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Vijaya Bhaskar Reddy.T et al. / Journal of Pharmacy Research 2012,5(7),3896-3899 Research Article ISSN: 0974-6943 Available online through http://jprsolutions.info A stability indicating RP-HPLC method for the determination of amitriptyline hydrochloride in pure and dosage forms 1 Vijaya Bhaskar Reddy.T1, Ramu.G1, Ravindra Babu.N1 and Rambabu.C1* *Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur, Andhra Pradesh, India Received on:07-04-2012; Revised on: 12-05-2012; Accepted on:16-06-2012 ABSTRACT A simple isocratic RP-HPLC method was developed for the determination of amitriptyline HCl in pure and dosage forms. A Waters HPLC equipped with Alliance 2695 separation module and 2487 dual wavelength UV-Visible detector and Symmetry C18 (4.6x150mm, 3.5 µm, Make: XBridge) HPLC column thermostated at 30oC was used in the present investigation. A mixture of potassium dihydrogen orthophosphate buffer of pH 3.0±0.05 and acetonitrile in the ratio 35:65 was used as mobile phase with a flow rate and injection volume were 0.8ml/min. and 20µl respectively. The data were acquired at 239nm. The proposed method was linear in the range of concentration 20-60µg/ml and the correlation coefficient is found to be 0.9990. The mean recovery of the substance was found to be 100.2%. The values of LOD and LOQ for amitriptyline HCl were found to be 0.015 and 0.052µg/ml respectively. The method has higher sensitivity towards the determination of the amitriptyline HCl. The developed method is found to be accurate and precise as indicated by recovery studies and % RSD not more than 2.0. The proposed method can be used as an alternative method for routine analysis in quality control. Keywords: RP-HPLC, Amitriptyline HCl, Linearity, LOD, LOD, Correlation coefficient INTRODUCTION Amitriptyline HCl (ATL) is used to treat depression, mainly melancholic, endogenous, or when anxiety or insomnia coexists. It is the most widely used tricyclic antidepressant(TCA) and has at least equal efficacy against depression as the newer class of SSRIs according to a study from early 2001.1 Amitriptyline is used for a number of medical conditions including: depressive disorders, anxiety disorders, attention deficit hyperactivity disorder, migraine prophylaxis, eating disorders, bipolar disorder, postherpetic neuralgia, and insomnia.2It is also used as a preventive for patients with recurring biliary dyskinesia .3 and in the treatment of nocturnal enuresis in children. ATL may be prescribed for other conditions such as cyclic vomiting syndrome post-traumatic stress disorder (PTSD), chronic pain, tinnitus, chronic cough,carpal tunnel syndrome (CTS), fibromyalgia, vulvodynia, interstitial cystitis, male chronic pelvic pain syndrome, irritable bowel syndrome (IBS), diabetic peripheral neuropathy, neurological pain, laryngeal sensory neuropathy, chronic fatigue syndrome and painful paresthesias related to multiple sclerosis. Typically lower dosages are required for pain modification of 10 to 50 mg daily. A randomized controlled trial published in June 2005 found that amitriptyline was effective in functional dyspepsia that did not respond to a first-line treatment (famotidineor mosapride).4 The chemical structure of the drug is 1-Propanamine,3-(10,11-dihydro-5Hdibenzo[a,d]cyclohepten-5-ylidene)-N,N-dimethyl-,hydrochloride with molecular formula and molecular weight C20H23N·HCl and 313.86g/mole respectively. It is available in different brand names such as Amitrol, Elavil, Endep, Levate, Tryptizol, Vanatrip. Amitriptyline Hydrochloride contains not less than 99.0percent and not more than 100.5percent of C20H23N·HCl, calculated on the dried basis. The chemical structure of the drug is given in Fig.1. Several techniques have been adopted for the determination of amitriptyline, including spectrophotometry 5,6 , high-performance liquid *Corresponding author. Rambabu.C Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur, Andhra Pradesh, India chromatography7,8gas chromatography9, capillary electrophoresis10 and voltammetry11 and potentiometric method using ionselective electrodes12,13 fluorescence polarization immunoassay method 14, UV spectrophotometric method 15, combination with other drugs 16,17 and UV method 18 MATERIALS AND METHODS Experimental A Waters HPLC equipped with Alliance 2695 separation module and 2487 dual wavelength UV-Visible detector was used for the separation and quantification. An analytical column; Symmetry C18 (4.6 mm ID x 150mm, 3.5 µm, Make: XBridge) was used in the analysis. Chromatographic software Empower-2 Fig.1 The chemical strucwas used for data collection and processing. ture of Amitriptyline HCl Elico-SL159 model, 2nm high resolution, double beam, 1cm length quartz coated optics and wavelength range1901100nm is used for measuring absorption spectrum. Chemicals and Reagents Acetonitrile and methanol of HPLC grade procured from Merck (India) were used. Potassium dihydrogen orthophosphate and orthophosphoric acid were all of AR grade (Merck, India) HPLC grade water obtained from Millipore system was used throughout the analysis. The investigated sample, amitriptyline HCl was obtained as a gift sample from Dr. Reddy’s Laboratory, Hyderabad, India. About 7.0 grams of potassium di hydrogen phosphate is transferred into a beaker dissolved in 1000ml with HPLC water, sonicated, filtered through 0.45µm filter and the pH of the solution was adjusted to 3.0±0.05 by adding a few drops of ortho phosphoric acid. The mobile phase was prepared by mixing about 350ml buffer and 650 ml of HPLC grade acetonitrile. The mixer was degassed in ultrasonic water bath for 5 minutes, filtered through 0.45µm filter. Journal of Pharmacy Research Vol.5 Issue 7.July 2012 3896-3899 Vijaya Bhaskar Reddy.T et al. / Journal of Pharmacy Research 2012,5(7),3896-3899 Table 1: System suitability parameters S.No. System suitability parameter Value of the parameter 1 2 3 Number of theoretical plates Tailing factor Retention time 2578.10 1.30 2.533 Table 2: Precision of the developed method Injection Concentration Peak Area Injection-1 Injection-2 Injection-3 Injection-4 Injection-5 40µg/ml 40µg/ml 40µg/ml 40µg/ml 40µg/ml Average Standard Deviation % RSD 2953021.000 2956623.000 2979601.000 2975186.000 2974030.000 2967692.000 11999.120 0.404 Table 3: Linearity of peak area against concentration of the drug S.No. Concentration Peak Area 1 2 3 4 5 6 Slope Intercept Correlation Coefficient LOD LOD 0.0 20.0 30.0 40.0 50.0 60.0 0 1503401 2288209 2975614 3745564 4372123 73363 35380 0.9990 0.015µg/ml 0.052 µg/ml Fig.2 Absorption spectrum of amitriptyline HCl in methanol Table 4: Accuracy of the proposed method %Concentration (at specification Level) Peak Area Amount Added (mg) Amount Found (mg) % Recovery Mean Recovery 50% 100% 150% 1514396 2999235 4344466 5.0 10.0 14.5 5.0 9.99 14.4 100.9% 99.9% 99.8% 100.2% Preparation of standard and sample About 10.0mg of 99.80 percent pure amitriptyline HCl was accurately weighed transferred into a 10ml volumetric flask, 7.0ml of diluent was added, sonicated to dissolve and filtered through 0.45µm filter. Further 0.4ml of the above solution was pipette out into 10ml volumetric flask and diluted to the mark with diluent, sonicated and filtered. The average weight of five amitriptyline HCl tablets was calculated, made them as homogeneous powder, an amount of the powder equivalent to 10 mg of amitriptyline HCl was weighed, transferred into a 10ml volumetric flask, dissolved in 7ml of diluent, sonicated to dissolve, made up to the mark, mixed well and filtered through through 0.45µm filter. Further 0.4ml of the sample solution was pipette out into 10ml volumetric flask and diluted to the mark with diluent, sonicated and filtered. Operating conditions The analysis was carried out under the isocratic conditions. The data were acquired at 239nm for 50min. and processed by use of Empower software data handling system. A mixture of buffer and acetonitrile in the ratio 35:65 (v/v) was used as diluents in the preparation of analytical solutions. Amitriptyline HCl working standard (40µg/ml) solution was used as system suitability solution. 20µl of system suitability solution, blank, five replicate injections of diluted standard solution and sample solution were separately chromatogramed. Fig.3Atypical chromatogram of standard drug Linearity The linearity plot of peak areas versus concentration was drawn for amitriptyline HCl and is presented in Fig.5. The linear regression data for the drug tested is given in Table 2. The data shown in Table 2 is confirmed that the detector response at 239 nm was linear over the range studied for the drug. The limit of detection (LOD) and limit of quantitation (LOQ) The limit of detection (LOD) and limit of quantitation (LOQ) were determined for amitriptyline HCl from the standard deviation of the peak area and slope of the linearity data. The values of LOD and LOQ for amitriptyline HCl were found to be 0.015 and 0.052µg/ml respectively. The results were depicted in Table 2. Precision and Accuracy System precision was verified using diluted solution standard solution, which was analysed for five times and R.S.D. of amitriptyline HCl peak areas was evaluated and found to be 0.404%. Precision of the method was studied for repeatability and intermediate precision. Repeatability was determined by analyzing five separate Amitriptyline HCl sample solutions the %R.S.D.was found to be 0.404% and given in Table 2. The intermediate precision of the method was determined on five separate sample solutions prepared from same lot by spiking by different days. The %R.S.D was evaluated and found to be 0.410% which was within the acceptance criterion of NMT 10% R.S.D. The results are presented in Table-5. Accuracy of the method was Journal of Pharmacy Research Vol.5 Issue 7.July 2012 3896-3899 Vijaya Bhaskar Reddy.T et al. / Journal of Pharmacy Research 2012,5(7),3896-3899 proposed conditions. Only one parameter was changed while the other was kept unaltered. The mean and R.S.D. were evaluated. The difference between the mean values from the repeatability mean results was found to be 10%.The studies indicated no effect on the determination of the drug. Therefore the test method is robust for the quantification of the drug. Table 5: Intermediate precision Injection Concentration Injection-1 40µg/ml Injection-2 40µg/ml Injection-3 40µg/ml Injection-4 40µg/ml Injection-5 40µg/ml Average Standard Deviation %RSD Area 3002113 2996759 3003927 3002104 3028438 3006668 12462.1 0.410 Table 6: Study of degradation of the drug in the presence of different degradation conditions Degradation Parameter Peak Area Peak Area of Standard % of Recovery % of drug Degraded 0.1M HCl 0.1 M NaOH Thermal Peroxide 2780828 2721026 2601420 2541617 2990138 2990138 2990138 2990138 92.99 91.00 86.99 84.99 7.00 9.00 13.00 15.00 Fig.4 A typical chromatogram of sample Study of degradation The percent of drug degraded in the presence of acid, base, thermal and peroxide conditions were studied. The drug standard was prepared in 0.1NHCl solution, 0.1N NaOH and 1% peroxide solutions and kept at room temperature for 48 hours. The drug standard in mobile phase is kept at 45oC for 36 hours. The amount of drug recovered of degraded is calculated by comparing the area of the standard with that of the area of the degraded sample. The results are presented in Table 6. RESULTS AND DISCUSSION The system suitable parameters such as tailing factor (1.3) and number of theoretical plates (2578) are found to be within the limits. A typical chromatogram for the standard and sample were presented in Fig.3 and Fig.4 respectively. The retention time of the component was found to be 2.256min. The intra-day precision or inter-day precision of a method was expressed in terms of statistical parameters such as standard deviation and %RSD. The %RSD was calculated for five replicate measurements and found to be less than 2.0. Inter-day precision of the method was determined by carrying out the experiment on different days using same instrument and same column under similar chromatographic conditions. The results are presented in Table2. The proposed method was linear in the range of concentration 20-60µg/ml and the correlation coefficient is found to be 0.9990. A calibration curve was constructed by plotting concentration against peak area (Fig.5). The correlation coefficient, slope and intercept were presented in Table-3.The accuracy of the method was determined from recovery experiments. The recovery studies were carried out at three different concentration levels (50%, 100% and 150% of target concentration). The percentage recovery of the drug at three different concentration levels and the mean percent of recovery are presented in Table-4. Robustness of the proposed method is checked by making slight deliberate change in the flow rate and mobile phase composition is made to evaluate the impact on the method. CONCLUSIONS A simple isocratic RP-HPLC method was developed for the determination of Amitriptyline HCl in pharmaceutical formulations as per the ICH guidelines. The method has higher sensitivity towards the determination of the Amitriptyline HCl. The developed method is found to be accurate and precise as indicated by recovery studies and % RSD not more than 2.0. Recovery studies are performed at 50%, 100% and 150% concentration levels are found to be within the limits mentioned as per ICH Guidelines. The proposed method was found to be simple, precise, accurate and robust. Therefore the method can be used for routine analysis in quality control. Fig. 5 Linearity plot of peak area against concentration of the drug. determined by analyzing Amitriptyline HCl sample spiked at three different concentration levels 50, 100 and 150% of each in triplicate at the specified limit. The mean recovery of the substance was found to be 100.2% and the data was given in Table 4. Robustness To evaluate the robustness of the method, the influence of small and premeditated alteration of analytical parameters on the quantification of the drug substance and selectivity was studied. The parameters selected were flow rate and mobile phase composition at 10% variation to that of the ACKNOWLEDGEMENTS The authors are grateful to Pharma Train, an analytical testing laboratory, Hyderabad for providing laboratory facilities, Dr. Reddy’s Laboratory for gifted samples and university authorities for providing provision for research work. REFERENCES 1. Barbui C and Hotopf M, “Amitriptyline”, The British Journal of Psychiatry: the Journal of Mental Science, 178 (2), 2001, 129– 144. 2. “Amitriptyline Hydrochloride”. The American Society of HealthSystem Pharmacists. Retrieved 3 April 2011. Journal of Pharmacy Research Vol.5 Issue 7.July 2012 3896-3899 Vijaya Bhaskar Reddy.T et al. / Journal of Pharmacy Research 2012,5(7),3896-3899 3. S. G. Hubscher et al. 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Patel, Indian J Pharm Sci. “Spectrophotometric and Chromatographic Simultaneous Estimation of Amitriptyline Hydrochloride and Chlordiazepoxide in Tablet Dosage Forms” , 71(4), 2009,472–476. Dhara Patel and Vivek Patel “simultaneous estimation of amitriptyline hydrochloride and prephenazine by ratio UV spectrophotometric method in combined tablet dosage form” IJPSR, 1 (12), 2010, 133-137. Source of support: Nil, Conflict of interest: None Declared Journal of Pharmacy Research Vol.5 Issue 7.July 2012 3896-3899