INTRODUCTION:

Mesalamine (5-aminosalicylic acid, 5-ASA, Fig 1) is used for its local effects in the treatment of inflammatory bowel disease, including ulcerative colitis and Crohn’s disease^1-2. Despite the fact that it has been used for over 50 years, the mechanism of action of this drug remains uncertain. 5-ASA has been shown to be a potent scavenger of reactive oxygen species that play a significant role in the pathogenesis of inflammatory bowel disease, inhibition of natural killer cell activity, inhibition of antibody synthesis, inhibition of cyclo-oxygenase and lipoxygenase pathways and impairment of neutrophil function^3-4. Literature reveals that that very few methods were developed for the estimation of mesalamine in pure and pharmaceutical dosage form. A HPLC method adopted by the British Pharmacopoeia (BP) is based on the mobile phase containing glacial acetic acid, methanol and methyl isobutyl ketone (10: 40: 50 v/v)⁵. A HPLC method available in United States Pharmacopoeia (USP) is based on the mobile phase containing tetrabutylammonium hydrogen sulphate as an ion-pairing agent, which shortens column life. Moreover, mobile phase preparation requires tedious procedures⁶. The spectrophotometric method was developed for the determination of MES in pure and its pharmaceutical formulations^7-8. Very few HPLC methods were developed for simultaneous determination of 5-aminosalicylic acid and its metabolite in human plasma⁹ and nitrosation method for the quantitization of MES in coated tablets¹⁰.In this present study, we developed simple and sensitive spectrophotometric method for Mesalamine.

Figure 1: Mesalamine

EXPERIMENTAL:

Instrumentation:

Spectral and absorbance measurements were made on SHIMADZU-1601 spectrophotometer by using 1 cm quartz cells. Ohaus Balance was used for weighing the samples. Commercially available tablets of Mesalamine were procured from the local market and estimated.

Optimization Scanning and determination of maximum wavelength (λ max):

In order to ascertain the wavelength of maximum absorption (λ max) of the drug, solution of the drug (10 μg/ml) in Phosphate buffer (pH- 6.8) was scanned using spectrophotometer within the wavelength region of 400 – 200 nm against Phosphate buffer (pH- 6.8) as blank. The resulting spectra were shown in fig (2) and the absorption curve showed characteristic absorption maxima at 331nm for Mesalamine.

Sample: Mesalamine (10 μg/ml)

Reference: Phosphate Buffer (pH-6.8)

Instrument: Shimadzu-1601 UV –Visible Spectrophotometer.

Figure 2: UV Spectrum for Mesalamine (10 μg/ml) at 331nm.

METHOD:

Preparation of Phosphate buffer solution (pH-6.8):

Dissolve 28.80gm of disodium hydrogen phosphate and 11.45gm of potassium dihydrogen phosphate in sufficient water to produce 1000ml.

Preparation of Stock Solutions:

Standard stock solution was prepared by dissolving 25 mg of drug in 25 ml of Phosphate Buffer (pH-6.8) to get concentration of 1mg/ml (1000 μg/ml) solutions.

Preparation of Working Standard Solutions and construction of standard graph:

The prepared stock solution was further diluted with Phosphate Buffer (pH-6.8) to get working standard solutions of 5 μg/ml to 125 μg/ml of mesalamine to construct Beer’s law plot for pure drug, different aliquots of Mesalamine were taken and diluted to 10 ml with Phosphate Buffer (pH-6.8). The absorbance was measured at 331 nm, against Phosphate Buffer (pH-6.8) as blank. The results were shown in table (1). The standard graph was plotted by taking concentration of drug on x-axis and absorbance on y-axis and was shown in Fig. (3) The drug has obeyed Beer’s law in the concentration range of 5-100μg/ml.

Figure3 : Standard Calibration curve of Masalamine

Table 1: Linearity Table for Mesalamine

Concentrations ( µgm/ml)	Absorbance
5	0.1387
10	0.2721
15	0.4407
20	0.5841
25	0.7124
30	0.8395
40	1.1573
50	1.395
60	1.675
70	1.955
80	2.235
90	2.515
100	2.795

Validation:

Precision:

The precision of the proposed method was ascertained by actual determination of ten replicates of fixed concentration of the drug within the Beer’s range and finding out the absorbance by the proposed method. From this absorbance, Mean, Standard deviation, % RSD was calculated. The readings were shown in Table (2).

Table 2: Precision Readings

Concentrations ( µgm/ml )	Absorbance	Statistical analysis
20	0.5841	Mean= 0.5844
20	0.5845	S.D= 0.00209
20	0.5842	%RSD= 0.3576
20	0.5844
20	0.5843
20	0.5845
20	0.5848
20	0.5846
20	0.5841
20	0.5845

Table 3 Optical Characteristics

Beer’s Law limit (μg/mL) 5-100 µgm/ml

Sandell’s sensitivity 0.03675

(μg/cm²/0.001absorbance unit)

Molar extinction coefficient 4.13×10³

(1 mole^-1 c.m^-1)

% Relative standard deviation 0.3576

Confidence limits

95% Confidence limits 0.1296

99% Confidence limits 0.1706

Correlation coefficient 0.999

Regression equation (Y*)

Slope (a) 0.027

Intercept (b) 0.012

* Y= a + bC where C is the concentration of mesalamine and Y is the peak area

Table 4: Recovery from the Formulation

Formu lation

Labeled amount (mg)

UV Method

Mesacol

(Tablets)

400 mg

Mean ± s.d

(amount mg recovered)

%Drug recovered

% RSD

398.88±1.134

99.72±0.274

0.274

Table 5: Accuracy Table

Sample ID	Concentration (µg/ml)		%Recovery of Pure drug	Statistical Analysis
Sample ID	Pure drug	Formulation	%Recovery of Pure drug	Statistical Analysis
S1 : 80 %	8	10	99.92	Mean	99.76
S2 : 80 %	8	10	99.58	SD	0.00701
S3 : 80%	8	10	99.79	% RSD	0.00702
S4 :100%	10	10	98.47	Mean	98.54
S5 :100%	10	10	98.79	SD	0.056
S6 :100%	10	10	98.36	% RSD	0.0568
S7 :120%	12	10	99.38	Mean	99.42
S8 :120%	12	10	99.53	SD	0.01
S9 :120%	12	10	99.37	% RSD	0.010

Accuracy:

To determine the accuracy of the proposed method, recovery studies were carried out by adding different amounts (80%, 100%, and 120%) of bulk samples of mesalamine within the linearity range were taken and added to the pre-analyzed formulation of concentration 20μg/ml. From that percentage recovery values were calculated. The results were shown in Table (5).

RESULTS AND DISCUSSION:

From the optical characteristics of the proposed method, it was found that Mesalamine obeys linearity within the concentration range of 5-100 μg/ml. From the results shown in Table (2) it was found that the % RSD is less than 2, which indicates that the method has good reproducibility. From the results shown in accuracy Table (5), it was found that the percentage recovery values of pure drug from the preanalyzed solution of formulation were in between 98.36 – 99.92, which indicates that the proposed method is accurate and also reveals that the commonly used excipients and additives in the pharmaceutical formulations were not interfering in the proposed method.

CONCLUSION:

The proposed method was simple, sensitive and reliable with good precision and accuracy. The proposed method is specific while estimating the commercial formulations without interference of excipients and other additives. Hence, this method can be used for the routine determination of Mesalamine in pure samples and pharmaceutical formulations.

ACKNOWLEDGEMENTS

The authors thank Wallace Pharmaceuticals, Goa for providing the gift sample of Mesalamine.

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