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PROJECT REPORT
"ANALYTICAL METHOD DEVELOPMENT AND
VALIDATION OF SOME ANTI-CANCER DOSES
FORMS'

UGC REFERENCE No. 47-114/07
SUBMITTED TO
UNIVERSITY GRANT COMMISSION
Dr. JWALANT J. VORA
M. G. SCIENCE INSTITUTE,
AHMEDABAD-380009
PRINCIPAL INVESTIGATOR,
PRINCIPAL,
MINOR RESEARCH PROJECT
M. G. SCIENCE INSTITUTE

RESEARCH PAPER PUBLISHED:
e-ISSN: 2249-7625
Print ISSN: 2249-7633
International Journal of
Pharmacy Practice & Drug Research
www.ijppdr.com
ANALYTICAL METHOD VALIDATION OF STABILITY-INDICATING HPLC
METHOD FOR DETERMINATION OF ASSAY OF MEMANTINE
HYDROCHLORIDE TABLETS

Introduction: This research work of the project deals with the
development and validation of stability indicating high performance
liquid chromatographic method for the quantitative determination of
Memantine hydrochloride. Study of an analytical method for the
determination of Memantine hydrochloride drug and also validate the
method as per the ICH guideline and required acceptance criteria. The
method was developed by using Hypersil BDS C8 column (250×4.6
mm id, 5 μm particle sizes) containing mobile phase Buffer and
acetonitrile in ratio (65:35). The flow rate was set at 1.0 mL/minute
and the injection volume was 10μL. The run time of injection is near
about 15 min. In this method use Waters HPLC system with
Reflective Index detector. The linearity of method was 50 % to 100
%, the correlation coefficient was found to be 0.9999. Eluents were
monitored by reflective index detector. The liquid chromatographic
method was validated with respect to specificity, precision (% RSD
about 0.71%). Stability an analytical solution (% deviation from
initial area count found in limit ±2%).
Origin of the Research Problem: Memantine hydrochloride,1-
amino3,5-dimethyl adamantine hydrochloride and CAS-19982-08-2,
is a tricyclic amine used for treatment of Cancer, Alzheimer,
Parkinson's, and spasmodic disease [1,2]. Memantine is promising
agent for treatment of all psychoactive drugs used as opiods, nicotine and cocaine [3,4]. Memantine act as blocker of N-Methyl-D-asparlate receptor complex. Memantine is a non-competitive NMDA antagonist in clinical use for many years in Europe. It produces few side effects, even among the geriatric patients [5,6] the NMDA receptor, a glutamate receptor subtype, may play a significant role in the development and maintenance of dependence of opoids, nicotine, and cocaine[7,8]. Memantine is approved by U.S.F.D.A and the European Medicines Agency for treatment of moderate-to-severe Alzheimer's disease [9]. Glutamate is produced and released by nerve cells in the brain, travels to nearby nerve cells where it attached to the NMDA receptor on the surface of the cells. Memantine blocks the receptor and there by decreases the effect of glutamate. Thus Memantine protect nerve cell from excess stimulation by glutamate. 1. Ditzler K, Arzneim. Efficacy and tolerability of Memantine patients with dementia syndrome, Forsch. Drug Res, 41, 1991, 773-780. 2. Schneider E, PA Fisher, R Clemens, F Balzereit, EW Fungfeld, HJ Haase, Dtsch Med Wochenschr. Effects of oral Memantine administration on Parkinson symptoms. Result of a placebo contolled ulticenter study, 109, 1984, 987-992. 3. Reiderer P, Lange KW, Kornhuber J, Danielczyk W, Harmacotoxic psychosis after memantine is Parkinson disease, Lancer, 338, 1991, 1022-1023. 4. Rabey J, Nissipeanu MP, Koreyzn AD. Efficacy of Memantine, an NMDA receptor antagonist, in the treatment of Parkinson disease, J. Neural Transm, 43, 1994, 91-104. 5. Kornhuber J, Weller M, Schoppmeyer K, Riederer P. Amantadine and Memantine are NMDA Receptor antagonist with neuroprotective properties. J Neural Transm Suppl, 43, 1994, 91-104. 6. Gortelmeyer R, Pantev M, Parsons CG, Quack G. Memantine in the treatment of mild to moderate dementia syndrome Kvon, Wild (Ed.) Spektrum der Neurorehabilitation W. Zuckschwerdt Verlag Munchen, 1993, 50-66. 7. Herman BH, Vocci F, Bridge P. The effect of NMDA receptor antagonist and nitric oxide synthesis inhibitors on opioid tolerance and withdrawal. Medication development issues for opiate addiction. Neuropsycopharmacology, 1995, 13:269. 8. Stephens DN. A glutametargic hypothesis of drug dependence: extrapolalation from benzodiazepine receptor ligands. Behav Pharmacol, 1995, 6, 425. 9. Mount C, Downton C. Alzheimer's disease: progress or profit, Nat Med, 7, 2012, 780. Present work:
Instruments and Apparatus The chromatography was performed on a Waters HPLC instrument equipped with Reflective index detector, Hypersil BDS C8; column (250×4.6 mm id, 5 μm particle size) was used as stationary phase. And in mobile phase Buffer and Acetonitrile in ratio of 65:35. (Buffer-1ml hexylamine in 1000 ml water and 1 ml trifluroacetic acid and pH adjust with dil. Ammonia. 7.0), and flow rate 1.0 ml/minute and temperature of reflective index detector cell is 40°C and column temperature 40°C. And injection volume is 10μl and run time is 15 minute. Mettler Toledo analytical balance and ultrasonic cleaner (Frontline FS 4, Mumbai, India) were used during the research work. Preparation of Standard Solution Accurately weighed of Memantine hydrochloride 62.5 mg standards was transferred to a 25 ml volumetric flask, dissolved in and diluted up to the mark with mobile phase. Preparation of Sample Solution For determination of the content of Memantine hydrochloride in tablets; twenty tablets were weighed and average weight was determined. The accurately weighed powder equivalent to 62.5 mg Memantine hydrochloride was transferred in a 25 ml volumetric flask and add about 15 ml mobile phase. The solution was sonicating for 15 min. The flask was allowed to stand for 5 min at room temperature and the volume was diluted up to the mark with mobile phase. The solution was filtered through 0.45μm-47mm membrane filter. Method Validation The methods were validated in compliance with ICH guidelines. The accuracy of the methods was determined by calculating recoveries of Memantine hydrochloride by the standard addition method. Recovery study was performed by spiking Memantine hydrochloride WRS in placebo at level 50%,100% and 150% of concentration in triplicate (total nine determination) and then proceeded with sample preparation as described under method. The result indicates that the recovery of Memantine hydrochloride from the sample by the proposed method is satisfactory. Mean Assay -100.33% Relative standard deviation -0.85% Intermediate Precision (Reproducibility) The intraday and interday precisions of the proposed methods were determined by estimating the corresponding responses 3 times on the same day and on 3 different days over a period of 1 week for 3 different concentrations of Memantine hydrochloride. Method Precision (Repeatability) The repeatability was checked by repeatedly injecting (n = 6) solution of Memantine hydrochloride. Mean Assay - 97.36% Relative standard deviation - 0.34% The robustness was studied by analyzing the same samples of Memantine hydrochloride by deliberate variation in the method parameters. The change in the responses was noted. Robustness of the method was studied by change in flow rate ± 0.2 ml/min. composition of mobile phase by ± 5 % of organic solvent, change in pH by ± 0.2 unit and column oven temperature by ± 2ºC. The parameters used in system suitability test were asymmetry of the chromatographic peak, peak resolution, theoretical plates and capacity factor, as RSD of peak area for replicate injections. Linearity
Prepare a series of standard preparation of Memantine hydrochloride WRS over the range of 50% to 150% using linearity solution. Solution Stability
A sample solution of memantine hydrochloride drug substance was prepared and kept at room temperature. It was analyse initially and at different type intervals. Result, discussion and conclusion:
The responses of sample solutions were measured by reflective index detector for quantitation of Memantine hydrochloride by the proposed methods. The amount of Memantine hydrochloride present in the sample solutions was determined. The mobile phase consisting of Buffer and Acetonitrile in ratio (65: 35), at a flow rate of 1.0 ml/min was found to be satisfactory to obtain good peak symmetry, better reproducibility and repeatability for Memantine hydrochloride. Linear correlation was obtained between peak area and concentration for Memantine hydrochloride in the range of 125.0-375.0 μg/ml (Table 1). The method was found to be specific as no significant change in the responses of Memantine hydrochloride was observed after 48 hrs. (Table 2)

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