Visible Spectrophotometric Methods Development for Quantification of Racecadotril in Commercial Formulations

Koruprolu. Raghubabu¹*, N. Mohan Rao¹, Buridi. Kalyana Ramu², C. Ramdas³

¹Department of Engineering Chemistry, AU College of Engineering (A), Andhra University,

Visakhapatnam-530003, AP (India)

²Department of Chemistry, Maharaja’s College (Aided & Autonomous), Vizianagaram-535002, AP (India)

³M/S Tychy Industries, R&D Division, Hyderabad (AP) India.

*Corresponding Author E-mail: drraghualways@yahoo.co.in, kalyanaramu23566@gmail.com

ABSTRACT:

Three direct, simple and sensitive visible spectrophotometric methods (M₁,M₂ and M₃) are described for the assay of Racecadotril in pure and solid dosage forms. The method M₁ is based on the formation of colored molecular complex with sodium nitroprusside in presence of acetaldehyde under alkaline conditions and exhibits λ _max at 560nm. The method M₂ involves oxidative coupling of drug with brucine in presence of sodium meta periodate and purple red colored species is formed and exhibits absorption maxima at 520nm. The method M₃ is based on the formation of yellowish brown colored species by the drug with Folin reagent and exhibits absorption maxima at 450nm.Regression analysis of Beer-Lambert plots showed good correlation in the concentration ranges (8.0-40) µg/ml for methods M₁, M₂ and (20-60) µg/ml for method M₃ respectively. The proposed methods are applied to commercial available formulations and the results are statistically compared with those obtained by the reported UV reference method (in methanol, λ _max at 231nm) and validated by recovery studies. The results are found satisfactory and reproducible. These methods are applied successfully for the estimation of the Racecadotril in the presence of other ingredients that are usually present in dosage forms.

KEYWORDS: Inner Molecular complex, Aromatic Nucleophillic substitution, Oxidative coupling, Statistical analysis, Regression equation.

INTRODUCTION:

Racecadotril (RCT) ¹(Figure1) is an anti diarrheal drug and oral enkephalinase inhibitor. Chemically it is (RS)-benzyl N-[3-(acetylthio)-2-benzyl propanoyl] glycinate with empirical formula of C₂₁H₂₃NO₄S and representing molecular weight of 385.476. It is a white crystalline powder that is soluble in methanol, ethanol, acetonitrile and dichloromethane. The drug is mainly used for the treatment of acute diarrhea of bacterial and viral aetiology. The drug acts by inhibition of enkephalinase, this produces an increases in the levels of enkepalins that act in the enterocyate, thus inhibits hyper secretion.

Figure 1: Chemical structure of Racecadotril

In literature, several analytical methods such as HPLC^2-6, HPLC tandem MS⁷, HPTLC⁸, NMR⁹, spectrophotometry and spectrofluorometry¹⁰, UV^11-12 and visibleSpectrometric^13-14 have been reported for the determination of RCT in bulk and formulations. Devala Rao et al reported three visible spectrophotometric methods using FC reagent, 1,10-phenanthroline(PTL)-FeCl₃and 2,2’-Bipyridine(BPN)-FeCl₃ for the determination of the drug in formulations and the functional groups present in the drug not fully exploited. For routine quality control analysis, cost effective visible spectrophotometric methods are required and preferred. Nevertheless, there still exists a need for development of sensitive accurate and flexible visible spectrophotometric methods for the determination of RCT in pharmaceutical preparations. So the authors have made some attempts in this direction and succeeded in developing three methods based on the reaction between the drug and SNP-ACD ^15-16(M₁) or BCN-IO₄^- reagent ¹⁷ (M₂) or Folin reagent ¹⁸ (M₃) under specified experimental conditions. These methods can be extended for the routine quality control analysis of pharmaceutical products containing RCT.

MATERIALS AND METHODS:

Apparatus and chemicals:

A Milton Roy UV/Visible spectrophotometer model-1201 with 10mm matched quartz cells was used for all spectral measurements. A Systronics digital pH meter mode-361 was used for pH measurements. All the chemicals used were of analytical grade.

Aqueous solutions of sodium nitro prusside (SNP, E. Merck, 1.0%, 3.35x10^-2M), acetaldehyde (10%), phosphate buffer of pH 8.0(prepared by mixing 30ml of 0.067M potassium hydrogen phosphate and 970ml of 0.067Mdisodium hydrogen phosphate and pH adjusted to 8.0), aqueous solution of brucine (Loba, 0.2%, 506.7x10^-3M prepared by dissolving 200mg of brucine initially in minimum amount of 0.16M sulphuric acid and then made up to 100ml with distilled water), sodium metaperiodate (BDH, 0.2%, 9.35x10^-3M prepared by dissolving 200mg of sodium metaperiodate in 100ml distilled water and standardized iodometrically) and sulphuric acid (Qualigens,1.2M prepared by diluting 126ml of conc. H₂SO₄ to 100ml of distilled water initially, followed by diluting to 1000ml with distilled water), Folin reagent (NQS) solution (Loba, 0.5%, 1.92x10^-2M prepared by dissolving 500mg of NQS in 100 ml of distilled water ) were prepared for methods M₁ M₂andM₃ respectively.

Preparation of Standard stock solution:

The standard stock solution (1mg/ml) of RCT was prepared by dissolving 100mg of RCT initially in 10 ml of 0.1MNaOH followed by dilution to 100ml with methanol for methods M₁andM₃. The standard stock solution (1mg/ml) of RCT was prepared by dissolving 100mg of RCT initially in 10 ml of methanol followed by dilution to 100ml with water for method M₂. The working standard solutions of RCT were obtained by appropriately diluting the standard stock solution with the same solvent (M₁and M₂- 200 µg/ml M₃- 400 µg/ml). The prepared stock solution was stored at 4⁰C protected from light. From this stock solution, a series of standards were freshly prepared during the analysis day.

Preparation of Sample solution:

About 20 sachets/tablets were weighed to get the average tablet weight and pulverized. The powder equivalent to 100mg of RCT was weighed, dispersed in 25ml of Isopropyl alcohol, sonicated for 15 minutes and filtered through Whatman filter paper No 41.The filtrate was evaporated to dryness and the residue was dissolved as under standard solution preparation.

Analytical procedures:

Method M₁:

Aliquots of standard RCT drug solution [1.0-5.0ml, 200µg/ml] were delivered into a series of 25ml calibrated tubes containing 15ml of buffer pH 8.0. Then 1.0ml each of SNP solution and acetaldehyde were added successively and shaken for 2 minutes and kept aside for 15 minutes at room temperature and made up to the mark with distilled water. The purple colored species was obtained and it was stable for 1 hour. The absorbance of the colored species was measured at 560nm against the reagent blank. The amount of drug was computed from its calibration curve.

Method M₂:

Aliquots of the standard RCT solution [1.0-5.0ml, 200µg/ml] were placed in a series of 25ml standard flask. Then 3.0ml brucine, 1.5ml of NaIO₄ solution and 2.0ml of sulphuric acid were added successively and the volume was brought up to 10ml with distilled water and kept in boiling water bath for 15min. for complete color development. The solution was cooled to room temperature and the volume was made up to the mark with distilled water. The absorbance was measured at 520nm against a reagent blank within the stability period 60min. The calibration graph was constructed by plotting the drug concentration versus absorbance. The amount of drug was computed from its calibration graph.

Method M₃:

To aliquots of the standard RCT solution [1.0-3.0ml, 500µg/ml] 1.0ml of folin reagent (1.092x10^-2M), 5.0 ml of buffer pH 8.0 and 1.5ml of distilled water were added and kept aside for 15 min for complete color development. Then the volume was made up to 25 ml using distilled water and sonicated for 1 min. The absorbance was measured at 450nm against a reagent blank within the stability period 30min. The calibration graph was constructed by plotting the drug concentration versus absorbance. The amount of drug was computed from its calibration graph.

RESULTS AND DISCUSSION:

In the present investigation, the presence of aliphatic secondary amine of RCT permits the development of visible spectrophotometric methods for its determination through inner molecular complex formation with SNP-ACD (M₁) or the oxidative coupling reaction with BCN-IO₄^- reagent (M₂) or the aromatic nucleophillic substitution with folin reagent (M₃).

Optimum operating conditions used in the procedure were established adopting variation of one variable at a time (OVAT) method. The effect of various parameters such as time, volume and strength of reagents, the order of addition of reagents, pH buffer solutions and solvent for final dilution of the colored species were studied. The other oxidants such as Fe (III), Cr (IV), IO₃^-, and S₂O₈^-2 were tried in place of NaIO₄ and found to be inferior incase of method M₂. Distilled water was found to be best solvent for final dilution. Other water miscible solvents like methanol, ethanol, propan-2-ol and acetonitrile have no additional advantage in increasing the intensity of the color in all three methods. The optical characteristics such as Beer’s law limit, Sandell‘s sensitivity, molar absorptivity, percent relative standard deviation, (calculated from the six measurements containing 3/4^th of the amount of the upper Beer’s law limits), Regression characteristics like standard deviation of slope (S_b), standard deviation of intercept (S_a), standard error of estimation (S_e) and % range of error (0.05 and 0.01 confidence limits) were calculated and the results are summarized in Table-1.

Commercial formulations containing RCT were successfully analyzed by the proposed methods. The values obtained by the proposed and reference methods for formulations were compared statistically by the t-and F-test and found not to differ significantly. As an additional demonstration of accuracy, recovery experiments were performed by adding a fixed amount of the drug to the pre analyzed formulations at three different concentration levels. These results are summarized in Table-2.

Chemistry of colored species:

Cullies and Waddington ¹⁵ found that many secondary but not primary or tertiary amines react with sodium nitroprusside and acetaldehyde under mild alkaline conditions. Wolfe and Swine hart¹⁶ have reported the formation of [Fe(CN)₅ H₂O]^3- in aqueous solution of sodium nitroprusside. The proposed method M₁ exploits structural features aliphatic secondary amine of the RCT molecule. The nature of colored species formation with sodium nitroprusside-acetaldehyde reagent is initial N-alkyl vinyl amine formation with acetaldehyde then followed by formation of colored inner molecular complex with sodium nitroprusside has been assumed in the scheme. Based on the analogy, the probable sequence of reactions is presented in scheme (Figure 2).

In method M₂, the dimethoxy benzene nucleus of brucine is attacked by IO₄^- with the formation of o-quinone (bruciquinone) which in turn undergo nucleophillic attack on the most electron-rich position of the coupler (secondary amino group of RCT) to give 1-monosubstituted bruciquinone derivative(purple red colored species). In method M_3, yellowish brown colored species (N-alkyl amino napthaquinone) was formed by replacement of the sulphonate group of the napthaquinone sulphonic acid by a secondary amino group of drug. The formation of colored species with these reagents may be assigned through above analogy as shown in Scheme (Figure 3and4).

Figure 2: Probable sequence of reaction for method M₁

Figure3: Probable scheme of reaction for method M2

Figure 4: Probable Scheme of reaction for method M₃

CONCLUSION:

The proposed methods applicable for the assay of drug, the advantage of wider range under Beer’s law limits, validated as per ICH guide lines and possess reasonable precision, accuracy, and simple, sensitive. The wavelength maximum (λ_max) and Sensitivity (€_max) order of the proposed methods is M₁>M₂>M₃. These methods can be extended for the routine quality control analysis of RCT in formulations.

Table 1: Optical Characteristics, precision and accuracy of proposed methods.

Parameter	Method M1	Method M2	Method M3
גmax (nm)	560	520	450
Beer’s law limit(µg/ml)	8-40	8-40	20-60
Sandell’s sensitivity(µg/cm^2/0.001 abs. unit	0.00372093	0.004102564	0.006530612
Molar absorptivity (Litre/mole/cm)	103597.75	93960.75	59026.625
Regression equation (Y)*
Intercept (a)	0.004	0.001	-0.086
Slope(b)	0.010	0.01	0.008
%RSD	2.0	1.74	1.91
% Range of errors(95% Confidence limits) 0.05 significance level 0.01 significance level
	2.1	1.8	2.0
	3.3	2.8	3.10

*Y= a + b x; Where Y= absorbance, x= concentration of RCT in µg/ml.

TABLE-2 ANALYSIS OF RCT BY PROPOSED AND REFERENCE METHODS.

Method	Formulation	Labeled Amount (mg)	Found by Proposed Methods			Found by Reference Method ± SD	#% Recovery by Proposed Method ± SD
			*Amount found ± SD	t	f
M1	Capsule	10	9.77 ± 0.10	2.17	1.61	9.86 ± 0.13	97.73± 1.0
M2	Capsule	10	9.82 ± 0.087	0.67	2.36	9.86 ± 0.13	98.17 ± 0.87
M3	Capsule	10	9.9± 0.10	0.63	1.77	9.86 ± 0.13	99.09±1.01

**Average ± Standard deviation of six determinations, the t- and f-values refer to comparison of the proposed method with reference method. (UV). Theoretical values at 95% confidence limits t =2.57 and F = 5.05.

# Recovery of 10mg added to the pre-analyzed sample (average of three determinations).

Reference method (reported UV method) using methanol (λ _max=231 nm).

ACKMOWLEDGEMENTS:

The authors thanks to R& D division Tychy Industries, Hyderabad AP India for providing gift sample of the drug and also thanks to University authorities for providing necessary facilities in this work.

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Received on 30.12.2013 Modified on 01.03.2014

Accepted on 11.03.2014 ©A&V Publications All right reserved

Res. J. Pharm. Dosage Form. and Tech. 6(3):July- Sept. 2014; Page 183-187