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.
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 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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DOI: 10.5958/2231-5691.2019.00011.X