INTRODUCTION:

Racecadotril is N-[(R, S)-3-acetylmercapto-2-benzyl]-glycine, benzyl ester, is an oral enkephalinase inhibitor for use in the treatment of acute diarrhea. Few UV¹, HPLC^2,3,4, NMR⁵ and LC-MS⁶ methods have been reported for the estimation of Racecadotril in bulk drug and pharmaceutical dosage forms. The proposed methods are based on the oxidation of drugs by Fe (III) in presence of 1, 10-phenanthroline or 2, 2' bipyridyl or potassium ferricyanide. The colored complex formed was measured at 510, 520 and 720 nm for method A, B and C respectively.

EXPERIMENTAL:

Apparatus:

A Systronics Double beam UV- visible spectrophotometer 2201 with 1cm matched quartz cells was used for all spectral and absorbance measurements. A Systronics digital pH meter was used for all pH measurements.

Materials and reagents:

All chemicals used were of analytical reagent grade. Racecadotril was obtained from Dr. Reddy’s pharmaceuticals private limited, Hyderabad. Zedott DT, Enuff DT are the commercial tablet formulations labeled to contain 10mg and 30 mg per tablet respectively.

1, 10-phenanthroline (0.198%) was prepared by dissolving 198mg of 1, 10-phenanthroline in 100ml of 0.1N hydrochloric acid. Ferric chloride (0.0033M) was freshly prepared by dissolving 162mg of Ferric chloride in 100ml of distilled water.33ml of the above solution further diluted to 100ml with distilled water. 0.2M orthophosphoric acid was prepared by diluting 1.3ml with 100 ml of distilled water. 2, 2' bipyridyl (0.156%) was prepared by dissolving 156mg of 2, 2' bipyridyl in 100ml of 0.1N hydrochloric acid. Potassium ferricyanide (0.2%) was prepared by dissolving 20mg of potassium ferricyanide in 100ml of distilled water. Stock reference solution (1000μg/ml) was freshly prepared from pure sample of Racecadotril by dissolving 0.1g in 100ml of acetonitrile (for method A) or methanol (for method B). Stock reference solution (2000μg/ml) was prepared by dissolving 0.2g of the Racecadotril in 100 ml of methanol (for method C).

Procedure for calibration curve:

In method A and B, different aliquots of stock reference solution (1000μg/ml) from 0.25-1 ml (for method A) or 0.2-1.2ml (for method B) were transferred in to a series of 10ml standard flasks. To each flask 1.0ml of ferric chloride and 1.0ml of 1, 10-phenanthroline or 2, 2’ bipyridyl were added and boiled in a water bath for 30min, then cooled to room temperature (25 ± 10c) and 2.0ml of orthophosphoric acid was added. The solutions were made up to volume with distilled water.

Table 1: Optical and Regression characteristics of Racecadotril

Parameters	Methods
Parameters	A	B	C
l_max(nm)	510	520	720
Beer’s law limits ( μg / ml)	25 - 100	20-120	200-1200
Molar absorptivity (L. mole^-1 cm^-1)	2.675 x 10³	2.399 x 10³	2.34x 10²
Detection limits ( μg / ml)	2.846	0.408	10.133
Sandell’s sensitivity (μg /cm ²/0.001 absorbance unit)	0.1441	0.16064	1.6461
Optimum photometric range	2.5-11.5	55-245	8-24
Regression equation (Y = a+ bc):
Slope (b)	0.0075	0.0063	6.1x10^-4
Standard deviation of slope (S_b)	9.92 x 10^-5	1.274 x 10^-5	3.078x 10^-6
Intercept (a)	0.001	0.002	0.00033
Standard deviation of intercept (S_a)	6.1 x 10^-3	7.7 x 10^-4	1.86x 10^-3
Standard error of estimation (S_e)	8.11 x 10^-5	2.68 x 10^-3	1.13x 10^-3
Correlation coefficient (r)	0.9994	0.9998	0.9998
% Relative standard deviation*	0.7014	0.5939	0.6029
% Range of Error (Confidence limits)* 0.05 level	0.736	0.6234	0.632
0.01 level	1.154	0.977	0.992
% Error in bulk samples**	-0.51	0.25	-0.19

* Average of six determinations, ** Average of three determinations

Method	Pharmaceutical Formulation		Labeled Amount	*Amount found ± S.D.**	Reference method	%Recovery ± R.S.D.
A	Zedott DT	Tablets I	10 mg	9.82 ± 0.023 t = 0.59, F = 2.35	10.05 ± 0.016	100.2 ± 0.229
	Zedott DT	Tablets II	30 mg	29.04 ± 0.033 t = 0.73, F = 1.82	30.01 ± 0.022	100.5 ± 0.358
	Enuff DT	Tablet I	10 mg	9.52 ± 0.12 t = 0.78, F = 2.06	10.05 ± 0.016	100.2 ± 0.229
	Enuff DT	Tablet II	30 mg	29.05 ± 0.036 t = 1.35, F = 2.67	30.01 ± 0.028	100.2 ± 0.229
B	Zedott DT	Tablets I	10 mg	9.77 ± 0.083 t = 0.71, F = 2.12	9.95 ± 0.016	99.72 ± 0.252
	Zedott DT	Tablets II	30 mg	27.08 ± 0.032 t = 1.35, F = 2.67	29.81 ± 0.022	96.95 ± 0.357
	Enuff DT	Tablet I	10 mg	9.02 ± 0.023 t = 0.78, F = 2.24	10.05 ± 0.016	99.81 ± 0.251
	Enuff DT	Tablet II	30 mg	29.85 ± 0.036 t = 1.32, F = 2.63	30.01 ± 0.028	99.5 ± 0.048
C	Zedott DT	Tablets I	10 mg	9.98.± 0.15 t = 0.78, F = 2.06	10.01 ± 0.037	98..2 ± 0.114
	Zedott DT	Tablets II	30 mg	29.79 ± 0.038 t = 1.35, F = 2.67	29.81 ± 0.021	99..5 ± 0.406
	Enuff DT	Tablet I	10 mg	9.87 ± 0.025 t = 0.78, F = 2.06	9.95 ± 0.017	98.92 ± 0.254
	Enuff DT	Tablet II	30 mg	29.95 ± 0.036 t = 1.35, F = 2.67	29.82 ± 0.025	99.02 ± 0.155

Table 2: Assay of Racecadotril

· Average ± standard deviation of eight determinations,

· t and F – values refer to comparison of the proposed method with reference method. Theoretical values at 95 % confidence limits t = 2.365 and F = 4.88.

· Reference method – in methanol

The absorbance of each solution was measured at 510 nm (method A) or 520 nm (method B) against the reagent blank. The calibration graph was constructed by plotting the absorbance versus concentration of the drug. The concentration of the unknown was read from the calibration graph or computed from the regression equation. In method C, different aliquots of stock reference solution (2000μg/ml) from 1- 6ml were transferred in to a series of 10ml standard flasks. To each flask 1ml of ferric chloride and 0.2 ml of potassium ferricyanide were added and kept aside for 5 minutes. The solutions were made up to volume with distilled water. The absorbance of each solution was measured at 720nm against the reagent blank. The calibration graph was constructed by plotting the absorbance versus concentration of the drug. The concentration of the unknown was read from the calibration graph or computed from the regression equation.

Procedure for pharmaceutical formulations:

Twenty tablets were weighed accurately and ground into a fine powder. An amount of powder equivalent to 10mg of Racecadotril was weighed and transferred in to a 100ml volumetric flask, 50ml of the methanol (method B and C) or acetonitrile (method A) was added and sonicated for 15min, then the volume was made up to the mark with the same solvent, mixed well and filtered. The assay of the tablets was carried out according to the general procedure.

RESULTS AND DISCUSSION:

Methods A, B and C are based on the oxidation of Racecadotril by excess of ferric salt (Fe III) .The reduced ferrous ion (Fe II) forms colored complex on treatment with 1, 10-phenanthroline or 2, 2' bipyridyl or potassium ferricyanide. As FeIII interfere even though to a little extent (especially in the lower range of beer’s law limits) in the determination of Fe II in methods A and B, the reactivity of interfering entity (Fe III) has to be made insignificant by complexing it with orthophosphoric acid. Optical characteristics such as Sandell’s sensitivity, molar absorptivity, precision and accuracy were found by performing six replicate determinations. The Relative standard deviation (RSD) and confidence limits were considered satisfactorily for all the three methods and are summarized in Table 1. The average drug content was determined for the proposed methods in Table 2.

CONCLUSION:

The proposed methods are simple, accurate and offer advantages of reagent availability and stability, less time consumption and high sensitivity. The commonly used excipients and additives in the preparation of formulations such as microcrystalline cellulose, lactose monohydrate, povidone, sodium starch glycolate, colloidal silicon dioxide and magnesium stearate were did not interfere with determination of racecadotril by the proposed methods even if they are present in large amounts than they usually exist.

ACKNOWLEDGEMENTS:

The authors are thankful to the management of Brown’s college of pharmacy for providing the facilities and Dr. Reddy’s pharmaceuticals private limited, Hyderabad for providing the gift sample of Racecadotril.

REFERENCES:

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2. Srinivasa Rao.T and Nappinnai.M Asian J of Chemistry.2007; 19(5): 3697-3702.

3. Prabu SL, Singh T, Joseph A, Kumar CD and Shirwaikar A. Indian J Pharm Sci.2007; 69:819-21.

4. The Merck Index, Merck and Co. Inc., New York, edn. 13, p.8182.

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Received on 21.03.2010 Modified on 25.05.2010

Asian J. Research Chem. 4(4): April 2011; Page 548-550