INTRODUCTION:

Ethamsylate^1-5 is an orally administered haemostatic drug. It is used in the treatment of capillary hemorrhage, hematemesis, hemoptysis, malena, hematuria, epistaxis, menorrhagia and post partum hemorrhage. It is believed to work by increasing capillary endothelial resistance and promoting platelet adhesion. It reduces capillary bleeding when platelets are adequate, probably exerts anti hyalurodinase actionimproves capillary wall stability. It is also claimed to inhibit PGI2 production and correct abnormal platelet function, but does not stabilize fibrin⁶. Ethamsylate is chemically 2,5-dihydroxy benzene sulfonic acid with N-ethylethanamine. The molecular formula of Ethamsylate is C₁₀H₁₇NO₅S. The molecular mass of Ethamsylate is 263.33 g/mol. It is official drug in British Pharmacopoeia. It is completely soluble in water, methanol, and ethanol but partially soluble in methylene chloride. Literature survey reveals that, only kinetic spectrophotometric determination⁷, Spectrophotometric simultaneous estimation from a Binary Mixture by Dual Wavelength⁸ and Simultaneous Equation Methods⁹ and some HPTLC methods¹⁰ have been reported. There is a need to develop and validate a new simple, rapid, reliable and precise UV spectrophotometric method for analysis of Ethamsylate in bulk and tablet formulation. Suitable statistical tests were performed on validation data ^11-12.

MATERIALS AND METHODS:

Instrument Used:

A Model: UV-Vis spectrophotometer 1700

Make: Shimadzu, Kyoto, Japan

Scan speed: 40nm/min

B. Bath Sonicator

Reagents and Solutions:

All the reagents used in this assay were of analytical grade. Tablets of Ethamsylate were purchased.

EXPERIMENTAL:

Determination of λmax: Weighed amount of Ethamsylate was dissolved in 0.1N HCl to obtain a 100µg/ml solution. This solution was subjected to scanning between 200-400 nm and absorption maximum was determined. The effect of dilution on absorption maxima was studied by diluting the above stock solution to 40μg/ml and scanned from 200-400nm.

Preparation of standard stock solution:

Standard drug solution of Ethamsylate was prepared by dissolving 10 mg Ethamsylate in 100 ml 0.1N HCl to obtain stock solution of 100 μg/ml concentration.

Preparation of calibration curve:

Calibration curve was prepared in 0.1N HCl at λmax 300 using UV-Vis spectrophotometer Model 1700.For this stock solution of 100 μg/ml was prepared. Serial dilution of 10, 15, 20, 25, 30, 35μg/ml were prepared and absorbance was taken at λmax 300.Averages of such 6 sets of values were taken for calibration curve, and solution were scanned in the range of 200-400 nm against blank. The calibration curve was plotted as shown in fig 2.and the data shown in Table no.2. The optical characteristics are summarized in Table no. 1and the overlain spectrum of Ethamsylate shown in fig.1.

Figure.1: results of analysis for recovery studies for marketed formulations

Table1: Optical parameters for Ethamsylate

Sr. No	Parameters	Data
1	λ-Max	300nm
2	Linearity	10-35ug/ml
3	Regression equation	y= 0.018x-0.0098
4	Correlation coefficient	R2= 0.999
5	Slop	0.018
6	Intercept	0.0098
7	LOD	0.277µg/ml
8	LOQ	0.861µg/ml

Table2: Calibration data

Concentration	Absorbance	SD	RSD
10	0.172	0.001472	0.84
15	0.26	0.001472	0.56
20	0.349	0.001612	0.33
25	0.432	0.001632	0.37
30	0.538	0.001211	0.22
35	0.618	0.001941	0.31

Assay:

Ten Tablets each containing of 250 mg of Ethamsylate was weighed and powdered. Powder equivalent to 100 mg of Ethamsylate was transferred into 100 ml volumetric flask dissolved in 0.1N HCl. The solution was then filtered through Whatman filter paper No 40 (0.45 micron).Aliquots of the sample were removed and diluted to 10 ml of 0.1N HCl to obtain strengths of 40µg/ml determined at the respective absorbance of 300nm against 0.1N HCl as a blank. (Table5)

Table 3: Accuracy data for analysis of Ethamsylate( recovery studies)

Amount of sample taken (µg/ml)	Amount of standard added (µg/ml)	total amount recovered (µg/ml)	SD	% recovery
10	0	9.96	0.015275	99.63
10	5	14.98	0.02	99.86
10	10	20.033	0.015275	100.01
10	15	24.96	0.02516	99.85

Recovery studies:

Recovery studies were performed to judge the accuracy of the method. Recovery studies were carried out by adding a known quantity of pure drug to the pre-analyzed formulation and the proposed method was followed. From the amount of drug found, percentage recovery was calculated. Recovery study was carried out by addition of standard drug to the sample at 3 different concentration levels. (Table 3)

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The LOD and LOQ of Ethamsylate were determined by using standard deviation of the response and slope approach as defined in International Conference on Harmonization (ICH) guidelines 7. The LOD and LOQ were found to be as in Table No 1. Limit of detection (LOD) and limit of quantification (LOQ) were calculated by Eq. (1) LOD 3.3 × s/S and (2) LOQ =10 × s/S respectively, where s is the standard deviation of blank and S is slope.

DISCUSSION:

The spectrum of Ethamsylate in 0.1N HCl showed the absorption maxima at 300nm. No effect of dilution was observed on the maxima, which confirmed the maxima at 300nm. The statistical analysis of data obtained for the calibration curve of Ethamsylate in pure solution indicated a high level of precision for the proposed method. The coefficient of correlation was highly significant. The linearity range was observed between 10–35µg/ml. The plot clearly showed a straight line passing through origin equation Y=0.018X - 0.0098 with correlation coefficient of 0.999. The assay method was validated by low values of standard deviation and standard error, indicating accuracy and precision (Table4) of the methods. Excellent recovery studies further proves the accuracy of the method.

Table4: Repeatabality data for Ethamsylate

Interday				Intraday
Concentration (ug/ml)	Absorbance	SD	%RSD	Absorbance	SD	%RSD
20	0.346	0.003785	1.093148	0.338	0.002	0.591716
25	0.429	0.001527	0.355514	0.419	0.001	0.238663
30	0.533	0.001154	0.216371	0.528	0.002	0.378788

Fig 2. Calibration graph of ethamsylate

Table 5: Determination of ethamsylate in marketed formulation

Tablet code	Label claim (mg/tab)	Amount found (mg)	%
ETHAM-1	250	249.8	99.92
ETHAM-2	250	249.95	99.98

CONCLUSION:

It can be concluded that the proposed method is simple, rapid, accurate, precise, economic and reproducible for UV spectrophotometric estimation of Etamsylate from pharmaceutical formulation. This method can be successfully applied for routine estimation of Ethamsylate in bulk, pharmaceutical dosage form.

RESULTS:

The UV scan of standard solution between 200 – 400 nm showed the absorption maxima at 300nm, shown in fig. 1. The Beer’s law was verified from the calibration curve by plotting a graph of concentration vs. absorbance. The plot is shown in fig. 2. Regression analysis showed very good correlation. The results thus obtained are depicted in table No.1 .The results of analysis for recovery studies for marketed formulations were studied and are shown in table no.3

ACKNOWLEDGEMENTS:

The authors wish to thank Prof. A.K. Saluja (Principal) A.R. College of Pharmacy and G.H. Patel Institute of Pharmacy for providing laboratory, store and library facility and last but not the least we wish to thank to all our friends and colleagues for their valuable support.

REFERENCES:

1. British Pharmacopoeia, Her Majesty’s Stationery Office, London, Ph Eur Monograph 1204; 2005 vol-1, 762.

2. www.cemolink.com/msds/2624-44-4_130.doc.

3. http://en.wikipedia.org/wiki/Ethamsylate.

4. ONeil M.J, The Merk Index- an encyclopedia of chemicals, Drugs and Biologicals. 13^th ed New Jersey, Merck and Co., 3757:664.

5. Sweetmann S C, Martindale – The Complete Drug Reference, 33rd ed London (UK), Pharmaceutical Press., 2002:732.

6. El-Enany Nahed., Belal Fathalla., Rizk Mohamed. Kinetic spectrophotometric determination of Ethamsylate in dosage forms. Journal of AOAC International 2007; 90(3):679-85.

7. Anju Goyal, Singhvi I. Spectrophotometric Estimation of simultaneous estimation of Etamsylate and Mefanamic acid from a Binary Mixture by Dual Wavelength and Simultaneous Equation Methods. Indian J. Pharm. Sci. 2008, 70(1), 105-108.

8. Roshan I., Kaushik KV., Diptish KN. Spectrophotometric methods for Simultaneous estimation of Ethamsylate and Tranexamic acid from combined tablet dosage form. Int J Chem Tech Res. 2010, 2(1), 74-8.

9. Roshan I., Kaushik KV., Diptish KN. Spectrophotometric methods for Simultaneous estimation of Ethamsylate and Tranexamic acid from combined tablet dosage form. Int J Chem Tech Res. 2010, 2(1),74-8.

10. Yogini SJ., Gokl ST., Sanjay JS. A simple and sensitive HPTLC method for quantitative analysis of Ethamsylate in tablets. J Planar Chrom. Sep/Oct; 2005, 18,380-3.

11. International conference on Harmonization, Guidance for Industry In; Q2B validation of analytical procedures, Methodology, Switzerland; IFPMA 1996;1-8

12. International conference on Harmonization, Guidance for Industry In; Q2A Text on validation of analytical methods, Switzerland; IFPMA 1994;1-4.

Received on 04.02.2013 Modified on 10.02.2013

Asian J. Research Chem. 6(2): February 2013; Page 166-168