Analytical Method Development and Validation for Simultaneous Estimation of _{Memantine and Donepezil} in Pharmaceutical Dosage form by RP-HPLC

 

C. Parthiban*, Vislavath Anjali, Vollala Supriya, Dhontam Bhanu Priya,

Sadineni Sri Nithya, M. Sudhakar

¹Professor, Malla Reddy College of Pharmacy, Maisammaguda,

Dhulapally, Secunderabad - 500100. Telangana, India.

²Department of Pharmaceutical Analysis, Malla Reddy College of Pharmacy,

Secunderabad - 500100, Telangana, India.

 

ABSTRACT:

_{A simple, precise and accurate method was developed for the simultaneous estimation of memantine and donepezil. The chromatogram was performed with a Thermo scientific C18 (150 x 4.6mm, 5µm). At a flow rate of 1ml/min, mobile phase used is 70:30% v/v mixture of buffer pH 4.5 and ACN. The best detection wave length for memantine and donepezil was at 265nm. The retention time was 2.325 and 3.088minutes.  Assay results of 100.87 percent and 99.31 percent, %RSD values are below 2 and linearity R2 values are 0.999 for both the drugs. The strategy appears to have been compact and worth, and it is proper for everyday Quality Control Estimates in Industry sections since Maintenance Times are Diminishing and that Gathered Code was reducing. Thus, the developed method was validated according to 1CH guidelines for routine analysis of the sample.}

 

 

INTRODUCTION:

Memantine is used to manage moderate to severe Alzheimer's dementia, a more recent systemic review and meta-analysis 6 indicates that memantine is beneficial as a first line drug for the treatment of Alzheimer's dementia. Cholinesterase inhibitors may be added to memantine for further beneficial effects on behavioral symptoms of dementia. Donepezil is indicated for the management of mild to moderate Alzheimer’s Disease at doses of 5mg or 10mg. It is also indicated for the management of moderate to severe Alzheimer’s Disease in a higher dose of 10mg or 23mg administered once daily.

 

Off-label uses include the management of vascular dementia, Parkinson's Disease-associated dementia, and Lewy body dementia, among others, When combined with memantine, the extended-release form of donepezil is indicated to treat the symptoms of moderate to severe dementia.

 

Different analytical methods have been reported in the literature for the assay of _{memantine and donepezil} pharmaceuticals and include spectrophotometry, HPLC and other techniques in individual analysis of these drugs^1-10. So no method was reported for analyzing the compounds combinely. The present study was to establish a simple, sensitive and low cost RP-HPLC method for simultaneous estimation of _{memantine and donepezil} in pharmaceutical dosage forms. The developed method was validated as per ICH    guidelines^11-16.

 

 

EXPERIMENTAL:

Reagents:

_{Memantine and donepezil} were kindly supplied by Ranbaxy. Acetonitrile, water and methanol (HPLC grade, Merck) and all the other reagents of AR grade were purchased from M R Enterprisers. A tablet Namzaric (Adamas Labouratories) containing 10mg of _Donepezil and 14mg of _Memantine and _{memantine and donepezil} pure drugs (API) were used.

 

Instruments:

The LC system consisted of a Waters model 515, PDA detector 2998 with 20µL sample loop. The output signals were monitored   using Empower 2 software and HPLC Agilent 1220 infinity were used.

 

Chromatographic conditions:

The elution was isocratic and the mobile phase consisted of a mixture of buffer (accurately weighed 1.41gm of sodium dihyrogen ortho phosphate in a 1000ml of volumetric flask add about 900ml of milli-Q water added and degas to sonicate and finally make up the volume with water then pH adjusted to 4.5 with dil. orthophosphoric acid solution and acetonitrile _{70:30% v/v. The best detection wave length for memantine and donepezil was at 265nm}. A ODS C₁₈ (150mm x 4.6 mm, 5mm) was used for determination. The flow rate was 1.0 ml/min. The volume of sample injected was 20 µL. Prior to  injection  of  the solutions,  column  was  equilibrated for  at least  30min  with  mobile phase flowing  through  the  system. The UV detector was set at wavelength of 265nm. A typical RP-HPLC chromatogram of _{memantine and donepezil} is shown in (Fig. 1).

 

Fig - 1: HPLC chromatogram of _{memantine and donepezil} optimized chromatographic condition.

 

Standard Preparation:

Accurately weighed and transferred 10mg of _Donepezil and 14mg of _Memantine working Standards into a 100 ml clean dry volumetric flask, add 70ml of diluent, sonicated for 30 minutes and make up to the final volume with diluent. From the above stock solution, 1ml was pipetted out in to a 10ml volumetric flask and then make up to the final volume with diluent.

Sample Preparation:

20 tablets were taken and their average weight was calculated. The tablets were crushed to a fine powder and drug equivalent to 10mg were transferred to a volumetric flask and dissolved in solvent. Transfer 1ml from the above solution into 10ml volumetric flask and filtered through 0.45μ membrane filter to get concentration of 14μg/ml and 10μg/ml for _{memantine and donepezil.}

 

Method Validation:

The developed method was validated as per ICH guidelines for its accuracy, linearity, precision, specificity, robustness, ruggedness, limit of detection and limit of quantification by using the following procedures.

 

Linearity:

Linearity of this method was evaluated by linear regression analysis and calculated by least square method and studied by preparing standard solutions of _{memantine and donepezil} different concentration levels. Absorbance of resulting solutions was measured and the calibration curve was plotted between absorbance vs concentration of the drug (Figure: 2 & 3). The response was found to be linear in the range 5-25µg/ml for _{memantine and} 5-25µg/ml for _donepezil. The data was given in table-1.

 

 

Fig – 2: Linearity for _{Memantine (}5-25µg/ml)

 

Fig – 3: Linearity for _{Donepezil (}5-25µg/ml)

 

Table 1: Linearity data of _{Memantine and Donepezil}

Table 2: Accuracy data

*n=6 (Average of 6 determinations)

 

Accuracy:

Accuracy was performed in triplicate for various concentrations of _{memantine and donepezil} equivalent to 50%, 100% and 150% of the standard amount were injected into the HPLC system per the test procedure. The average % recovery was calculated. The data was given in table-2.

 

Precision:

A) Method Repeatability:

Six sample solutions of the same concentration (100%) were prepared and injected into the HPLC system as per test procedure. The results were given in table-3.

 

Table 3: Precision data of _{Memantine and Donepezil}

 

Limit of detection and Limit of Quantification:

LOD and LOQ were calculated from the average slope and standard deviation from the calibration curve as per ICH guidelines. The LOD and LOQ of _memantine found to be 0.13µg/ml and 0.429µg/ml respectively. The LOD and LOQ of _donepezil were found to be 0.11µg/ml and 0.363µg/ml respectively.

 

Robustness:

Robustness was done by small deliberate changes in the chromatographic conditions and retention time of _{Memantine and Donepezil} in were noted. The factors selected were flow rate and variation in the mobile phase composition. The results remained unaffected by small variations in these parameters as shown in table-4 and 5.

 

Table 4: Robustness data relating to change in flow rate (1.0ml/min)

*n=3 (Average of 3 determinations)

 

Table 5: Robustness data relating to change in mobile phase composition

*n=3 (Average of 3 determinations)

 

Assay:

The assay and % purity were calculated for brandsStorvas-EZ (Ranbaxy) and Atofast-EZ(Intralabs) with label claim 10mg and 10mg. The observed value was compared with that of standard value without interference from the excipients used in the tablet dosage form. The results were given in table-6.

 

Table-6: Results of analysis of laboratory samples (Assay)

*n=3 (Average of 3 determinations)

 

 

 

RESULTS:

A reverse-phase column procedure was proposed as a suitable method for the simultaneous estimation of _{memantine and donepezil} in dosage form. The chromatographic conditions were optimized by changing the mobile phase composition. Different ratios were experimented to optimize the mobile phase. Finally, buffer and acetonitrilein the ratio 70:30v/v was used as mobile phase, which showed good resolution of _{memantine and donepezil} in peak. The wavelength of detection selected was 265nm, as the drug showed optimized absorbance at this wavelength. By our proposed method the retention time of _{memantine and donepezil} were about 2.367mins and 3.417mins and none of the impurities were interfering in its assay.

 

DISCUSSION:

The statistical analysis of data and the drug recovery data showed that the method was simple, rapid, economical, sensitive, precise and accurate. It can thereby easily adopt for routine quality control analysis. The results of this analysis confirmed that the proposed method was suitable for determination of drug in pharmaceutical formulation with virtually no interference of additives. Hence the proposed method can be successfully applied in simultaneous estimation of _{memantine and donepezil} marketed formulation.

 

CONCLUSION:

The proposed method is rapid, accurate and sensitive. It makes use of fewer amounts of solvents and change of set of conditions requires a short time. This method can be suitably analyzed for the routine analysis of _{memantine and donepezil} bulk and its pharmaceutical dosage forms. It does not suffer from any interference due to common excipients present in pharmaceutical preparation and can be conveniently adopted for quality control analysis.

 

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Received on 01.07.2023         Modified on 08.08.2023

Accepted on 12.09.2023   ©Asian Pharma Press All Right Reserved