Development and Validation of Memantine Hydrochloride by RP-HPLC Method
 
Ayesha Anees1, Asra Ali Bahazeq2, MD. Muzaffar-Ur-Rehman2*, Syed Akbar2, Juveria Mehveen3

1Department of Pharmacology, Sultan-ul-uloom College of Pharmacy, Banjara Hills, Hyderabad, Telangana, India-500034.

2Department of Pharmaceutical Chemistry, Sultan-ul-uloom College of Pharmacy, Banjara Hills, Hyderabad, Telangana, India-500034.

3Department of Pharm D, Sultan-ul-uloom College of Pharmacy, Banjara Hills, Hyderabad, Telangana, India-500034.

*Corresponding Author E-mail: m.muzaffar687@gmail.com

 

ABSTRACT:

Memantine hydrochloride is a class of N-Methyl-D-Aspartase receptor antagonist used in the treatment of Alzheimer’s disease. The main aim of the present study was to develop a method to determine Memantine hydrochloride in tablet formulation which is simple, sensitive, precise, robust and economical as per ICH guidelines. The separation of the drug was achieved on Inertsil ODS 3V (250x4.6mm) 5µm column. The mobile phase used was a mixture of KH2PO4, Acetonitrile and methanol in the ratio 30:40:30. The detection of Memantine hydrochloride was done at λ max of 260nm. A good linear relationship with correlation co-efficient of 0.999 was obtained between the concentration range of 60µg/ml -140µg/ml and the LOD and LOQ values were 1.82µg/ml and 5.50µg/ml respectively. The method was also employed on the tablet formulation (Mentadem 10mg) and the recovery was found to be 99.15%.

 

KEYWORDS: Memantine hydrochloride, Alzheimer’s disease, ICH guidelines, Validation

 

 


INTRODUCTION:

Memantine hydrochloride, chemically known as 3,5-dimethyadamantan-1-amine;hydrochloride, with the molecular formula of C12H21N.HCl and molecular weight 215.765g/mol, is the first drug to be approved by US FDA, manufactured as Torrent Pharmaceuticals Ltd.[1-2] It has been widely used to treat cognitive symptoms of Alzheimer’s disease such as memory loss, confusion, problems with thinking and reasoning.[3-7] It acts as antagonist to Glutamatergic NMDA receptor [8-9] which is responsible for cognitive symptoms and also at 5-HT3 [10] where it acts as anti-emetic.

 

Literature study reveals several assay methods for determining the drug by HPLC [11] RP-HPLC [12-13], and spectrofluorimetry [14] techniques. Its determination in the rat and human plasma were also noted by derivatized flourimetric [15], mass spectroscopic [16] and gas chromatographic [17] methods respectively. In the present study, the developed method was validated for all the parameters [18-22] as per ICH guidelines [23-24] to determine the presence Memantine hydrochloride in bulk and tablet formulation (Mentadem 10mg) using combination of potassium di-hydrogen phosphate, acetonitrile and methanol in the ratio of 30:40:30v/v/v as a solvent. The method developed was found to be simple, accurate, precise and robust as there was no significant change upon changing the conditions.

 

 

Figure 1: Structure of Memantine Hydrochloride

 

MATERIALS AND METHODS:

Chemicals and Reagents:

Memantine hydrochloride was obtained as a gift sample from Chandra labs, Hyderabad, India. Methanol and Acetonitrile used were of HPLC grade where as potassium dihydrogen phosphate used was of AR grade and purchased from Merk (India). Memantine hydrochloride formulation (Mentadem 10mg) was purchased from local pharmacy.

 

Instrumentation:

HPLC system used in the study was of Shimadzu (LC 20 AT VP) empowered with Spin chrome (LC SOLUTIONS) software. Analytical column used was Inertsil ODS 3V with the dimensions 250x4.6mm packed with particle size of 5µm. Other equipments used were ultra sonicator of Citizen, Digital Ultrasonic Cleaner, pH meter of Global digital, electronic balance of Shimadzu and syringe of Hamilton.

 

Method development:

Literature reveals analytical methods for the determination of Memantine hydrochloride, but was not economical. Therefore, it was found to be significant to develop and validate a method using RP-HPLC method to determine the drug using Inertsil ODS (250x4.6x5µ) column. To optimize the determination, various trials were conducted using the same column with various combinations of the mobile phases. The mobile phase was premixed and filtered using 0.45µ filter. Various parameters were considered for the optimization of the method such as retention time, number of theoretical plates and resolution.

 

Preparation of Memantine Hydrochloride standard solution

Weigh accurately 50 mg of Memantine hydrochloride in 50 ml of volumetric flask and dissolve in 10ml of mobile phase and make up the volume with mobile phase. From above stock solution 100 µg/ml of Memantine is prepared by diluting 1ml to 10ml with mobile phase. This solution is used for recording chromatogram.

 

Preparation of Memantine Hydrochloride sample solution

10 Tablets (each Tablet contains 10 mg of Memantine) were weighed and taken into a mortar uniformly mixed. Test stock solutions of Memantine (100μg/ml) and was prepared by dissolving weight equivalent to 10 mg of Memantine and dissolved in sufficient mobile phase. After that filtered the solution using 0.45-micron syringe filter and Sonicated for 5 min and dilute to 50ml with mobile phase. Further dilutions are prepared in 5 replicates of 100 μg/ml of Memantine was made by adding 1 ml of stock solution to 10 ml of mobile phase.

 

Preparation of pH 6.0 buffer:

2.72 gm of potassium di hydrogen phosphate (KH2PO4) was weighed and dissolved in 100ml of water and volume was made up to 1000ml with water. pH was adjusted to 6.0 using ortho phosphoric acid. To remove all fine particles and gases the buffer was filtered through 0.45µ filters.

 

Mobile Phase

A mixture of 30 volumes of Potassium Dihydrogen phosphate buffer pH 6.0, 40 volumes of Acetonitrile and 30volumes of Methanol was prepared. To remove gases the mobile phase was sonicated for 10min.

 

Procedure:

Mixture of mobile phase of potassium di-hydrogen phosphate, acetonitrile and methanol in the ratio 30:40:30 at pH 6.0 was used for the detection of Memantine Hydrochloride. Mobile phase was pumped into the column 30 minutes prior to the injection of sample to equilibrate it. 20 µl of the standard and sample were injected into the system at a flow rate of 1ml/min at 25oC and the peak was measured. The drug was detected at λ max of 260nm. Run time was set to 8 minutes and the retention time under these conditions was found to be 3.490 minutes. Typical chromatograms of the standard and sample are shown in figure 2 and 3 from which % assay was calculated.

 

RESULTS AND DISCUSSION:

Method optimization

The method adopted was optimized by evaluating combination of solvents in different compositions. Mobile phase of potassium di hydrogen phosphate buffer pH 6.0, acetonitrile and methanol in the ration of 30:40:30v/v/v on Inertsil ODS column with the dimensions of 250x4.6mm and particle size of 5µm, gave a peak with good resolution with the retention time of 3.49 minutes.

 

System suitability:

After column equilibration, the system suitability parameters which include theoretical plates, resolution and asymmetric factor were evaluated. The evaluation was done by preparing the standard solution as per the test method and injecting 20µl of it into the chromatographic system in a replicate of five. The results are shown in Table 1.

 

 



Figure 2: Chromatogram of optimized trial


 

TABLE 1: Results for system suitability of Memantine

Injection

Retention time (min)

Peak area

Theoretical plates (TP)

Tailing factor (TF)

1

3.460

5671.643

2702

1.806

2

3.450

5587.778

2805

1.806

3

3.473

5700.527

2723

1.806

4

3.467

5510.683

2832

1.774

5

3.467

5579.223

2832

1.806

6

3.460

5671.643

2702

1.806

Mean

3.4628

5620.250

 

SD

0.0080

72.759

 

%RSD

0.23

1.29

 

 


Method validation:

ICH guidelines were strictly followed to validate the method to determine the parameters such as system suitability, linearity, specificity, accuracy, Limit of detection and quantitation (LOD and LOQ), precision and robustness of the method. No interference by the excipients was found during specificity of the analytical peak. Linearity of the method was determined by plotting concentration (in µg) and absorbance on X-axis and Y-axis respectively. A good linear relationship was obtained between the concentration ranges of 0.2 µg-0.6 µg with the correlation co-efficient of 0.9984. Purity of the method was found to be 99.31% and the results of assay are shown in table 2. Accuracy of the method was determined by analyzing at different concentration levels of 80%, 100% & 120% and the mean recovery was found to be 99.15%. Precision study was performed by injecting sample solution at working concentration in five replicates into the chromatographic system and its percentage relative standard deviation (%RSD) was found to be less than 2% which meets the acceptance criteria, which determines the method to be precise. From the response obtained from calibration curve, limit of detection and limit of quantitation values were found to be 1.82µg/ml and 5.50µg/ml respectively. Robustness of the method was determined by making small modifications in the standard method.

 

 

TABLE 2: ASSAY RESULTS

 

Standard Area

Sample Area

Injection-1

5578.661

5577.355

Injection-2

5580.073

5629.969

Injection-3

5578.661

5667.162

Injection-4

5580.863

5589.047

Injection-5

5625.083

5577.355

Average Area

5579.132

5608.178

Standard weight

50

Sample weight

760

Average Wt.

152

Label claim

10

std.purity

98.8

Assay in mg

9.93

%Assay

99.31

 

Specificity:

Specificity of the method was determined by injecting blank, sample and standard solutions into the chromatographic system and their peaks were compared to each other. There was no interference observed due to excipients from the tablet formulation. A good correlation was obtained between the retention times of the standard and the sample. Chromatograms are shown in the figure 3 and 4 respectively.

 

 

Figure 3: Chromatogram for specificity of Memantine sample

 

 

Figure 4: Chromatogram for Specificity of Memantine standard

 

 

Figure 5: Blank chromatogram for specificity by using mobile phase

 

Linearity:

From the standard stock solution prepared, serial dilutions were made to get the concentrations of 60, 80, 100, 120 and 140µg/ml and injected into the chromatographic system twice. The respective peak area obtained were recorded and a graph was plotted between concentration and peak area on X and Y axis respectively as shown in figure 6. A good correlation was obtained with the correlation co-efficient value of 0.999, the results of which are shown in table 3.

 

TABLE 3: RESULTS OF LINEARITY

S. No.

Concentration µg/ml

Peak Area

1

60

3499.675

2

80

4638.465

3

100

5697.355

4

120

6931.177

5

140

8101.848

S.D.

31.62

1818.36

Slope

57.49

Intercept

25.175

 

 

Figure 6: Calibration curve of Memantine hydrochloride

 

Accuracy:

Accuracy was determined by Recovery studies. To the pre analyzed sample, the reference standards of the drugs were added at the level of 80%, 100%, 120%. Recovery studies were carried out thrce and the percentage recovery and percentage mean recovery were calculated for drug is shown in table. For checking accuracy of the method, recovery studies were carried out by addition of standard drug solution to pre-analyzed sample solution at three different levels 80%, 100%, 120%. Results are shown in table 4.

 

Precision:

Prepared sample preparations of the same batch of tablets as per test method were analyzed for precision studies by injecting the sample 6 times in to the chromatographic column. % RSD was less than 2 which was found to be within the acceptance range. The results are shown in table 5.

 

 

 

 


TABLE 4: RECOVERY RESULTS OF MEMANTINE HYDROCHLORIDE

Recovery level

Amount taken (mcg/ml)

Area

Average area

Amount recovered (mcg/ml)

% Recovery

Average % recovery

80%

100

5688.661

5690.26

98.14

98.14

99.15%

100

5690.661

100

5691.355

100%

120

6731.177

6758.941

118.63

98.86

120

6783.889

120

6761.758

120%

140

8101.848

8124.750

140.66

100.47

140

8113.313

140

8159.089

 


TABLE 5: RESULTS FOR METHOD PRECISION OF MEMANTINE HYDROCHLORIDE

S.No.

Retention time

Area

1

3.460

5671.643

2

3.450

5587.778

3

3.473

5700.527

4

3.467

5510.683

5

3.467

5579.223

6

3.460

5671.643

Average

3.4628

5620.250

St dev

0.0080

72.759

%RSD

0.23

1.29

 

 

Limit of Detection (LOD) and limit of Quantitation (LOQ):

Limit of detection is the lowest concentration of the analyte that can be detected in a given sample where as Limit of quantitation is the lowest concentration at which the method performance system is acceptable for specific use. LOD and LOQ values were calculated from the linearity results from the following formulae and the results are shown in the table 6.

 

Limit of Detection (LOD)=3.3x /S

Limit of Quantitation (LOQ)=10x /S

Where, σ = the standard deviation of the response

S = the slope of the calibration curve

 

TABLE 6: RESULTS OF LOD AND LOQ

Standard deviation

Slope

LOD

LOQ

31.62

57.49

1.82µg/ml

5.50µg/ml

 

Robustness:

Robustness of the method was demonstrated by injecting the prepared solution as per the method and changing the chromatographic conditions such as flow rate and wavelength. The results indicate no significant changes which are shown in table 7.

 

 

TABLE 7: ROBUSTNESS DATA FOR MEMANTINE HYDROCHLORIDE

Parameter

Retention time (min)

Tailing factor

Flow rate

 

 

0.8ml/min

4.003

2.000

1.2ml/min

3.040

1.690

Wavelength

 

 

258nm

3.447

1.833

262nm

3.483

1.839

 

CONCLUSION:

The method that was developed was selective, accurate, precise and robust as the method remains unaffected by changes in the chromatographic conditions such as flow rate and wavelength. Therefore, the method can be employed for the quality control analysis of Memantine hydrochloride in pharmaceutical formulations.

 

ACKNOWLEDGEMENT:

All the authors are thankful to sultan-ul-uloom college of pharmacy for providing required facilities to undergo the research work.

 

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Received on 17.12.2018       Accepted on 23.01.2019

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2019; 9(2): 69-74.

DOI: 10.5958/2231-5691.2019.00011.X