DEVELOPMENT OF NEW ANALYTICAL METHODS FOR THE ESTIMATION OF OPIOID ANTAGONIST NALTREXONE HYDROCHLORIDE

114 P a g e International Standard Serial Number (ISSN): 2319-8141 International Journal of Universal Pharmacy and Bio Sciences 4(1): January-February 2015 INTERNATIONAL JOURNAL OF UNIVERSAL PHARMACY AND BIO SCIENCES IMPACT FACTOR 2.093*** Pharmaceutical Sciences ICV 5.13*** RESEARCH ARTICLE!!! DEVELOPMENT OF NEW ANALYTICAL METHODS FOR THE ESTIMATION OF OPIOID ANTAGONIST NALTREXONE HYDROCHLORIDE Jabeen Farhana, Wajeed Khan, Vinayak K Durgakar, Asiya Begum KEYWORDS: Naltrexone hydrochloride; MBTH; Potassium ferricyanide; 1, 10 Phenanthroline; 2, 2' Bipyridyl; Folin- Ciocalteu reagent; RP- HPLC. For Correspondence: Asiya Begum * Address: CMR college of pharmacy, Moinabad. Email: asiyabegum889@gmail.c om CMR college of pharmacy,moinabad. ABSTRACT In the present work, six simple and sensitive spectrophotometric methods (method A, B, C, D, E, F) and RP HPLC method have been developed for the quantitative determination of Naltrexone hydrochloride. Method A is a UV spectrophotometric method in which Naltrexone hydrochloride was dissolved in distilled water and distilled alcohol and it exhibited absorption maximum at 281 nm and obeyed Beer s law in the concentration range of 50-250 g/ml. Method B based on oxidation followed by coupling of 3-methyl-2-benzothiazolinone hydrazone (MBTH) in presence of ferric chloride to form green colored chromogen. Method C, D and E is based on Oxidation followed by complex formation with potassium ferricyanide, 1,10-phenanthroline and 2, 2' Bipyridyl in presence of ferric chloride to form blue colored, blood red and orange red colored chromogen respectively. Method F is based on formation of stable blue colored chromogen due to reduction of Folin- ciocalteau reagent in presence of sodium hydroxide. The above methods were exhibited absorption maximum at 661, 715, 509.6, 520.8, 734 nm and obeying beers law in the concentration range of 20-100, 2-10, 2-10, 2-10 and 5-25 µg/ml respectively. In RP-HPLC mode the chromatographic separation was achieved by using column oyster C 18 (250 x 4.6mm, 5 ). The mobile phase consists of combination of ammonium acetate buffer (ph 5.8) and acetonitrile in the ratio 60:40 v/v respectively and was pumped at a rate of 1.0 ml/min at 30 0 C and the injection volume was 10 µl. The detection was carried out at 220nm and the calibration curve was linear in the range of 12-36 µg/ml. Retention time was 5.198 min. The correlation coefficient was found to be 0.9999. There was no significant difference in intra-day and inter-day analysis; the RSD value 0.769% indicates high precision of analytical method. The proposed methods are economical, simple, sensitive and accurate for quantitative determination of Naltrexone hydrochloride in bulk drug and pharmaceutical formulations.

115 P a g e International Standard Serial Number (ISSN): 2319-8141 INTRODUCTION: Analytical chemistry is the science of making quantitative and qualitative measurements. In practice, quantifying an analyte in a complex sample becomes an exercise in problem solving. To be efficient and effective, an analytical chemist must know the tools that are available to tackle a wide variety of problems. Analytical chemistry is divided into two branches qualitative and quantitative. A qualitative method provides information about the identity of atomic or molecular species or functional groups in sample. A quantitative method provides numerical information as to the relative amount of one or more of the components 1, 2 Quality control is a concept, which strives to produce a perfect product by series of measures designed to prevent and eliminate errors at different stages of production. The decision to release or reject a product is based on one or more types of control action. With the growth of pharmaceutical industry during last several years, there has been rapid progress in the field of pharmaceutical analysis involving complex instrumentation. Providing simple analytical procedure for complex formulation is a matter of most importance. It becomes necessary, therefore, to develop new analytical method for such drugs. In brief the reasons for the development of newer methods of drugs analysis are: The drug or drug combination may not be official in any pharmacopoeias. A proper analytical procedure for the drug may not be available in the literature due to patent regulations. Analytical methods may not be available for estimation of drug in the form of formulation excipients. Pharmaceutical analysis deals not only with medicaments (drugs and formulations), but also with their precursors i.e. with the raw material whose degree of purity, which in turn decides the quality of medicaments. The quantity of a drug is determined, after establishing its authenticity, which is carried by testing its purity and the quality of the pure substance in the drug and its formulations. Varieties of analytical methods are used for the analysis of drugs in bulk, formulations and biological samples. In pharmaceutical industry, spectrophotometric and chromatographic methods have gained the significance in recent years.

116 P a g e International Standard Serial Number (ISSN): 2319-8141 ULTRAVIOLET-VISIBLE ABSORPTION SPECTROSCOPY SPECTROPHOTOMETRIC METHODS It is defined as a method of analysis that embraces the measurement of absorption by chemical species of radiant energy at definite and narrow wavelength approximating monochromatic radiation. The fundamental law that governs the quantitative spectrophotometric analysis is the Beer- Lambert s law which is stated as; When a beam of monochromatic light is passed through a transparent cell containing a solution of an absorbing substance, reduction of intensity of the light may occurs; the rate of reduction in intensity with the thickness of the medium is proportional to the intensity of the light and the concentration of the absorbing substances. Mathematically Beer-Lamberts law is expressed as; A = a b c Where, A = absorbance or optical density, a = absorptivity or extinction coefficient, b = path length of radiation through sample (cm), c = concentration of solute in solution. This deals with the absorption of electromagnetic radiation in the wavelength region of 160 to 780 nm. UV absorption spectroscopy deals with absorption of light by a sample in the Ultra Violet (UV) region (190-380 nm), while Visible region (380-780 nm). Absorption spectroscopy (colorimetry) deals with absorption of light by a sample in the visible region (380-780 nm). Currently, research grade UV-VIS absorption instruments come in two configurations. The first is called scanning spectrophotometer because it measures the intensity of transmitted light of a narrow band pass, and scans the wavelength in time in order to collect a spectrum. Because absorption is a ratiometric measurement, these instruments generally require the user to measure two spectra, one sample and one blank. The blank should be identical to the sample in every way except that the absorbing species of interest is not present. This can be done either consecutively with a single beam instrument followed by the ratio calculation, or simultaneously with a dual beam instrument. The dual beam method is faster, and has the added advantage that lamp drift and other slow intensity fluctuations are properly accounted for in the

117 P a g e International Standard Serial Number (ISSN): 2319-8141 DRUG PROFILE Naltrexone hydrochloride is an opioid receptor antagonist used primarily in the management of alcohol dependence and opioid dependence. It is marketed as generic from as its hydrochloride salt, Naltrexone Hydrochloride 18. It is official in Martindale 19, Merck Index 20, USP 21,.BP 22. Chemical structure : Chemical Name : (5α)-17-(cyclopropylmethyl)-4,5-epoxy-3,14-dihydroxy morphinan -6-one hydrochloride. Empirical formula : C 20 H 23 N0 4.HCl Molecular weight : 377.86 Physical properties : A white or almost white, very hygroscopic powder. Melting point : 276 0 c Solubility : It is freely soluble in water; slightly soluble in alcohol, practically insoluble in dichloromethane. Storage : Store at room temperature in a cool place. Protect from light. Mechanism of action Naltrexone binds to the opioid mu (µ) receptor antagonistically, thereby preventing conventional opiate (heroin, morphine) drugs from binding and inducing opioid neural responses. The mechanism of action of Naltrexone in alcoholism is not understood; however, involvement of the endogenous opioid system is suggested by preclinical data. Naltrexone competitively binds to such receptors and may block the effects of endogenous opioids. Pharmacological action Opioid receptor antagonist markedly attenuating or completely blocking, reversibly, the subjective effects of IV administered opioids.

118 P a g e International Standard Serial Number (ISSN): 2319-8141 Indications and Usage Alcohol dependence; blockade of exogenously administered opioids (oral); treatment of alcohol dependence in patients able to abstain from alcohol in an outpatient setting (IM injection). Unlabeled Uses Eating disorders; postconcussional syndrome unresponsive to other treatments. DRUG PROFILE Naltrexone hydrochloride is an opioid receptor antagonist used primarily in the management of alcohol dependence and opioid dependence. It is marketed as generic from as its hydrochloride salt, Naltrexone Hydrochloride 18. It is official in Martindale 19, Merck Index 20, USP 21,.BP 22. Chemical structure : Chemical Name : (5α)-17-(cyclopropylmethyl)-4,5-epoxy-3,14-dihydroxy morphinan -6-one hydrochloride. Empirical formula : C 20 H 23 N0 4.HCl Molecular weight : 377.86 Physical properties : A white or almost white, very hygroscopic powder. Melting point : 276 0 c Solubility : It is freely soluble in water; slightly soluble in alcohol, practically insoluble in dichloromethane. Storage : Store at room temperature in a cool place. Protect from light. Mechanism of action Naltrexone binds to the opioid mu (µ) receptor antagonistically, thereby preventing conventional opiate (heroin, morphine) drugs from binding and inducing opioid neural responses. The mechanism of action of Naltrexone in alcoholism is not understood; however, involvement of the endogenous opioid system is suggested by preclinical data. Naltrexone competitively binds to such receptors and may block the effects of endogenous opioids.

119 P a g e International Standard Serial Number (ISSN): 2319-8141 Pharmacological action Opioid receptor antagonist markedly attenuating or completely blocking, reversibly, the subjective effects of IV administered opioids. Indications and Usage Alcohol dependence; blockade of exogenously administered opioids (oral); treatment of alcohol dependence in patients able to abstain from alcohol in an outpatient setting (IM injection). Unlabeled Uses Eating disorders; postconcussional syndrome unresponsive to other treatments. UV-VISIBLE SPECTROPHOTOMETERIC METHODS. Method A : Uv Spectrophotometeric Method (Reference Method By Cosolvancy ). Method B : Visible Spectrophotometeric Method By Using Mbth Reagent. Method C : Visible Spectrophotometeric Method By Using Potassium Ferricyanide Reagent. Method D : Visible Spectrophotometeric Method By Using 1,10 Phenanthroline Reagent. Method E : Visible Spectrophotometeric Method By Using 2,2' Bipyridyl Reagent. Method F : Visible Spectrophotometeric Method By Using Folin Cio-Calteu Reagent. INSTRUMENTS USED 1. A systronics 119 UV/visible spectrophotometer with 1 cm matched quartz cells were used. CHEMICALS AND REAGENTS: All chemicals and reagent used were of Analytical grade. Sr. No. Name Source 1. Distilled ethanol SD Fine Chemical, Mumbai. 2. Ferric chloride SD Fine Chemical, Mumbai. 3. MBTH Loba Chemicals, Mumbai. 4. Potassium Ferricyanide SD Fine Chemical, Mumbai. 5. 1, 10 Phenanthroline SD Fine Chemical, Mumbai. 6. 2, 2' Bipyridyl Qualigens, Mumbai. 7. Folin-Ciocalteu reagent (FC) Loba Chemicals, Mumbai. 8. Sodium Hydroxide Qualigens, Mumbai. Bulk drug of Naltrexone Hydrochloride was obtained from Intas Pharmaceutical Pvt Ltd, Ahmedabad. Formulations of Naltrexone Hydrochloride used for the study are Naltima (Intas pharma Pvt. Ltd) and Nodict (Sun Pharm Ltd) both containing 50 mg were procured from local market.

120 P a g e International Standard Serial Number (ISSN): 2319-8141 Preparation of Reagents 1. 1% aqueous Ferric chloride solution 1 gm of ferric chloride reagent was accurately weighed and dissolved in distilled water in 100mL volumetric flask, volume was made upto the mark with distilled water. 2. 0.5% aqueous Ferric chloride solution 0.5 gm of ferric chloride reagent was accurately weighed and dissolved in distilled water in 100mL volumetric flask, volume was made upto the mark with distilled water. 3. 0.4% aqueous Ferric chloride solution 0.4 gm of ferric chloride reagent was accurately weighed and dissolved in distilled water in 100mL volumetric flask, volume was made upto the mark with distilled water. 4. 0.5% aqueous MBTH Reagent solution 0.5 gm of MBTH reagent was accurately weighed and dissolved in distilled water in 100mL volumetric flask, volume was made upto the mark with distilled water. 5. 0.1% aqueous Potassium ferricyanide solution 0.1 gm of potassium ferricyanide was accurately weighed and dissolved in distilled water in 100mL volumetric flask, volume was made upto the mark with distilled water. 6. 0.2% alcoholic 1, 10 Phenanthroline solution 0.2 gm of 1, 10 phenanthroline reagent was accurately weighed and dissolved in distilled ethanol in 100mL volumetric flask, volume was made upto the mark with distilled ethanol. 7. 0.2% alcoholic 2, 2' Bipyridyl solution 0.2 gm of 2, 2' Bipyridyl reagent was accurately weighed and dissolved in distilled ethanol in 100mL volumetric flask, volume was made upto the mark with distilled ethanol. 8. 1N FC Reagent solution The available 2N FC reagent is diluted in the ratio of 1:1 with distilled water. 9.1N aqueous Sodium Hydroxide solution 4 gm of sodium hydroxide was accurately weighed and dissolved in distilled water in 100 ml volumetric flask, volume was made upto the mark with distilled water. METHOD A (UV Spectrophotometric method; Reference method by Cosolvancy). A. Preparation of standard calibration curve of bulk drug. 1. Solvent Used Distilled water and ethanol were used as solvent.

121 P a g e International Standard Serial Number (ISSN): 2319-8141 2. Preparation of standard stock solution Accurately weighed 100 mg of Naltrexone Hydrochloride (bulk drug or its formulation) was dissolved in 40 ml of distilled water in 100mL volumetric flask and volume was made upto the mark with distilled ethanol (i.e.1000 µg/ml). 3. Preparation of calibration curve Fresh aliquots of Naltrexone Hydrochloride ranging from 0.5 to 2.5 ml (1 ml-1000µg/ml) were transferred into a series of 10 ml volumetric flasks to provide final concentration range of 50 to 250 μg/ml. The solution in each tube were made upto the mark with distilled ethanol. The absorbance of solution was measured at 281 nm against the reagent blank (Figure 3.1). The amount of Naltrexone Hydrochloride present in the sample solution was computed from its calibration curve (Figure 3.2). B. Analysis of tablet formulation The sample of the powder tablet claimed to contain 50 mg. The accurate quantity equivalent to 100 mg of active ingredient was extracted with distilled water (3 x 15 ml) and filtered through a 0. 45 µm membrane filter, followed by adding distilled ethanol upto 100 ml to get the stock solution of 1 mg/ml. Subsequent dilutions of this solution were made with distilled ethanol to get concentration of 50-250 µg/ml and were prepared as above and analyzed at the selected analytical wavelengths, 281 nm and their results were statically validated.

122 P a g e International Standard Serial Number (ISSN): 2319-8141 METHOD A (UV SPECTROPHOTOMETERIC METHOD BY COSOLVANCY) Figure 3.1:- Absorption spectra for Naltrexone hydrochloride in distilled water and alcohol (150 g/ml, λmax 281 nm) Figure 3.2:- Calibration curve for Naltrexone hydrochloride at 281 nm with distilled water and ethanol Table 3.1:- Data for absorption spectrum of Naltrexone hydrochloride for UV method (Drug concentration = 150 g/ml) 215 0.288 230 0.353 250 0.412 265 0.460 281 0.510 310 0.437 330 0.364 350 0.227

123 P a g e International Standard Serial Number (ISSN): 2319-8141 Table 3.2:- Result of calibration curve for Naltrexone hydrochloride by UV spectroscopy Concentration Absorbance (μg/ml) Mean ± S.D* % CV 0 0.000 ± 0.0000 0 50 0.171±0.002 1.16 100 0.338±0.004 1.18 150 0.510±0.002 0.392 200 0.679±0.004 0.589 250 0.854±0.002 0.234 *Average of three determinations. Table 3.3:- Optical characteristic and precision by UV spectroscopy UV(BY COSOLVANCY Parameter METHOD) max (nm) 281 Beer s law limits ( g/ml) (c) 50-250 Molar absorptivity (lit. mol -1 cm -1 ) 1.288 x 10 3 Limit of Detection (LOD/ mcgml -1 ) 3.23 Limit of Quantification (LOQ/ mcgml -1 ) 9.79 Sandell s sensitivity ( g/ml 0.001 abs unit) 0.00295 Regression equation (Y*) Slope (b) 3.406 x 10-3 Intercept (a) 3 x 10-4 Standard error of estimation (Se) 0.0016 Correlation coefficient (r) 0.9999 % RSD 0.0047 Range of Errors** Confidence limit with 0.05 level Confidence limit with 0.01 level 0.0039 0.0058 % Error in bulk Samples*** 0.0248 *Y=bC+a, where C is the concentration of Naltrexone hydrochloride in g/ml and Y is the absorbance at the respective maximum absorbency, **Average of eight determination, ***Average of three determination.

124 P a g e International Standard Serial Number (ISSN): 2319-8141 Table 3.4:- Assay and Recovery of Naltrexone hydrochloride in Pharmaceutical dosage form (Tablet) by UV Method Sample Labeled amount (mg) Amount obtained (mg) Proposed method* % recovery T1 50 48.76 97.52 T2 50 49.21 98.42 T1 and T2 are tablets from different manufactures, *Average of 5 determinations (50 mg of Naltrexone hydrochloride was added and recovered). Table 3.5:- Determination of Accuracy of Naltrexone hydrochloride Level of % Recovery Amount of drug in Formulation* (mg) Amount of standard drug added (mg) Total amount of drug found (mg) % Recovery of pure drug 80 % 49.76 40 88.01 97.78 49.07 40 88.89 98.76 48.85 40 88.76 98.62 100 % 49.95 50 99.80 99.80 48.82 50 98.31 98.31 49.05 50 98.87 98.87 120 % 49.87 60 109.72 99.74 49.28 60 108.55 98.68 49.56 60 109.38 99.43 *Pre-analyzed sample taken from T 1 and T 2. Table 3.6:- Statistical data for accuracy determination Level of % Recovery Total amount found (mg) Mean* Standard deviation* Coefficient of variation* (%) Standard error* 80 % 88.55 0.4750 0.5364 0.2742 100 % 98.99 0.7526 0.7602 0.4345 120 % 109.2 0.6019 0.5511 0.3745 *The results are the mean of three readings at each level of recovery.

125 P a g e International Standard Serial Number (ISSN): 2319-8141 Table 3.7:- Repeatability Data for Naltrexone hydrochloride at 281nm Concentration Std. Abs 1 Abs 2 Abs 3 Mean (μg/ml) Deviation* 50 0.169 0.171 0.173 0.171 0.002 100 0.339 0.334 0.343 0.338 0.004 150 0.509 0.508 0.513 0.510 0.002 200 0.675 0.678 0.684 0.679 0.004 250 0.851 0.855 0.856 0.854 0.002 *Average of three determinations. METHOD B (Visible Spectrophotometric method by MBTH Reagent). EXPERIMENTAL A. Preparation of standard calibration curve of bulk drug. 1. Solvent Used Distilled ethanol and distilled water were used as solvent. 2. Preparation of standard stock solution Accurately weighed 100 mg of Naltrexone Hydrochloride (bulk drug or its formulation) was dissolved in 40 ml of distilled water in 100mL volumetric flask and volume was made upto the mark with distilled ethanol (i.e.1000 µg/ml). 3. Preparation of calibration curve Fresh aliquots of Naltrexone Hydrochloride ranging from 0.2 to 1 ml (1 ml-1000 µg/ml) were transferred into a series of 10 ml volumetric flasks to provide final concentration range of 20 to 100 µg/ml. To each flask 1 ml of aqueous ferric chloride (1 %) solution and 0.2 ml of MBTH reagent (0.5% in distilled water) were added. Aliquotes were kept aside for 10 mins. The solutions in each tube were made upto mark with distilled water. The absorbance of green colored chromogen was measured at 661 nm against the reagent blank (Figure 3.3). The color species was stable for 1.2 hour. The amount of Naltrexone Hydrochloride present in the sample solution was computed from its calibration curve (Figure 3.4). B. Analysis of tablet formulation The sample of the powder tablet claimed to contain 50 mg. The accurate quantity equivalent to 100 mg of active ingredient was extracted with distilled water (3 x 15mL) and filtered through a 0. 45 µm membrane filter, followed by adding distilled ethanol upto 100mL to get the stock

126 P a g e International Standard Serial Number (ISSN): 2319-8141 solution of 1 mg/ml. Subsequent dilutions of this solution were made with distilled water to get concentration of 20-100 µg/ml and were prepared as above and analyzed at the selected analytical wavelengths, 661 nm and their results were statically validated. Figure 3.3:- Absorption spectra for Naltrexone hydrochloride by using MBTH reagent (60 g/ml, λmax 661nm) Figure3.4:- Calibration curve for Naltrexone hydrochloride at 661 nm with 1% FeCl 3 and 0. 5% MBTH reagent Table3.8:- Data for absorption spectrum of Naltrexone hydrochloride for MBTH method (Drug concentration = 60 g/ml) Wavelength (nm) Absorbance 620 0.452 630 0.591 640 0.673 650 0.739 661 0.812 670 0.783 680 0.643 690 0.503

127 P a g e International Standard Serial Number (ISSN): 2319-8141 Table3.9:- Result of calibration curve for Naltrexone by MBTH reagent method Concentration Absorbance (μg/ml) Mean ± S.D* % CV 0 0.000 ± 0.0000 0 20 0.203±0.004 1.97 40 0.403±0.004 0.992 60 0.609±0.004 0.656 80 0.812±0.003 0.369 100 1.015±0.003 0.295 *Average of three determinations. Table3.10:- Optical characteristic and precision by MBTH Method Parameter max (nm) 661 Beer s law limits ( g/ml) (c) 20-100 MBTH Method Molar absorptivity (lit. mol -1 cm -1 ) 3.8350 x 10 4 Limit of Detection (LOD/ mcgml -1 ) 0.501 Limit of Quantification (LOQ/ mcgml -1 ) 1.52 Sandell s sensitivity ( g/ml 0.001 abs unit) 0.0012 Regression equation (Y*) Slope (b) 0.01021 Intercept (a) -1.334 x 10-4 Standard error of estimation (Se) 0.287 Correlation coefficient (r) 0.9999 % RSD 0.190 Range of Errors** Confidence limit with 0.05 level Confidence limit with 0.01 level 0.0019 0.0013 % Error in bulk Samples*** 2.27 *Y=bC+a, where C is the concentration of Naltrexone hydrochloride in g/ml and Y is the absorbance at the respective maximum absorbency, **Average of eight determination, ***Average of three determination.

128 P a g e International Standard Serial Number (ISSN): 2319-8141 Table3.11:-Assay and Recovery of Naltrexone hydrochloride in Pharmaceutical dosage form (Tablet) by MBTH Method Sample Labeled amount (mg) Amount obtained* (mg) Proposed Method Reference method (UV Method by cosolvancy) % recovery T1 50 49.96 48.76 98.9 T2 50 48.96 49.21 98.96 T1 and T2 are tablets from different manufactures, *Average of 5 determinations (50 mg of Naltrexone hydrochloride was added and recovered). Table3.12:- Determination of Accuracy of Naltrexone hydrochloride Level of % Recovery Amount of drug in formulation* (mg) Amount of standard drug added (mg) Total amount Of drug found(mg) % Recovery of pure drug 49.96 40 89.78 99.75 80 % 49.83 40 89.78 99.75 49.62 40 89.01 98.90 49.85 50 99.70 99.7 100 % 49.92 50 99.85 99.85 49.27 50 99.17 99.17 49.95 60 109.88 99.89 120 % 49.79 60 109.58 99.61 49.82 60 109.72 99.74 *Pre-analyzed sample taken from T 1 and T 2. Table3.13:- Statistical data for Accuracy determination Level of % Recovery Total amount found (mg) Mean* Standard deviation* Coefficient of variation* (%) Standard error* 80 % 89.52 0.4446 0.4966 0.2567 100 % 99.57 0.3573 0.3588 0.2063 120 % 109.70 0.1501 0.1368 0.0866 *The results are the mean of three readings at each level of recovery.

129 P a g e International Standard Serial Number (ISSN): 2319-8141 Table3.14:- Repeatability Data for Naltrexone hydrochloride at 661 nm Concentration Std. Abs 1 Abs 2 Abs 3 Mean (μg/ml) Deviation* 20 0.203 0.199 0.207 0.203 0.004 40 0.404 0.398 0.407 0.403 0.004 60 0.613 0.605 0.609 0.609 0.004 80 0.809 0.816 0.811 0.812 0.003 100 1.015 1.018 1.012 1.015 0.003 *Average of three determinations. Nature of colored Species An attempt has been made to indicate the nature of colored species in the proposed method for Naltrexone hydrochloride (which contain phenolic hydroxyl group), tentatively based on analogy (reactive functional groups in drug, reagents nature) and probability (relative reactivities and impact of functional moieties one over the other). This method is based on the oxidative coupling of Naltrexone hydrochloride with MBTH in the presence of ferric chloride. MBTH on oxidation with ferric chloride looses two electrons and one proton to form an electrophilic intermediate, which is the active coupling species, reacts with the coupler (Naltrexone hydrochloride) by electrophilic attack on the most nucleophilic site of Naltrexone hydrochloride to form a bluish green colored species (as shown in Scheme-3.1) with absorption maximum at 661 nm (Figure-3.3) and Beer s law is obeyed in the concentration range of 20-100 g/ml (Figure-3.4.).

130 P a g e International Standard Serial Number (ISSN): 2319-8141 Naltrexone hydrochloride reaction with MBTH Scheme 3.1 METHOD C (Visible Spectrophotometric method by Potassium ferricyanide) EXPERIMENTAL A. Preparation of standard calibration curve of bulk drug. 1. Solvent Used Distilled water and ethanol are used as solvent. 2. Preparation of standard stock solution Accurately weighed 100 mg of Naltrexone Hydrochloride (bulk drug or its formulation) was dissolved in 40 ml of distilled water in 100 ml volumetric flask and volume was made upto the mark with distilled ethanol (i.e.1000 µg/ml). Stock solution A.

131 P a g e International Standard Serial Number (ISSN): 2319-8141 From the above stock solution A 10 ml of solution was pipetted out in 100 ml volumetric flask and the volume was made up to the mark with distilled ethanol to obtain the final concentration of 100 µg/ml. Stock solution B. 3. Preparation of calibration curve Fresh aliquots of Naltrexone Hydrochloride ranging from 0.2 to 1.0 ml (1 ml-1000µg/ml) were transferred into a series of 10 ml volumetric flasks to provide final concentration range of 2 to 10 μg/ml. To each flask 1ml of aqueous Ferric chloride (1%) solution and 1 ml of potassium ferricyanide (0.1% in distilled water) were added. The solution in each tube were made upto the mark with distilled water. The absorbance of bluish green colored chromogen was measured at 715 nm against the blank (Figure 3.5). The color species was stable for one hour. The amount of Naltrexone Hydrochloride present in the sample solution was computed from its calibration curve (Figure 3.6). B. Analysis of tablet formulation The sample of the powder tablet claimed to contain 50 mg. The accurate quantity equivalent to 100 mg of active ingredient was extracted with distilled water (3 x 15mL) and filtered through a 0. 45 µm membrane filter, followed by adding distilled ethanol upto 100mL to get the stock solution of 1 mg/ml. Subsequent dilutions of this solution were made with distilled water to get concentration of 2-10 µg/ml and were prepared as above and analyzed at the selected analytical wavelengths, 715 nm and their results were statically validated.

132 P a g e International Standard Serial Number (ISSN): 2319-8141 Figure3.5:- Absorption spectra for Naltrexone hydrochloride by using potassium ferricyanide and ferric chloride (6 g/ml, λmax 715 nm) Figure3.6:- Calibration curve for Naltrexone hydrochloride at 715 nm with 1% FeCl 3 and 0.1% potassium ferricyanide reagent Table3.15:-Data for absorption spectrum of Naltrexone hydrochloride for potassium ferricyanide method (Drug concentration = 6 g/ml) Wavelength (nm) Absorbance 675 0.35 685 0.489 695 0.654 705 0.796 715 0.846 725 0.786 735 0.633 745 0.503

133 P a g e International Standard Serial Number (ISSN): 2319-8141 Table 3.16:- Result of calibration curve for Naltrexone hydrochloride by Potassium ferricyanide method Concentration Absorbance (μg/ml) Mean ± S.D* % CV 0 0.00 ± 0.00 0 2 0.213 ± 0.005 2.347 4 0.426 ± 0.002 0.469 6 0.639 ± 0.004 0.625 8 0.852 ± 0.006 0.704 10 1.065 ± 0.005 0.469 *Average of three determinations. Table 3.17:- Optical characteristic and precision by potassium ferricyanide method Parameter Potassium ferricyanide method max (nm) 715 Beer s law limits ( g/ml) (c) 2-10 Molar absorptivity (lit. mol -1 cm -1 ) 4.0244 x 10 3 Limit of Detection (LOD/ mcgml -1 ) 0.052 Limit of Quantification (LOQ/ mcgml -1 ) 0.159 Sandell s sensitivity ( g/ml 0.001 abs unit) 0.0042 Regression equation (Y*) Slope (b) 0.10594 Intercept (a) 5.333 x 10-4 Standard error of estimation (Se) 0.0059 Correlation coefficient (r) 0.9999 % RSD 0.264 Range of Errors** Confidence limit with 0.05 level Confidence limit with 0.01 level % Error in bulk Samples*** 2.4 0.0020 0.0014 *Y=bC+a, where C is the concentration of Naltrexone hydrochloride in g/ml and Y is the absorbance at the respective maximum absorbency, **Average of eight determination, ***Average of three determination.

134 P a g e International Standard Serial Number (ISSN): 2319-8141 Table 3.18:- Assay and Recovery of Naltrexone hydrochloride in Pharmaceutical dosage form (Tablet) by potassium ferricyanide method Sample Labeled amount (mg) Amount obtained* (mg) Proposed Method Reference method (UV Method by cosolvancy) % recovery T1 50 48.8 48.76 99.02 T2 50 49.7 48.21 98.57 T1 and T2 are tablets from different manufactures, *Average of 5 determinations (50 mg of Naltrexone hydrochloride was added and recovered). Table 19:- Determination of Accuracy of Naltrexone hydrochloride Level of % Recovery Amount Of drug in formulation* (mg) Amount of standard drug added (mg) Total amount Of drug found (mg) % Recovery of pure drug 49.93 40 89.74 99.71 80 % 49.35 40 89.30 99.22 49.81 40 89.48 99.42 49.89 50 99.55 99.55 100 % 49.72 50 99.55 99.55 49.45 50 99.37 99.37 49.25 60 109.07 99.15 120 % 49.82 60 109.37 99.42 49.56 60 109.31 99.37 *Pre-analyzed sample taken from T 1 and T 2. Level of % Recovery Table 3.20:- Statistical data for Accuracy determination Total amount found (mg) Mean* Standard deviation* Coefficient of variation* (%) Standard error* 80 % 89.51 0.2212 0.2481 0.1277 100 % 99.49 0.1039 0.1044 0.0600 120 % 109.30 0.1587 0.1451 0.0915 *The results are the mean of three readings at each level of recovery.

135 P a g e International Standard Serial Number (ISSN): 2319-8141 Table 3.21:- Repeatability Data for Naltrexone hydrochloride at 715 nm Concentration Std. Abs 1 Abs 2 Abs 3 Mean (μg/ml) Deviation* 2 0.208 0.218 0.219 0.213 0.005 4 0.425 0.424 0.429 0.426 0.002 6 0.635 0.643 0.639 0.639 0.004 8 0.845 0.853 0.858 0.852 0.006 10 1.059 1.067 1.069 1.065 0.005 *Average of three determinations. Nature of Colored species An attempt has been made to indicate the nature of colored species in the proposed method for Naltrexone hydrochloride (which contain phenolic hydroxyl group), tentatively based on analogy (reactive functional groups in drug, reagents nature) and probability (relative reactivities and impact of functional moieties one over the other). This method is based on the reaction of drug with ferric chloride and potassium ferricyanide, which forms blue colored chromogens (as shown in scheme-3.2) exhibiting absorption maximum at 715 nm (Fig.-3.5) against reagent blank and obeyed Beer s law in the concentration range of 2-10 g/ml (Fig.-3.6). In this method, Naltrexone hydrochloride exhibits reducing property due to the presence of functional moieties (one or more) vulnerable to oxidation selectively with oxidizing agents such as Fe (III) under controlled experimental conditions when treated with known excess of oxidant, Naltrexone hydrochloride undergoes oxidation, giving products of oxidation [inclusive of reduced form of oxidant Fe (II) from Fe (III)] besides unreacted oxidant. It is possible to estimate the drug content colorimetrically, which is equivalent to either reduced oxidant or reduced form of oxidant formed. The reduced form of Fe (III) (i.e., Fe (II) has a tendency to give a colored complex on treatment with K 3 Fe [(CN) 6 ] 3-. Reaction of Naltrexone hydrochloride with Potassium ferricyanide Naltrexone hydrochloride + Fe (III) Oxidation products + Fe (III) + Unreacted Fe (III) 3 Fe 2 + + 2 [ Fe (CN) 6 ] -3 Fe 3 [ Fe (CN) 6 ] 2 Scheme 3.2

136 P a g e International Standard Serial Number (ISSN): 2319-8141 METHOD D (Visible Spectrophotometric method by 1, 10 Phenanthroline) EXPERIMENTAL A. Preparation of standard calibration curve of bulk drug. 1. Solvent Used Distilled water is used as solvent. 2. Preparation of standard stock solution Accurately weighed 100 mg of Naltrexone Hydrochloride (bulk drug or its formulation) was dissolved in 40 ml of distilled water in 100 ml volumetric flask and volume was made upto the mark with distilled water (i.e.1000 µg/ml). Stock solution A. From the above stock solution A 10 ml of solution was pipetted out in 100 ml volumetric flask and the volume was made up to the mark with distilled water to obtain the final concentration of 100 µg/ml. Stock solution B. 3. Preparation of calibration curve Fresh aliquots of Naltrexone Hydrochloride ranging from 0.2 to 10 ml (1 ml-100µg/ml) were transferred into a series of 10 ml volumetric flasks to provide final concentration range of 2 to 10 μg/ml. To each flask 0.5 ml of ferric chloride (0.4%) solution and 1 ml of alcoholic 1, 10 phenanthroline (0.2%) were added and heated at 40 0 C for 10 mins. The solutions were cooled to room temperature and made upto the mark with distilled water. The absorbance of blood red colored chromogen was measured at 509.6 nm against the reagent blank (Figure 3.7). The color species was stable for 1.2 hour. The amount of Naltrexone Hydrochloride present in the sample solution was computed from its calibration curve (Figure 3.8). B. Analysis of tablet formulation The sample of the powder tablet claimed to contain 50 mg. The accurate quantity equivalent to 100 mg of active ingredient was extracted with distilled water (3 x 15mL) and filtered through a 0. 45 µm membrane filter, followed by adding distilled water upto 100mL to get the stock solution of 1 mg/ml. Subsequent dilutions of this solution were made with distilled water to get concentration of 2-10 µg/ml and were prepared as above and analyzed at the selected analytical wavelengths, 509.6 nm and their results were statically validated.

137 P a g e International Standard Serial Number (ISSN): 2319-8141 Figure 3.7:- Absorption spectra for Naltrexone hydrochloride by 1, 10 Phenanthroline reagent (6 g/ml, λmax 509.6 nm) Figure 3.8:- Calibration curve for Naltrexone hydrochloride at 509.6 nm by ferric chloride (0.4%) and 1, 10 Phenanthroline reagent (0.2%) Table 3.22:- Data for absorption spectrum of Naltrexone hydrochloride for 1, 10 Phenanthroline method (Drug concentration = 150 g/ml) Wavelength (nm) Absorbance 470 0.56 480 0.602 490 0.638 500 0.669 509.6 0.709 520 0.678 530 0.508 540 0.485

138 P a g e International Standard Serial Number (ISSN): 2319-8141 Table 3.23:- Result of calibration curve for Naltrexone hydrochloride by 1, 10 Phenanthroline method Concentration Absorbance (μg/ml) Mean ± S.D* % CV 0 0.00 ± 0.00 0 2 0.219±0.003 1.36 4 0.438±0.003 0.684 6 0.657±0.002 0.304 8 0.876±0.002 0.228 10 1.095±0.003 0.273 *Average of three determinations. Table 3.24:- Optical characteristic and precision by 1, 10 Phenanthroline method Parameter 1, 10 Phenanthroline method max (nm) 509.6 Beer s law limits ( g/ml) (c) 2-10 Molar absorptivity (lit. mol -1 cm -1 ) 4.139 x 10 4 Limit of Detection (LOD/ mcgml -1 ) 0.022 Limit of Quantification (LOQ/ mcgml -1 ) 0.068 Sandell s sensitivity ( g/ml 0.001 abs unit) 0.0024 Regression equation (Y*) Slope (b) 0.1097 Intercept (a) -5.999 x 10-4 Standard error of estimation (Se) 0.0002672 Correlation coefficient (r) 0.9999 % RSD 0.115 Range of Errors** Confidence limit with 0.05 level 0.00632 Confidence limit with 0.01 level 0.00935 % Error in bulk Samples*** 0.12 *Y=bC+a, where C is the concentration of Naltrexone hydrochloride in g/ml and Y is the absorbance at the respective maximum absorbency, **Average of eight determination, ***Average of three determination.

139 P a g e International Standard Serial Number (ISSN): 2319-8141 Table 3.25:- Assay and Recovery of Naltrexone hydrochloride in Pharmaceutical dosage form (Tablet) by 1, 10 Phenanthroline reagent method. Sample Labeled amount (mg) Amount obtained* (mg) Proposed Method Reference method (UV Method by cosolvancy) % recovery T1 50 49.31 48.76 98.89 T2 50 49.34 49.21 98.92 T1 and T2 are tablets from different manufactures, *Average of 5 determinations (50 mg of Naltrexone hydrochloride was added and recovered). Table 3.26:- Determination of Accuracy of Naltrexone hydrochloride Level of % Recovery Amount Of drug in formulation* (mg) Amount of standard drug added (mg) Total amount Of drug found (mg) % Recovery of pure drug 49.65 40 89.37 99.30 80 % 49.77 40 89.22 99.13 49.92 40 89.84 99.82 49.83 50 99.66 99.66 100 % 49.76 50 99.68 99.68 49.45 50 99.20 99.20 49.82 60 109.71 99.73 120 % 49.78 60 109.45 99.50 49.53 60 109.31 99.37 *Pre-analyzed sample taken from T 1 and T 2. Table 3.27:- Statistical data for Accuracy determination Level of % Recovery Total amount found (mg) Mean* Standard deviation* Coefficient of variation* (%) Standard error* 80 % 89.48 0.3235 0.3615 0.1868 100 % 99.51 0.2715 0.2728 0.1568 120 % 109.50 0.2030 0.1853 0.1172 *The results are the mean of three readings at each level of recovery.

140 P a g e International Standard Serial Number (ISSN): 2319-8141 Table 3.28:- Repeatability Data for Naltrexone hydrochloride at 509.6 nm Concentration (μg/ml) Abs 1 Abs 2 Abs 3 Mean Std. Deviation* 2 0.216 0.222 0.219 0.219 0.003 4 0.441 0.435 0.438 0.438 0.003 6 0.656 0.660 0.655 0.657 0.002 8 0.872 0.877 0.879 0.876 0.002 10 1.098 1.095 1.092 1.095 0.003 *Average of three determinations. Nature of Colored Species An attempt has been made to indicate the nature of colored species in the proposed method for Naltrexone hydrochloride (which contain phenolic hydroxyl group), tentatively based on analogy (reactive functional groups in drug, reagents nature) and probability (relative reactivities and impact of functional moieties one over the other). In this method Naltrexone hydrochloride was made to react with excess of ferric chloride under specified experimental conditions into ferrous salt, the amount of conversion corresponds to the drug concentration. The ferrous ions thus formed in the reactions were made to react with 1,10 PTL to form blood red colored complex (as shown in scheme 3.3) due to the formation of six coordinate bonds between ferrous ion and six nitrogen atoms of three molecules of 1,10 PTL which have got a lone pair of electron each, with absorption maximum ( max) at 509.6 nm (Fig.- 3.7) and obeyed Beer s law in the concentration range of 2-10 g/ml (Fig.-3.8). The first step in the method is the oxidation of Naltrexone hydrochloride with oxidant. Naltrexone hydrochloride + Fe (III) Oxidation products + Fe (II). The second step concerns with the estimation of Fe (II) with 1,10-PTL. Reaction of Naltrexone hydrochloride with 1,10-Phenanthroline Scheme 3.3

141 P a g e International Standard Serial Number (ISSN): 2319-8141 METHOD E (Visible Spectrophotometric method by 2,2' Bipyridyl) EXPERIMENTAL A. Preparation of standard calibration curve of bulk drug. 1. Solvent Used Distilled water is used as solvent. 2. Preparation of standard stock solution Accurately weighed 100 mg of Naltrexone Hydrochloride (bulk drug or its formulation) was dissolved in 40 ml of distilled water in 100 ml volumetric flask and volume was made upto the mark with distilled water (i.e.1000 µg/ml). Stock solution A. From the above stock solution A 10 ml of solution was pipetted out in 100 ml volumetric flask and the volume was made up to the mark with distilled water to obtain the final concentration of 100 µg/ml. Stock solution B. 3. Preparation of calibration curve Fresh aliquots of Naltrexone Hydrochloride ranging from 0.2 to 10 ml (1 ml-100µg/ml) were transferred into a series of 10 ml volumetric flasks to provide final concentration range of 2 to 10 μg/ml. To each flask 0.5 ml of ferric chloride (0.5%) solution and 0.5 ml of alcoholic 2, 2' Bipyridyl (0.2%) were added and heated at 40 0 C for 20 mins. The solutions were cooled to room temperature and made upto the mark with distilled water. The absorbance of orange red colored chromogen was measured at 520.8 nm against the reagent blank (Figure 3.9). The color species was stable for 1.2 hour. The amount of Naltrexone Hydrochloride present in the sample solution was computed from its calibration curve (Figure 3.10). B. Analysis of tablet formulation The sample of the powder tablet claimed to contain 50 mg. The accurate quantity equivalent to 100 mg of active ingredient was extracted with distilled water (3 x 15mL) and filtered through a 0. 45 µm membrane filter, followed by adding distilled water upto 100mL to get the stock solution of 1 mg/ml. Subsequent dilutions of this solution were made with distilled water to get concentration of 2-10 µg/ml and were prepared as above and analyzed at the selected analytical wavelengths, 520.8 nm and their results were statically validated.

142 P a g e International Standard Serial Number (ISSN): 2319-8141 Figure 3.9:- Absorption spectra for Naltrexone hydrochloride by 2, 2' Bipyridyl method (6 g/ml, λmax 520.8 nm) Figure 3.10:- Calibration curve for Naltrexone hydrochloride at 520.8nm by Ferric chloride (0.5%) and 2, 2' Bipyridyl (0.2%) 3.29:- Data for absorption spectrum of Naltrexone Table hydrochloride for 2, 2' Bipyridyl (Drug concentration = 150 g/ml) Wavelength (nm) Absorbance 480 0.351 490 0.389 500 0.432 510 0.467 520.8 0.498 530 0.472 540 0.401 550 0.337

143 P a g e International Standard Serial Number (ISSN): 2319-8141 Table 3.30:- Result of calibration curve for Naltrexone hydrochloride by 2, 2' Bipyridyl method Concentration Absorbance (μg/ml) Mean ± S.D* % CV 0 0.00 ± 0.00 0 2 0.166 ± 0.002 1.204 4 0.332 ± 0.003 0.906 6 0.498 ± 0.003 0.602 8 0.665 ± 0.002 0.30 10 0.828 ± 0.003 0.362 *Average of three determinations. Table 3.31:- Optical characteristic and precision by 2, 2' Bipyridyl method Parameter max (nm) 520.8 Beer s law limits ( g/ml) (c) 2-10 2, 2' Bipyridyl method Molar absorptivity (lit. mol -1 cm -1 ) 3.1636 x 10 4 Limit of Detection (LOD/ mcgml -1 ) 3.60 Limit of Quantification (LOQ/ mcgml -1 ) 10.92 Sandell s sensitivity ( g/ml 0.001 abs unit) 0.0011 Regression equation (Y*) Slope (b) 0.0681 Intercept (a) 0.0583 Standard error of estimation (Se) 0.00186 Correlation coefficient (r) 0.9987 % RSD 1.055 Range of Errors** Confidence limit with 0.05 level 0.0044 Confidence limit with 0.01 level 0.0065 % Error in bulk Samples*** 0.0056 *Y=bC+a, where C is the concentration of Naltrexone hydrochloride in g/ml and Y is the absorbance at the respective maximum absorbency, **Average of eight determination, ***Average of three determination.

144 P a g e International Standard Serial Number (ISSN): 2319-8141 Table 3.32:- Assay and Recovery of Naltrexone hydrochloride in Pharmaceutical dosage form (Tablet) by 2, 2' Bipyridyl method Amount obtained* (mg) Sample Labeled amount (mg) Proposed Method Reference method (UV Method by cosolvancy) % recovery T1 50 49.45 48.76 97.52 T2 50 49.72 49.21 99.44 T1 and T2 are tablets from different manufactures, *Average of 5 determinations (50 mg of Naltrexone hydrochloride was added and recovered). Level of % Recovery 80 % 100 % 120 % Table 3.33:- Determination of Accuracy of Naltrexone hydrochloride Amount Of drug in formulation* (mg) *Pre-analyzed sample taken from T 1 and T 2. Amount of standard drug added (mg) Total amount Of drug found (mg) % Recovery of pure drug 49.82 40 89.31 99.32 49.56 40 89.41 99.34 49.85 40 89.77 99.74 49.72 50 99.31 99.31 49.85 50 99.74 99.74 49.92 50 99.83 99.83 49.85 60 109.10 99.18 49.39 60 109.21 99.28 49.79 60 109.54 99.58

145 P a g e International Standard Serial Number (ISSN): 2319-8141 Level of % Recovery Table 3.34:- Statistical data for Accuracy determination Total amount found (mg) Mean* Standard deviation* Coefficient of variation* (%) Standard error* 80 % 89.50 0.2419 0.2702 0.1397 100 % 99.63 0.2779 0.2789 0.1605 120 % 109.30 0.2290 0.2095 0.1322 *The results are the mean of three readings at each level of recovery. Table 3.35:- Repeatability Data for Naltrexone hydrochloride at 520.8 nm Concentration (μg/ml) Abs 1 Abs 2 Abs 3 Mean Std. Deviation* 2 0.164 0.165 0.169 0.166 0.002 4 0.329 0.332 0.335 0.332 0.003 6 0.495 0.498 0.501 0.498 0.003 8 0.663 0.668 0.664 0.665 0.002 10 0.825 0.831 0.828 0.828 0.003 *Average of three determinations. Nature of Colored Species An attempt has been made to indicate the nature of colored species in the proposed method for Naltrexone hydrochloride (which contain phenolic hydroxyl group), tentatively based on analogy (reactive functional groups in drug, reagents nature) and probability (relative reactivities and impact of functional moieties one over the other). This method was also worked out on the principle where 2,2'-bipyridyl forms complex with ferric (III) ions followed by the oxidation of the drug by this complex which results into the formation of orange red colored chromogen (as shown in scheme-3.4). This colored species exhibited absorption maxima ( max) at 520.8 nm (Fig.-3.9) and Beer s law is obeyed in the concentration range of 2-10 g/ml (Fig.- 3.10). The first step in the methods mentioned above is the oxidation of Naltrexone hydrochloride with oxidant. Naltrexone hydrochloride + Fe (III) Oxidation products + Fe (II) The second step concerns with the estimation of Fe (II) with 2,2'-BPL.

146 P a g e International Standard Serial Number (ISSN): 2319-8141 Reaction of Naltrexone hydrochloride with 2,2'-Bipyridyl Scheme 3.4 METHOD F (Visible Spectrophotometric method by FC reagent) EXPERIMENTAL A. Preparation of standard calibration curve of bulk drug. 1. Solvent Used Distilled water is used as solvent. 2. Preparation of standard stock solution Accurately weighed 100 mg of Naltrexone Hydrochloride (bulk drug or its formulation) was dissolved in 40 ml of distilled water in 100 ml volumetric flask and volume was made upto the mark with distilled water (i.e.1000 µg/ml). Stock solution A. From the above stock solution A 10 ml of solution was pipetted out in 100 ml volumetric flask and the volume was made up to the mark with distilled water to obtain the final concentration of 100 µg/ml. Stock solution B. 3. Preparation of calibration curve Fresh aliquots of Naltrexone Hydrochloride ranging from 0.5 to 2.5 ml (1 ml-100µg/ml) were transferred into a series of 10 ml volumetric flasks to provide final concentration range of 5 to 25 μg/ml. To each flask 1 ml of aqueous sodium hydroxide (1 N) solution and 0.5 ml of FC reagent (1N) were added. The solutions were cooled to room temperature and made upto the mark with distilled water. The absorbance of blue colored chromogen was measured at 734 nm against the reagent blank (Figure 3.11). The color species was stable for 1.2 hour. The amount of Naltrexone Hydrochloride present in the sample solution was computed from its calibration curve (Figure 3.12).

147 P a g e International Standard Serial Number (ISSN): 2319-8141 A. Analysis of tablet formulation The sample of the powder tablet claimed to contain 50 mg. The accurate quantity equivalent to 100 mg of active ingredient was extracted with distilled water (3 x 15mL) and filtered through a 0. 45 µm membrane filter, followed by adding distilled water upto 100mL to get the stock solution of 1 mg/ml. Subsequent dilutions of this solution were made with distilled water to get concentration of 5-25 µg/ml and were prepared as above and analyzed at the selected analytical wavelengths, 734 nm and their results were statistically validated. Figure 3.11:- Absorption spectra for Naltrexone hydrochloride by FC reagent method (15 g/ml, λmax 734 nm) Figure 3.12:- Calibration curve for Naltrexone hydrochloride at 734 nm by FC reagent method Table 3.36:- Data for absorption spectrum of Naltrexone hydrochloride for FC reagent method (Drug concentration = 15 g/ml) Wavelength (nm) Absorbance 694 0.537 704 0.542 714 0.564 724 0.587 734 0.672 744 0.637 754 0.588 764 0.514

148 P a g e International Standard Serial Number (ISSN): 2319-8141 Table 3.37:- Result of calibration curve for Naltrexone hydrochloride by FC reagent method Concentration Absorbance (μg/ml) Mean ± S.D* % CV 0 0.00 ± 0.00 0 5 0.224 ± 0.003 1.339 10 0.448 ± 0.002 0.446 15 0.672 ± 0.003 0.446 20 0.896 ± 0.002 0.223 25 1.118 ± 0.002 0.178 *Average of three determinations. Table 3.38:- Optical characteristic and precision by FC reagent method Parameter FC reagent method max (nm) 734 Beer s law limits ( g/ml) (c) 5-25 Molar absorptivity (lit. mol -1 cm -1 ) 1.699 x 10 4 Limit of Detection (LOD/ mcgml -1 ) 0.140 Limit of Quantification (LOQ/ mcgml -1 ) 0.426 Sandell s sensitivity ( g/ml 0.001 abs unit) 0.0035 Regression equation (Y*) Slope (b) 0.0447 Intercept (a) 9.333 x 10-4 Standard error of estimation (Se) 0.006745 Correlation coefficient (r) 0.9999 % RSD 0.283 Range of Errors** Confidence limit with 0.05 level 0.00159 Confidence limit with 0.01 level 0.00236 % Error in bulk Samples*** 0.31 *Y=bC+a, where C is the concentration of Naltrexone hydrochloride in g/ml and Y is the absorbance at the respective maximum absorbency, **Average of eight determination, ***Average of three determination.

149 P a g e International Standard Serial Number (ISSN): 2319-8141 Table 3.39:- Assay and Recovery of Naltrexone hydrochloride in Pharmaceutical dosage form (Tablet) by FC reagent method Sample Labeled amount (mg) Amount obtained* (mg) Proposed Method Reference method (UV Method by cosolvancy) % recovery T1 50 49.77 48.76 98.99 T2 50 49.21 49.21 98.96 T1 and T2 are tablets from different manufactures, *Average of 5 determinations (50 mg of Naltrexone hydrochloride was added and recovered). Table 3.40:- Determination of accuracy of Naltrexone hydrochloride Level of % Recovery 80 % 100 % 120 % Amount Of drug in formulation* (mg) *Pre-analyzed sample taken from T 1 and T 2. Amount of standard drug added (mg) Total amount of drug found (mg) % Recovery of pure drug 49.85 40 89.66 99.62 49.27 40 89.19 99.10 49.91 40 89.16 99.06 49.72 50 99.07 99.07 49.91 50 99.48 99.48 49.85 50 99.46 99.46 49.65 60 109.79 99.49 49.82 60 109.66 99.69 49.27 60 109.90 99.00