly

precise. The amount of Mesalamine from preanalysed sample containes known amount of

the drug are shown in Table 4 about 99.76% Mesalamine could be recovered from the pre

analyzed sample indicating high accuracy of the proposed method.

The absence of additional peak indicates no interference of the excipients used in

the tablet formulations. The tablets were found to contain 99.37 to 99.54% (Table-5). The

low %RSD indicates reproducibility of the assay in the tablet dosage forms. The

proposed method is highly accurate, precise and simple.

Figure 3 Linearity Plot of Mesalamine by HPLC Method

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TABLE 1: CALIBRATION OF THE PROPOSED HPLC METHOD

S.NO Concentration of Mesalamine (μg/ml)

Mean Peak Area (n=6)

1 20 221.38 2 40 440.18 3 60 690.58 4 80 950.11 5 100 1183.24

TABLE 2: REGRESSION CHARACTERISTICS OF THE LINEARITY PLOT OF

MESALAMINE

Parameters Method

Linearity Range 20-100µg/ml

Slope (a) 12.168

Intercept (b)* -32.970

Correlation coefficient (r ) 0.9996

Standard deviation 0.8829

% RSD 0.2004

No. of Theoretical plates 3441

LOD 0.2394

LOQ 0.7255

HETP 0.0435

Range of errors**

Confidence limits with 0.05 level Confidence limits with 0.01 level

0.9338 1.3816

*Y=bC+a, where Y is the absorbance unit and C is the concentration of Mesalamine in g/ml, **Average

of six determinations,

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TABLE 3: INTRA AND INTER-DAY PRECISION FOR MESALAMINE ASSAY

IN PHARMACEUTICAL DOSAGE FORMS BY THE PROPOSED HPLC

METHOD

Intra-day Precision Inter-day Precision Concentration of

Mesalamine (μg/ml)

Mean amount

found(n=3)

Percent amount found

Percent RSD

Mean amount

found(n=3)

Percent amount found

Percent RSD

20 19.98 99.90 0.18 19.92 99.60 0.24

40 40.03 100.07 0.26 39.86 99.65 0.32

60 59.96 99.93 0.42 59.90 99.83 0.47

TABLE 4: ACCURACY DATA (TRIPLICATE VALUES AT DIFFERENT

CONCENTRATION LEVELS)

Amount taken (μg/ml)

Amount found (μg/ml)

Percent recovery Mean % recovery

% RSD

20+10=30 29.98 99.93

20+10=30 29.86 99.53

20+10=30 29.95 99.83

99.76

0.20

20+20=40 40.09 100.22

20+20=40 39.95 99.87

20+20=40 39.97 99.92

100.00

0.18

20+30=50 49.96 99.92

20+30=50 49.92 99.84

20+30=50 49.84 99.68

99.88

0.12

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TABLE 5: EVALUATION OF MESALAMINE IN TABLET DOSAGE

FORMULATIONS BY PROPOSED HPLC METHOD

*mean of six determinations, M1= Mesacol (Uni pharma ), M2= Walasa (Wallace Pharma)

CONCLUSION

The proposed HPLC method for the estimation of Mesalamine is simple,

sensitive, accurate and can be used for the routine quality analysis of the drug in bulk as

well as its pharmaceutical formulations.

ACKNOWLEDGEMENTS

The Authors are thank full to Principal, Management, HKES’s College of

Pharmacy, Gulbarga Karnataka (India) for providing necessary laboratory facilities to

carry out the present work and Cosmo Pharma Pvt Ltd, Goa for providing gift sample of

drug for research.

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5. Bertam G Katzung: Basic and Clinical Pharmacology, 9th edn. Mc. Graw Hill,

Singapore 2007; 1030.

Label Claim (mg)

Amount of drug obtained by proposed method (mg)

% Recovery*

M1 400 398.14 99.37

M2 400 399.92 99.54

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6. Tripathi KD: Essentials of Medical Pharmacology. Jaypee Brothers Medical

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For Correspondence: Venugopal Darak Department of Pharmaceutical Analysis, H.K.E.S’s College of Pharmacy, Sedam Road, Gulbarga-585104.(Karnataka) Email: Venu.darak@gmail.com