Development and Validation of Difference Spectrophotometric Method for the Quantitative Estimation of Mesalamine in Bulk Drug and Dosage forms.

 

P Ravi Kumar*, Y Padmavathi , P Niveditha ,  N. Raghavendra Babu.

Department of Pharmaceutical Analysis and Quality Assurance, G. Pulla Reddy College of Pharmacy, Osmania University, Mehdipatnam, Hyderabad, Telangana, India.

*Corresponding Author E-mail: pravikumar@gprcp.ac.in

 

ABSTRACT:

A sensitive, precise, economic spectrophotometric method i.e. a difference spectroscopic method was developed for the estimation of mesalamine in bulk and dosage (tablet and suppository) forms. Difference spectroscopic method was developed by measuring the difference absorbance of the drug in 0.1N HCl and in 0.1 N NaOH showing maximum absorbance at 330nm. Beer’s law was obeyed in the concentration range of 5-25 μg/ml. The LOD and LOQ were found to be 0.3 μg/ml and 0.9 μg/ml respectively. Correlation coefficient was observed as 0.999. The method was validated as per ICH guideline Q2R1 and the results were within the official limits. The developed difference spectroscopic method was applied for quantitative analysis of the drug in bulk and pharmaceutical dosage forms.

 

KEYWORDS: Mesalamine, COX inhibitor, Difference spectroscopic method, ICH guidelines.

 


 

INTRODUCTION:

Mesalamine is used to treat inflammatory bowel disease and some forms of arthritis. It acts by inhibiting the production of cyclo-oxygenase and prostaglandin, thromboxane synthetase, platelet activating factor synthetase, and interleukin-1 by macrophages. so reduces the acute inflammatory response in inflammatory bowel disease. Production of immunoglobulin by plasma cells is also decreased. It also used to treat ulcerative colitis, proctitis and crohn's disease.1

Mesalamine is chemically known as 5-amino-2-hydroxy benzoic acid. Molecular weight of the drug is CHNO and molecular weight is 153.14. It is white powder slightly soluble in water, soluble in dilute HCL and in dilute alkali hydroxides. Practically insoluble in chloroform, ether and butyl alcohol. It should be preserved in tight, light resistant container2. Absorption of mesalamine is similar in fasted and fed subjects. The absorbed mesalamine is rapidly acetylated in the gut mucosal wall and by the liver. It is excreted mainly by the kidney as N-acetyl-5-aminosalicylic acid.3

 

Figure 1chemicalmstructure of mesalamine.

 

A substance whose spectrum is unaffected by changes of pH may be determined by a difference spectrophotometric procedure if it can be quantitatively converted by means of a suitable reagent to a chemical species that has different spectral properties to its unreacted parent substance. The A between equimolar solutions of the unreacted substance and its derivative is free of interference if the irrelevant absorption is unaffected by the reagent. The measured value in a quantitative difference spectrophotometric assay is the ∆A at any suitable wavelength measured to the baseline, e.g. ∆A1 at λ1 or amplitude between an adjacent maximum and minimum, e.g. ∆A2 at λ2 and  λ1.  At λ1 ∆A= Aalk – Aacid. Where Aalk and Aacid are the individual absorbances at λ1 in 0.1M sodium hydroxide and 0.1M hydrochloric acid solution respectively. If the individual absorbances, Aalk and AAcid are proporational to the concentration of the analyte and pathlength, the ∆A also obeys the Beer-Lambert Law and a modified equation may be written as  ∆A=∆ abc 4-8.

 

MATERIALS AND METHODS:

Chemicals and Reagents:

Mesalamine working standard was a gift sample form Neuheit pharmaceuticals, Hyderabad. Hydrochloric acid (AR grade) obtained from Sisco Labs, Mumbai and Sodium hydroxide (AR grade) procured from sd fine chemicals Ltd (SDFCL), Mumbai. Pentasa-500 mg tablets manufactured by Ferring Pharmaceuticals and Mesacol-500 suppositories manufactured by Sun Pharma Ltd. were purchased from local market.

 

Instruments:

UV-Visible spectrophotometer (Shimadzu 1800), Digital balance (Shimadzu BL-220H), Ultrasonic bath sonicator (PCI Analytics 6.5li200H).

 

Method development:9-12

Preparation of working standard solutions:

100 mg of mesalamine weighed accurately and transferred into a 100 volumetric flask, few ml of 0.1N HCl was added to dissolve the drug. Volume was made up to the mark with the same solvent to get 1000 μg/ml solution. 1.0 ml of above solution taken into another 100ml volumetric flask and diluted to 100ml with same solvent to get 100 μg/ml solution. Same procedure was followed to prepare drug solution in 0.1N NaOH.

 

Determination of Absorption maximum:

1.0 ml of 100μg/ml solution in 0.1N NaOH was taken into two 10ml volumetric flask and made to 10ml with same solvent to get 10μg/ml solution. 10μg/ml solution of drug in 0.1N HCl also prepared in the same manner. NaOH solution placed in sample cell and Hcl solution was placed in reference cell of the instrument, scanned in the range of 200-400nm, difference absorption spectrum was recorded and 330nm selected as absorption maximum. The spectrum shown in the figure2.

 

 

Figure 2 Difference absorption spectrum of mesalamine

 

 

Figure 3 Overlay difference absorption specta of mesalamine(5-25μg/ml)

 

Construction of calibration curve:

From the working standard stock solution of mesalamine (100µg/ml), appropriate aliquots like 0.5, 1.0, 1.5, 2.0, 2.5ml solutions were pipetted into 10ml volumetric flasks. The solution in each flask was made up with 0.1N NaOH in one set and with 0.1N HCL in another set to obtain working standard concentration ranging from 5-25µg/ml. The difference absorbance of these solutions was measured at 330nm by taking 0.1N NaOH prepared concentrations in sample cell and 0.1 N HCL prepared concentrations in reference cell. Overlay spectra of all concentrations is shown in figure 3. The calibration curve was constructed between concentration of drug and absorbance and is shown in figure 4 and calibration curve data is represented in table1.

 

 

Figure 4 Calibration curve of mesalamine working standard

 

Table 1 Calibration curve data of mesalamine

S. No

Concentration (μg/ml)

Absorbance*

1

5

0.156

2

10

0.312

3

15

0.470

4

20

0.630

5

25

0.781

*Average of 3 determinations

Method validation:13

The developed method was validated as per ICH Q2 (R1) guidelines.

 

Linearity:

Five points calibration curve were obtained in a concentration range from 5-25μg/ml for mesalamine. The response of the drug was found to be linear in the investigation concentration range and the linear regression equation was y = 0.032x with correlation coefficient 0.999.

 

Limit of Detection (LOD) and Limit of Quantization (LOD):

The LOD and LOQ reported as a means to analyse the sensitivity. The LOD and LOQ for mesalamine were found to be 0.3μg/ml and 0.9μg/ml respectively and were calculated by using standard deviation method.

 

Precision:

The precision of the developed analytical method was assessed for repeatability, intra-day precision and inter-day precision. Repeatability is the precision of the instrument and was checked by repeatedly measuring the absorbance of mesalamine solution of 15μg/ml concentration. The intraday precision was determined by analysing three concentrations (12μg/ml, 15μg/ml and18μg/ml) i.e at three levels (80%, 100% and120%), 3 times on the same day. Interday precision was also performed in same manner but on different days. All the results were reported in terms of relative standard deviation and expressed in table 2, 3 and 4.

 

Acceptance criteria:

% RSD of repeatability, intra and inter day precision should not be more than 2.

 

Table 2 Repeatability data for mesalamine

S. No

Concentration (μg/ml)

Absorbance

1

15

0.477

2

15

0.473

3

15

0.479

4

15

0.476

5

15

0.479

6

15

0.479

Avg

 

0.477

SD

 

0.00098

%RSD

 

0.25


 

Table 3 Intra-day precision data for mesalamine

S. No

Concentration (μg/ml)

Mean Absorbance*

Mean±SD

%RSD

Morning

Afternoon

Evening

1

12

0.368

0.377

0.371

0.372±0.003

0.80

2

15

0.442

0.451

0.459

0.450±0.006

1.33

3

18

0.525

0.529

0.536

0.530±0.004

0.75

*Average of 3 determinations

 

Table 4 Inter-day precision data for mesalamine

S. No

Conc. (μg/ml)

Mean Absorbance

Mean*±SD

%RSD

Morning

Afternoon

Evening

1

12

0.372

0.381

0.392

0.381±0.006

1.57

2

15

0.450

0.462

0.469

0.460±0.008

1.73

3

18

0.530

0.543

0.552

0.541±0.008

1.47

*Average of 3 determinations

 


The results shows that all the calculated %RSD values are below 2%, therefore the method is precise. This indicates that the method is repeatable and reproducible on different days.

 

Accuracy:

The analytical method accuracy is the nearness of the results obtained against the real values at each level of mesalamine concentration. The results obtained for accuracy studies for the drug substance reported in terms of %RSD and are represented in table 5. The accuracy for drug product was assessed in terms of recovery by determining the standard concentration and added known concentration to the sample. The test was done by spiking the tablet powder with pure mesalamine at three different levels (80%, 100% and 120%).The results are shown in table 6.

 

Acceptance criteria:

%RSD of accuracy for the drug substance should not be more than 2. % recovery for drug product should be 95%-105%.


 

Table 5 Accuracy data for mesalamine drug substance

Concentration (μg/ml)

Absorbance

Mean

Standard Deviation

%RSD

 

16

0.481

0.489

0.0085

1.73

0.488

0.498

 

20

0.610

0.606

0.004

0.6

0.606

0.602

 

24

0.677

0.677

0.005

0.07

0.678

0.678

From the above data it is found that the %RSD values are within the acceptable limits indicating that the method is accurate.

 

Table 6 Recovery data for mesalamine drug product (pentasa tablet 500 mg)

S. no

Amount of standard (μg/ml)

Amount of sample (μg/ml)

Total concentration (μg/ml)

Total concentration found ( μg/ml)

% Recovered

1

20

16

36

35.5

98.6

2

20

20

40

39.8

99.7

3

20

24

44

43.4

98.6

*Average of 3 determinations

 


Table 7 optimized UV conditions for mesalamine

λmax

330nm

Beer’s law range

5-25 μg/ml

Sandell’s sensitivity

0.285μg/ml/cm3

Molar absorptivity

3.6 X 106 lit/mol/cm

Limit of detection (μg/ml)

0.3 μg/ml

Limit of quantification (μg/ml)

0.9 μg/ml

Regression equation (y)

y = 0.032x

Coefficient of correlation (r2)

0.999

 

Assay of mesalamine tablets and Suppositories:

10 tablets mesalamine (PENTASA-500mg) were weighed, triturated and powder equivalent to 100 mg taken into 100 ml volumetric flask, distilled water added to dissolve the drug, and volume made upto the mark to get 1000μg/ml. Serial dilutions were carried out separately in 0.1 N HCl and 0.1 N NaOH to obtain 20μg/ml solution. The difference absorbance of three dilutions of 20μg/ml concentrations was measured, average is considered and amount of drug in tablet and its percentage was calculated. 10 suppositories, 1 in each tube containing 10 ml of water 370 C. After stirring in a constant temperature water bath the tubes are withdrawn at 10 minutes intervals then immediately placed in an ice bath to solidify the melted excipient. The supernatant liquid is collected and diluted to get 100μg/ml solution. Further dilutions and analysis carried out as in case of tablet. The results are shown in Table 8.

 

Acceptance criteria: 95-105%

 

Table 8 Assay of mesalamine drug products

Formulation

Labelled amount (mg)

Amount obtained (mg)

% Recovery

PENTASA (Tablet)

500

492

98

MESACOL (Suppository)

500

489

97

 

CONCLUSION:

A sensitive, precise and accurate difference spectroscopic method was developed for the quantitative estimation of mesalamine pure drug as well as in formulation. A linear response was observed between 5-25μg/ml concentration range at the absorption maximum of 330nm.The coefficient of correlation was observed as 0.999. The developed method validated as per ICH guidelines and successfully applied for the assay of tablets and suppositories. The method can be used for routine analysis of mesalamine in bulk and in formulations.

 

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Received on 06.07.2017                Accepted on 16.09.2017               

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2017; 7(4): 225-228.

DOI: 10.5958/2231-5675.2017.00036.9