Simultaneous Estimation of Donepezil and Memantine by Reverse Phase HPLC in Bulk and Pharmaceutical Dosage Form

 

Kamepalli Sujana¹*, D. Gowri Sankar², K. Abbulu³

¹Pharmaceutical Analysis Division, University College of Pharmaceutical Sciences, Acharya Nagarjuna University, Guntur-522510, A.P, India

²Pharmaceutical Analysis Division, Andhra University, Visakhapatnam

³Department of Pharmaceutics, MRIPS, Hyderabad

*Corresponding Author E-mail: sujana_36@yahoo.co.in

ABSTRACT:

This investigation describes a new precise, sensitive and accurate RP-HPLC method for the Simultaneous estimation of Donepezil and Memantine in Bulk and Tablets. The resolution of two drugs was achieved on Agilent C8 (150mm x 4.6mm i.d., 3.5mm particle size) column with UV detection at 230nm and the mobile phase consist of Buffer, water and Acetonitrile (50:5:45 v/v).Using chromatographic conditions described Donepezil and Memantine were well resolved with mean retention times of 3.707 and 6.976 min, respectively. Linear response (r>0.999) was observed over the range of 50-70µg/ml for Donepezil and 100-140µg/ml for Memantine. The lower limit of quantification and lower limit of detection was 9.95 and 3.0 for Donepezil and 10.01 and 3.14 for Memantine. The Validation parameters were performed according to the ICH guidelines and the proposed method can be useful in the routine analysis for the determination of Donepezil and Memantine in Pharmaceutical dosage forms.

 

 

INTRODUCTION:

Donepezil Hydrochloride (DNP)^[1,2] is a reversible inhibitor of the enzyme acetyl cholinesterase, known chemically as (±)-2,3-dihydro-5,6-dimethoxy2-[[1-(phenylmethyl)-4-piperidinyl]methyl]-1H-inden-1-onehydrochloride.

 

Memantine Hydrochloride (MEM)^[3] is an orally active NMDA receptor antagonist, chemically designated as 1-amino-3,5-dimethyladamantane hydrochloride. Both drugs in combination used in the treatment of Alzheimer's disease. Literature survey reveals many methods are reported for estimation of DNP^[4-7] and MEM^[8-11]individually and no methods are reported in combination of DNP and MEM. In this communication, a new simple, rapid and precise RP-HPLC method have been reported for simultaneous determination of  DNP and MEM which can be used for its routine analysis in normal laboratories.

 

MATERIALS AND METHODS:

Chromatograms were made on Waters (Alliance) with Auto Sampler and Ultraviolet detector. The data acquisition was performed by Empower Software.

 

Glass wares used in each step were rinsed thoroughly with double distilled water, dried in hot air oven. DNP and MEM was obtained from Pharma Train Institution, Hyderabad. The pharmaceutical preparation of combination of DNP and MEM is Donamem (Sun Pharmaceutical Industries Ltd.) was obtained from local market. Acetonitrile used is HPLC grade obtained from Merck (India) and water used is double distilled water. Other reagents were of AR grade.

 

Chromatographic Conditions

The used analytical column was Agilent C₈ (150mm x 4.6mm i.d., 3.5mm particle size) column. The mobile phase consists of mixture of Buffer, water and Acetonitrile (50:5:45 v/v), filtered through 0.22µm Millipore filter and degassed by sonication. Separation was carried out isocratically, at ambient temperature (23±1°C), and a flow rate of 0.8 ml/min with Ultraviolet detection at 230nm.The injection volume was 20 ml.

 

Preparation of Standard solutions

Accurately weigh and transfer 10 mg of DNP and MEM into a 10mL clean dry volumetric flask separately. Add about 7mL of Diluent (Mobile phase) and sonicate to dissolve it completely and make volume up to the mark with the same solvent (Stock solution).Further pipette 0.6 ml of DNP and 1.2 ml of MEM from the above stock solution into a 10ml volumetric flask and dilute up to the mark with diluent.

 

Analysis of marketed formulation

Twenty tablets of DNP and MEM were crushed and made into powder. Accurately weigh and transfer equivalent to 10 mg of DNP and MEM sample into a 10mL clean dry volumetric flask. Add about 7mL of Diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent (Stock solution). From this working standard of 120 µg/ml was prepared. 20 µL of the standard, sample were injected into the chromatographic system and the areas were measured for the DNP and MEM peaks. The content of DNP and MEM were calculated and found to be 98.2 for both drugs respectively.

 

RESULT AND DISCUSSION:

Optimization of Chromatographic Conditions

Chromatographic conditions were optimized by changing the mobile phase composition and buffers used in the mobile phase. Different experiments were performed to optimize the mobile phase but adequate separation of drugs could not be achieved. By altering the pH of mobile phase a good separation was achieved. The optimized mobile phase consisting of 0.01 M Potassium dihydrogen Phosphate (pH 3.0 with Orthophosporic acid), water and Acetonitrile mixed in the ratio of 50:5:45 v/v and flow rate of 0.8 ml/min.DNP and MEM were eluted at 3.707 and 6.976 minutes respectively with a run time of 10 min under the above optimized chromatographic conditions. Typical chromatograms for simultaneous estimation of DNP and MEM are shown in Figure 1and2.

 

Method Validation

System Suitability Results

For DNP and MEM peaks the tailing factor were found to be 1.49 and0.97 respectively and the Theoretical Plates obtained were found to be 2377.13 and 2194.57 respectively.

Linearity

The calibration curves obtained by plotting Peak Area against Concentration for DNP and MEM. The linearity was obtained in the concentration range of 50-70 μg/ ml for DNP and 100-140 μg/ml for MEM. The regression coefficient values (R²) for DNP and MEM found to be 0.999 and 0.999 respectively.

 

Accuracy and precision

The accuracy of the RP-HPLC method was determined by calculating Recoveries of DNP and MEM for 50%, 100% and 150% with respect to target concentration and results are tabulated in Table 1 and 2 respectively. The System precision of the proposed method was determined by injecting standard solution for five times and measured the area for them in HPLC. The Method Precision of the proposed method was determined by injecting six sample solutions into HPLC prepared individually. The %RSD for the areas of system precision and method precision data were calculated and given in Table 3.

 

 

Figure 1: Chromatogram of  DNP and MEM Standard

 

Figure 2: Chromatogram of  DNP and MEM Sample

 

TABLE 1: Recovery Results for DNP

% Concentration (at specification Level)	Area	Amount Added (mg)	Amount Found (mg)	% Recovery	Mean Recovery
50%	4245471	10.0	10.42	101.2%	100.16%
100%	8099172	20.0	19.88	99.4%
150%	11387715	30.0	29.00	99.9%

 

TABLE 2: Recovery Results for MEM

%Concentration (at specification Level)	Area	Amount Added (mg)	Amount Found (mg)	% Recovery	Mean Recovery
50%	9037	10.0	10.96	99.64%	99.45%
100%	16532	20.0	20.05	100.25%
150%	23944	30.0	29.04	98.44%

 

Table 3: Precision of DNP and MEM

S.NO.	PRECISION	DNP	MEM
1.	System precision (Average Area and %R.S.D)	4339776and 0.03	14276 and 0.45
2.	method precision (Average Area and %R.S.D)	4431453 and 0.02	11590 and 1.70

 

Limits of Detection and Quantitation

For determining the limit of detection (LOD), 10mg of DNP and MEM was transferred in10mL clean dry volumetric flask .Add about 7mL of Diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent(Stock solution) separately. From this a working standard of 0.1µg/ml DNP and MEM was prepared and injected separately. The LOD was found to be 3.0 for DNP and 3.1for MEM.

 

For determining the limit of Quantitation, from the above stock solution, prepared 0.4µg/ml solution of DNP and 100 µg/ml solution of MEM and injected. The LOQ was found to be 9.95 for DNP and 10.01 for MEM respectively.

 

Robustness

As part of the Robustness, deliberate change in the flow rate, mobile phase composition was made to evaluate the impact on the method. The results reveal that the method is robust enough. The results are summarized in Table 4, 5, 6 and 7

 

Table 4: System suitability results for DNP (flow rate varied)

S.No	Flow Rate (ml/min)	System Suitability Results
		USP Plate Count	USP Tailing
1	0.7	2772.32	1.53
2	0.8	2555.1	1.51
3	0.9	2609.53	1.51

 

 

 

Table 5: System suitability results for MEM (flow rate varied)

S.No	Flow Rate (ml/min)	System Suitability Results
		USP Plate Count	USP Tailing
1	0.7	3353.84	1.01
2	0.8	2821.27	1.12
3	0.9	3431.17	1.44

 

Table 6: System suitability results for DNP (mobile phase varied)

S.No	Change in Organic Composition in the Mobile Phase	System Suitability Results
		USP Plate Count	USP Tailing
1	1.0 % less	2586.10	1.48
2	*Actual	2555.1	1.51
3	1.0% more	2533.79	1.54

 

Table 7: System suitability results for MEM (mobile phase varied)

S.No	Change in Organic Composition in the Mobile Phase	System Suitability Results
		USP Plate Count	USP Tailing
1	1.0% less	2584.71	0.98
2	*Actual	2821.27	1.12
3	1.0% more	3138.39	1.22

* Results for actual Mobile phase composition (50:5:45 Buffer: Water: Acetonitrile) have been considered from Assay standard

 

CONCLUSION:

A new HPLC method was developed and validated for simultaneous determination of DNP and MEM in combined pharmaceutical dosage form and assured the satisfactory precision and accuracy and also determining lower concentration of each drug in its solid dosage form. The method has been found to be better, because of use of a less economical and readily available mobile phase, lack of extraction procedures, no internal standard, and use of the same mobile phase for washing of the column. All these factors make this method suitable for quantification of DNP and MEM in bulk drugs and in pharmaceutical dosage forms. It can therefore be concluded that use of the method can save much time and money and it can be used in small laboratories with very high accuracy and a wide linear range.

 

ACKNOWLEDGEMENTS:

The authors are grateful to Dr. D. Gowri Sankar, Dr. K. Abbulu and Mr. Chandrasekhar for the continuous support throughout the work.

 

REFERENCES:

1.     United States pharmacopoeia35 and National Formulary (30).2; 2012: 2967.

2.       Indian Pharmacopoeia.2; 2010:1248.

3.       Martindale, The complete drug reference. A(37);2011:397.2

4.       Paresh P Chothe et al. Stability Assessment Of Donepezil Hydrochloride Using Validated RP-HPLC Method. Research Journal of Pharmaceutical, Biological and Chemical Sciences.1 (3); 2010:296-305.

5.       Zhiyong  Xie et al. Rapid and sensitive determination of donepezil in human plasma by liquid chromatography/tandem mass spectrometry: application to a pharmacokinetic study. Rapid Commun. Mass Spectrom.20; 2006: 3193–3198.

6.       Mahasen A. Radwan et al. Stereoselective HPLC assay of donepezil enantiomers with UV detection and its application to pharmacokinetics in rats. Journal of Chromatography B.830; 2006:114–119.

7.       Pappa H et al. A new HPLC method to determine Donepezil hydrochloride in tablets. Journal of Pharmaceutical and Biomedical Analysis.64; 2012:16-25.

8.       Sai Praveen P et al. Extractive spectrophotometric method for the determination of Memantine. Research Journal of Pharmaceutical, Biological and Chemical Sciences.1 (3); 2010:222-225.

9.       Zargh A et al. Sensitive and Rapid HPLC Method for Determination of Memantine in Human Plasma Using OPA Derivatization and Fluorescence Detection: Application to Pharmacokinetic Studies. Sci Pharm. 78; 2010: 847–856.

10.    Bhavil N et al. A Validated Stability-indicating Reverse Phase Hplc Assay Method for the Determination of Memantine Hydrochloride Drug Substance with Uv-Detection Using Precolumn Derivatization Technique. Analytical Chemistry Insights. 5; 2010: 37–45

11.    Mohamed G et al. Determination of memantine in rat plasma by HPLC-fluorescence method and its application to study of the pharmacokinetic interaction between memantine and methazolamide. Biomedical Chromatography.26 (2); 2012: 214–219.

 

 

Accepted on 04.07.2012      © RJPT All right reserved