INTRODUCTION:

Eprosartan Mesylate (EPM)^{1 – 5}a new drug and it is used as anti hypertensive agent which is chemically mono methane sulfonate of (E) – 2 – butyl – 1 - (p-carboxybenzyl) – α – 2 – thienyl methyl imidazole – 5 – acrylic acid. (Fig.1). EPM is not official in any pharmacopoeia. EPM, a potent vasoconstrictor, is the principal pressor agent of rennin - angiotensin system. Literature survey revealed that SPE – HPLC – UV^{6 – 7} and HPTLC⁸ methods were reported for the estimation of EPM in plasma. However, there is no UV spectrophotometric method has been reported for the estimation of EPM. Hence the present work aims to develop a simple, precise, accurate and validated UV spectrophotometric method for the estimation of EPM in pure and in tablet dosage forms. Confirmation of the applicability of the developed method was validated according to the International Conference on Harmonization (ICH)^{9 – 10}guidelines for the determination of EPM in pure and in tablet dosage forms.

MATERIALS:

Instruments:

Shimadzu UV-1700 UV/VIS spectrophotometer with 1cm matched quartz cells was used for spectral and absorbance measurements.

Drug Material and Chemicals:

Pharmaceutically pure sample of EPM was obtained as generous gift samples from Sairam Organics Pvt. Ltd., Hyderabad, India. 0.1M Sodium hydroxide AR grade was used as solvent in this study. Shimadzu model 1700 Double Beam UV – Visible spectrophotometer with a pair of 10 mm matched quartz cells was used for absorbance measurements.

The tablet formulations Teveten containing 400 mg of EPM tablet formulation– I and 600 mg of EPM tablet formulation – II (Solvay Pharmaceuticals, Mumbai, India) was procured from the local market. All the chemicals and reagents used were obtained from Qualigens India Pvt. Ltd., Mumbai, India.

Preparation of Reagents¹⁹

0.1M Sodium hydroxide

4 Gms of sodium hydroxide was accurately weighed and dissolved in distilled water and the volume was made up to 1000 ml with distilled water.

Fig.1. Chemical structure of Eprosartan Mesylate

METHOD:

Simple UV Spectroscopic Method:

This method is based on the measurements of absorbance of EPM at its λ max was found to be 292 nm. 0.1M Sodium hydroxide of analytical reagent grade was selected as solvent for developing spectral characteristics of drug. The selection was made after assessing the solubility of the drug in different solvents.

Preparation of standard stock solution:

50 mg of EPM was accurately weighed and transferred to 100 ml volumetric flask. This drug was dissolved in 0.1 M sodium hydroxide and made up to the mark with 0.1 M sodium hydroxide. The standard stock solutions contain 500 mg/ ml of EPM. This solution was further diluted to obtain (10 mg/ ml) of drug solution.

Preparation of calibration graph:

0.5 – 2.5 ml of standard stock solution of EPM was transferred into a series of five 50 ml volumetric flasks and made up to mark with 0.1M sodium hydroxide. The absorbance of different concentration solutions was measured at 292 nm against 0.1 M sodium hydroxide as blank. The calibration curve was plotted using concentration against absorbance. The solutions were found to be linear with the concentration range of 5 – 25 mg/ ml of EPM. The curve was linear with the concentration range of 5 - 25 μg/ ml. The procedure was repeated for six times.

Application of the proposed procedure for the determination of dosage form:

Marketed tablet formulations Teveten I and II were used for analysis. Twenty tablets were weighed and their average net weight was calculated. The tablet was made to a fine powder.

Method:

The powder equivalent to equivalent to 50 mg of Eprosartan Mesylate formulations were transferred into a 100 ml volumetric flasks separately, added 0.1M Sodium Hydroxide to dissolve and made up to the volumes individually. Then the solutions were sonicated for 10 minutes. After sonication the solutions were centrifuged at 100 rpm for 15 minutes. The solutions were filtered through Whatmann filter paper No.41. From the clear solutions, 1.5 ml of the solution was transferred into a 50 ml standard flask and made up to the mark with 0.1M Sodium Hydroxide to produce 15 mg/ ml concentration for tablet formulation –I and tablet formulation –II the absorbance measurements were made six times for these two formulations at 292 nm. The procedure was repeated for six times for both formulations. The amount of Eprosartan Mesylate present in formulations was determined by using slope and intercept values from calibration graph.

Recovery studies:

The accuracy of the proposed method was examined by determining the recovery of the drug by standard addition technique. To the preanalysed formulations a known amount of raw materials were added and it can be analyzed by proposed method.

Method:

50 mg equivalent of Eprosartan Mesylate formulations were taken into a series of three 100 ml standard flasks. To that 10 mg, 20 mg and 30 mg of raw materials were added in to series of standard flasks 1, 2 and 3, respectively. Dissolved with 0.1M Sodium Hydroxide and made up to volume with 0.1M Sodium Hydroxide .The solutions were sonicated for 10 minutes. After sonication, the solution was centrifuged at 100 rpm for 15 minutes. The solutions were filtered through Whatmann filter paper No. 41. From each standard flask, 1.5 ml of the clear filtrate was transferred into a series of three 50 ml standard flasks and made up to volume with 0.1M Sodium Hydroxide individually for both formlations. The amount of drug recovered from each formulations were calculated. Each concentration was repeated for three times.

RESULTS AND DISCUSSION:

Two simple precise and accurate methods were developed for the estimation of EPM in pure and in formulations, (Fig 2). The linearity of the methods was found to be 5 - 25 μg/ ml (Fig-3). The optical characteristics such as correlation co-efficient, slope, intercept, molar absorptivity, LOD and LOQ were calculated and are shown in Table 1. The correlation co-efficient was found to be 0.9998. The calibration graph was shown in figure 2. The absorbance was measured and the amount of EPM in tablet formulations were found to be 100.04 ± 0.4414 and 100.29 ± 0.9004 for formulations – I and formulations – II respectively, and which are shown in (Table 2) and (Table 3). To study the precision of the method the analysis of formulations were carried out for six times. The % RSD values were found to be 0.4410 and 0.9026 for formulations – I and formulations – II respectively. By the low % RSD values, the precision of the method were confirmed. Further the precision was confirmed by intermediate precision. The analysis of formulations was carried out for three times in the same day and on three successive days. The % RSD values for interday and intraday analyses of formulations were found to be less than 2% and are shown in table 4 and 5 for formulations – I and formulations – II respectively. The accuracy of method was confirmed by recovery studies.

Fig 2 Simple UV spectrum of Eprosartan Mesylate containing 10 µg/ ml

Fig 3. Calibration Curve of Eprosartan Mesylate by UV Method

Table 1: Optical characteristics

PARAMETERS

EPM

λmax (nm)

Beers Law Limit (µg/ ml)

Molar absorptivity (mol.l^-1cm^-1)

Régression equation (Y= mx+c)

Slope (m)

Intercept (c)

Corrélation coefficient (r)

Standard error

LOD(µg/ml)

LOQ(µg/ml)

292

5 - 25

18444.21

Y = 0.03441x+0.01019

0.03441

0.01019

0.9998

0.000543

0.2422

0.7337

* Mean of six observations

Table 2: Assay of commercial formulation - I

Label amount mg/tablet)	*Amount found (mg/tablet)**	%Label Claim	SD	% RSD	SE
400	400.16	100.04	0.4414	0.4410	0.1802

* Mean + SD of six observations

Table 3: Assay of commercial formulation - II

Label amount mg/tablet)	*Amount found (mg/tablet)**	%Label Claim	SD	% RSD	SE
600	601.72	100.29	0.9004	0.9026	0.3676

* Mean + SD of six observations

Table 4: Intraday and Interday precision of the method for formulation - I

Amount Found (Percentage Obtained)		% RSD
Intraday*	Interday*	Intraday*	Interday*
99.35	100.38	0.0355	0.01955

* Mean + SD of three observations

Table 5: Intraday and Interday precision of the method for formulation - II

Amount Found (Percentage Obtained)		% RSD
Intraday*	Interday*	Intraday*	Interday*
100.14	99.73	0.1559	0.9527

* Mean + SD of three observations

A known amount of standard drug materials were added with pre-analyzed formulations in different levels. The amount of drug recovered was calculated and the percentage recovery was found to be in the range of 101.00% - 102.33 and 99.33 % - 101.00 %for formulations – I and formulations – II respectively %. The procedure was repeated for 3 times for each concentration and the % RSD values were calculated. The low % RSD values ensure that the excipients used in formulations are not interfering in the analysis of EPM. The results of recovery studies are shown in table 6 and 7.

Table 6: Recovery studies for formulation -I

SI. No	Amount Present (µg/ml)	*Amount added (µg/ml)**	*Amount found (µg/ml)**	*Amount recovered (µg/ml)**	*Percentage recovery %**	Average ± S.D	% RSD	S.E
1	14.96	3	18.03	3.07	102.33	101.48 ± 0.7382	0.7274	0.4262
2	14.96	6	21.02	6.06	101.00
3	14.96	9	24.06	9.10	101.11

* Mean + SD of three observations

Table 7: Recovery studies for formulation -II

SI. No	Amount Present (µg/ml)	*Amount added (µg/ml)**	*Amount found (µg/ml)**	*Amount recovered (µg/ml)**	*Percentage recovery %**	Average ± S.D	% RSD	S.E
1	15.06	3	18.09	3.03	101.00	100.11 ± 0.8404	0.8395	0.4853
2	15.06	6	21.02	5.96	99.33
3	15.06	9	24.06	6.00	100.00

* Mean + SD of three observations