INTRODUCTION:

Mesalamine is chemically known as 5–amino–2– hydroxy benzoic acid, is an anti-inflammatory drug used to treat inflammation of the digestive tract (crohn’s disease) and mild to moderate ulcerative colitis. Regular consumption of Mesalamine, structurally related to NSAIDs^1-7, seems to reduce the risk of CRC in patients with ulcerative colitis. It is a bowl-specific amino salicylate drug that is metabolized in the gut and has its predominant actions there, thereby having fewer systemic side effects. Few spectrophotometric^8-9, chromatographic^10-26 and other methods^27-29 for the estimation of Mesalamine have been reported. The present work describes a precise RP-HPLC method for the estimation of Mesalamine in drug formulations.

MATERIALS AND METHODS:

Instrument:

A PEAK LC – P 7000 Isocratic pump equipped with UV detector was used for the analysis.

Chemicals and Reagents:

HPLC grade methanol, A.R. grade potassium dihydrogen phosphate and ortho phosphoric acid were used for the analysis.

Chromatographic conditions:

Chromatographic separation was carried out in an isocratic mode utilizing kromosil C18 column with dimensions (5 µm, 250 mm x 4.6 mm) as stationary phase with injection volume of 20 µl. The mobile phase composed of methanol and 0.025 M potassium dihydrogen phosphate (pH adjusted to 2.0 with ortho phosphoric acid) in the ratio 65:35 (v/v) at a flow rate of 1.0 ml/min. with detection at 250 nm using a UV detector. The chromatogram of Mesalamine is showed in Fig.1.

Preparation of standard drug solution:

50 mg of Mesalamine was accurately weighed and transferred into a 100 ml volumetric flask, dissolved in mobile phase to obtain a stock solution containing 500 µg/ml. From this, suitable dilutions were made to obtain the working standard concentrations ranging from 0-150 µg/ml.

Preparation of sample solution:

The formulation powder equivalent to 50 mg of drug was accurately weighed and transferred to a 100 ml volumetric flask. About 20 ml of mobile phase was added and sonicated for 10 min., filtered through 0.45 µm membrane filter and the volume was made to the mark with mobile phase to get the stock solution. From this, suitable dilutions were made to obtain the working concentrations ranging from 0-150 µg/ml.

Assay procedure:

Various standard concentrations of Mesalamine ranging from 0-150 µg/ml were prepared in mobile phase and 20 µl of each of the drug solution was injected for five times. Quantitative determinations were made by comparison of the peak area from a standard injection. The amount of Mesalamine present in the sample was calculated through the calibration curve (Fig.2). For comparing accuracy of the results obtained by the proposed method, the author has developed a sensitive UV-spectrophotometric method (Fig.3 and 4) and adapted it as a reference method.

Fig.1. Chromatogram of Mesalamine

Fig.2 Calibration curve of proposed method

Fig.3 Absorption spectrum UV reference method

Fig.4 Calibration curve of UV reference method

Table-1. System suitability parameters, regression characteristics and precision of the proposed method

S.No.	Parameter	Proposed Method
1	Wave length (nm)	250
2	Retention Time (t), (min.)	2.85
3	Theoretical plates (n)	3659
4	Plates per meter (N)	14636
5	Height Equivalent to Theoretical Plate (HETP), (mm)	6.8325×10^-5
6	Peak Asymmetry	0.98
7	Resolution factor	-
8	Linearity range (µg/ml)	20.0 – 100.0
9	Regression equation (Y=mX+b)
	Slope (m)	1369.26
	Intercept (b)	3277.60
10	Correlation coefficient (r)	0.9996
11	Relative Standard Deviation (%) *	0.5537
12	Percent range of error (confidence limits)
	0.05 level	± 0.6874
	0.01 level	± 1.1401
13	Limit of Detection	3.1341
14	Limit of Quantification	10.4469
* Mean of five determinations

Table-2 Assay of Mesalamine in commercial formulations.

Formulation	Labeled Amount (mg)	Proposed Method			Reference method	% Recovery**
Formulation	Labeled Amount (mg)	Amount found*	t-Value	F-Value	Reference method	% Recovery**
Tablet-I	400	399.89 ± 0.068	1.6276	1.5286	399.96 ± 0.055	99.98 ± 0.032
Tablet-II	500	499.83 ± 0.067	1.1294	2.7944	499.91 ± 0.112	99.67 ± 0.189
Tablet-III	800	799.89 ± 0.051	1.2030	3.2541	799.96 ± 0.092	98.81 ± 0.158

* Mean ± standard deviation of five determinations. The t-test and F-test values refer to comparison of the proposed method with the reference method. Theoretical values at 95% confidence limits, t=2.306, F=6.39.

**Mean ± standard deviation of three determinations.

RESULTS:

The system suitability tests were carried out on freshly prepared standard stock solution of Mesalamine under the optimized chromatographic conditions, which include number of theoretical plates, HETP, linearity range, resolution, peak asymmetry, LOD and LOQ have been calculated with the observed readings and the results are presented in table-1. The precision of the method was ascertained from the peak area values obtained by actual determination of five replicates of a fixed amount of the drug. The percent relative standard deviation and percent range of errors (confidence limits 0.05 and 0.01 levels) were calculated and are presented in table-1. To find out the suitability of the proposed method for the assay of formulations, different dosage forms containing selected drug were analyzed by proposed method and the reference method. The results obtained from the proposed and reference methods were compared statistically by the t-test and F-test and are summarized in table-2. In order to check the accuracy of the proposed method, recovery experiments were performed by adding known amount of pure drug to the pre-analyzed formulation and percent recovery values obtained are listed in table-2.

DISCUSSION:

This method is developed based on the use of a C18column with a suitable mobile phase without use of internal standard. The system suitability parameters reveal that the proposed method has good sensitivity. Statistical analysis of the results shows that the proposed procedure has good precision and accuracy. The results obtained in the determination of Measalamine in commercial formulations and recovery experiments reveal that the proposed method is sensitive and suitable for the analysis of active ingredient present in the pharmaceutical formulations.

ACKNOWLEDGEMENTS:

I would like to acknowledge the contribution of my research supervisor Prof. K. Saraswathi with whose help I was able to incorporate this work in my thesis. I tender grateful thanks to the Management and Principal, R.V.R. & J.C. College of Engineering, Guntur for their encouragement to me for doing this work.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 30.06.2020 Modified on 29.12.2020

Research J. Pharm. and Tech 2022; 15(1):301-304.

DOI: 10.52711/0974-360X.2022.00049