Two spectrophotometric methods^15,16and one spectrofluorimetric¹⁷ method have also been reported. One stability indicating RP-HPLC method with precolumn derivatization¹⁸ have also been reported which suffer from long chromatographic run time and also high retention time.

The objective of this study was to develop stability indicating reverse phase high performance liquid chromatography method for the estimation of Memantine HCl in pure and tablet dosage form without any derivatization and having short retention time. This method was found to be linear, precise, accurate, sensitive, specific, and robust, and therefore suitable for routine analysis.

MATERIALS AND METHOD

Chemicals and Reagents:

Memantine was obtained as a gift from Hetero Drugs Ltd, Hyderabad. HPLC grade acetonitrile and analytical grade Sodium dihydrogen phosphate, Disodium hydrogen phosphate, Ortho phosphoric acid obtained from SD Fine Chemicals Ltd, Mumbai. Hydrochloric acid, sodium hydroxide, hydrogen peroxide of analytical grade was obtained from Merck Chemicals Ltd, Mumbai. HPLC grade water was used to prepare all solutions.

HPLC Instrumentation and Chromatographic conditions:

The analytical separations were carried out on a Shimadzu HPLC system equipped with Photo Diode Array detector. The output of signal was monitored and integrated using LC – solutions 2000 software. The analytical column was Intersil ODS C₁₈ (150 × 4.6mm, 5µ). Mobile phase consisted of 0.2M Sodium Dihydrogen Ortho Phosphate, 0.1M Di Sodium Hydrogen Ortho Phosphate (pH adjusted to 3 with phosphoric acid) and Acetonitrile in the ratio of 50:50. Mobile phase was mixed, filtered through 0.45µmembrane filter and degassed under ultrasonication. The mobile phase was used as diluent. The flow rate was 1 ml/min and runtime was 7 minutes. The column was maintained at ambient temperature. UV detection was measured at 272 nm and the volume of sample injected was 10 μl.

Preparation of standard stock solution:

50mg of Memantine HCl was weighed accurately and dissolved in 50ml of mobile phase to get the concentration of 1000 µg/ml. Resultant solution was filtered through Whatman filter paper. The standard chromatogram for memantine HCl (100μg/ml) was shown in figure 2.

Preparation of working standard solution:

Working standard solutions of Memantine HCl were prepared by accurately transferring the (0.2, 0.4, 0.6, 0.8. 1.0 and 1.2 ml) aliquots of the standard stock solution into a series of six 10 ml volumetric flasks. The volume was made upto mark with mobile phase to obtain concentration range of 20 – 120 µg/ml.

Preparation of sample solutions:

Ten tablets were finely powdered and weighed. A portion of the powder equivalent to about 10mg of Memantine hydrochloride was weighed accurately and transferred into100mL volumetric flask and mixed thoroughly for 20minutes for complete dissolution of Memantine hydrochloride and then the sample solution was filtered and diluted to 100ml with mobile phase to get concentration of 100µg/ml and used for analysis.

Figure 2: Standard chromatogram for Memantine HCl

RESULTS AND DISCUSSION:

HPLC method development and optimization:

To optimize the chromatographic conditions, different columns, mobile phases, flow rates etc., were tested. 0.2M Sodium Dihydrogen Ortho Phosphate, 0.1M DiSodium Hydrogen Ortho Phosphate (pH adjusted to 3 with Phosphoric acid) and Acetonitrile in the ratio of 50:50 was preferred as mobile phase because it resulted in a greater response to Memantine after several preliminary investigatory runs compared with the different mobile phase combinations. The effect of the flow rate was studied in the range 0.9 to 1.2 ml/min and 1ml/min was preferred to be effective. Under these conditions, the analyte peak obtained was well-defined and free from tailing. The retention time (RT) was found to be 3.337 min. The optimized chromatographic parameters were listed in table 1.

Table 1: Optimized chromatographic parameters

Optimized Chromatographic parameters

Elution Isocratic

Mobile phase 0.2M NaH₂PO₄ + 0.1M Na₂HPO₄

(pH-3) adjusted with H₃PO₄ and

Acetonitrile (50:50)

Column Intersil ODSC₁₈column

Flow rate 1ml/min

Detection 272nm

Injection volume 10μl

Temperature Ambient

Retention time 3.337 min

Run time 7 min

Concentration 20-120μg/ml

Validation of the method:

When method development and optimization are complete, it is necessary to accomplish method validation. The validation studies include linear range (correlation coefficient), method precision (RSD, %), method accuracy (% recovery and RSD, %), sensitivity studies (LOD & LOQ), and robustness.

System suitability studies:

System-suitability tests are an integral part of method development and are used to ensure adequate performance of the chromatographic system. Retention time (RT), number of theoretical plates (N), tailing factor (T), and peak asymmetry (AS), resolution (RS) were evaluated. The system suitability test was performed using five replicate injections of standards before analysis of samples. The system suitability method acceptance criteria set in each validation run were: capacity factor > 2.0, tailing factor ≤ 2.0 and theoretical plates > 2000. In all cases, the relative standard deviation (R.S.D) for the analytic peak area for two consecutive injections was < 2.0%. System suitability parameters were shown in table 2.

Table 2: System suitability parameters

Parameters	Values
Retention time	3.337 min
Theoretical plates	5421.000
Peak asymmetric factor	1.360

Linearity:

The linearity of the method was evaluated by preparing six series of standard solutions of Memantine HCl in the range of 20–120 µg/ml in mobile phase and injecting the solutions into the HPLC system. Excellent correlation between Memantine peak area and concentration was observed with R² = 0.999 (Figure.3). The regression equation was found to be Y = 22.13x + 13.04. Statistical data are presented in table 3 and the calibration curve was shown in figure 3.

Table 3: Linearity results for Memantine

S.No	Concentration (μg/ml)	Area (mV.sec)(n=6)
1	20	475.632
2	40	868.252
3	60	1323.707
4	80	1809.351
5	100	2248.266
6	120	2659.204

Figure 3: Calibration curve of Memantine

Precision:

System precision: (Repeatability)

To study precision, five replicate standard solutions of Memantine HCl (100µg/ml) were prepared and analyzed using the proposed method. The percent relative standard deviation (% RSD) for peak responses was calculated. Results of system precision studies were shown in table 4.

Table 4: Results of system precision for memantine

S.No	Rt (min)	Peak Area (mV.sec)
1	3.313	2359.407
2	3.31	2338.546
3	3.333	2342.458
4	3.327	2340.143
5	3.328	2344.523
MEAN	3.3202	2345.015
S.D	0.0082	8.3590
%RSD	0.2478	0.3564

Method precision: (Reproducibility)

The intraday and inter-day precision of the proposed method was determined by analyzing the corresponding responses 5 times on the same day and on different days for concentration of sample solutions of 100µg/ml. The result was reported in terms of relative standard deviation (% RSD). Results of method precision studies were shown in table 5.

Table 5: Results of Method precision for Memantine

S.No	MEMANTINE HCl Standard area= 2345.015
S.No	Peak Area (mV.sec)	% Labelled claim
1	2342.445	99.890
2	2349.55	100.193
3	2349.803	100.204
4	2358.859	100.590
5	2367.816	100.972
MEAN	2353.695	100.369
S.D	9.8102	0.4183
%RSD	0.4168	0.4402

Intermediate precision

The intermediate precision of the proposed method was determined by performing the method by two analysts (Analyst 1 and Analyst 2) for concentration of sample solutions 100µg/ml. The percent relative standard deviation (% RSD) for peak responses was calculated. The results for intermediate precision were shown in table 6.

Table 6: Results of Intermediate precision for Memantine

S.NO	ANALYST – 1		ANALYST – 2
S.NO	Rt(min)	Area (mV.sec)	Rt(min)	Area (mV.sec)
1	3.357	2419.347	3.357	2419.247
2	3.356	2414.347	3.357	2417.921
3	3.356	2414.143	3.355	2417.921
4	3.357	2419.227	3.357	2419.642
5	3.357	2419.428	3.356	2416.237
MEAN	3.357	2417.299	3.357	2418.194
S.D	0.0005	2.6228	0.0008	2.2471
% RSD	0.0163	0.1085	0.0266	0.0929

Accuracy:

Accuracy of the method was confirmed by the standard addition method, which was carried out by performing recovery studies at 3 different concentrations 80%, 100% and 120% of these expected, in accordance with ICH guidelines, by replicate analysis (n=3). Known amount of standard drug solution (10µg/ml) was added to a pre analyzed sample solution (80, 100, 120 µg/ml) and percentage drug content was measured. The closeness of obtained value to the true value indicates that the proposed method is accurate. Recovery studies were shown in table 7.

%Recovery = [(Ct –Cpa)/ Cs] × 100.

Where,

· Ct = Total concentration of analyte

· Cpa = Concentration of pre-analysed sample

· Cs = Concentration of standard added to pre-analysed sample.

Robustness:

The robustness study was performed to evaluate the influence of small but deliberate variation in the chromatographic condition. The robustness was checked by changing parameters like flow rate of mobile phase and detection wavelength

· Change in the detection wavelength by ± 2nm (270nm and 274nm)

· Change in flow rate by ± 0.1 ml/minute (0.9 ml/min and 1.1 ml/minute)

After each change, sample solution was injected and % assay with system suitability parameters were checked.

Robustness values were given in table 8.

Limit of Detection and Quantitation:

Detection and Quantitation limit were calculated by the method based on the standard deviation (s) and slope of the calibration plot, using the formula

Limit of Detection = s × 3.3/S

Limit of Quantitation = s × 10/S

Where

s = the standard deviation of the response.

S = the slope of the calibration curve (of the analyte).

Results of LOD & LOQ were shown in table 9.

Table 7: Results of recovery studies for Memantine

Name

Pre Analysed Sample Concentration (µg/ml)

Spiked amount

(µg/ml)

Area (mV.sec)

Amount Recovered

(µg/ml)

% Recovery

Average %

Recovery

MEMANTINE HCl

( Mean Standard Area = 2365.174 mv.Sec)

2123.052

2122.972

2132.03

89.76

89.75

89.71

99.7

99.6

99.66 + 0.05

100

2612.51

2612.745

2612.263

110.54

110.46

110.44

100.4

101.1 + 0.05

120

3077.549

3078.220

3077.381

130.11

130.14

130.12

100.1

100.3

100.1

102.5 + 0.05

Table 8: Results of Robustness for Memantine

Parameter	Rt (min)	Area(mV.sec) (n=5)	% RSD
Flow rate (ml/min)
0.9ml/min	3.927	2856.323	0.3463
1.1ml/min	3.220	2357.382	0.3779
Wavelength (nm)
270nm	3.373	1938.697	0.510
274nm	3.380	2425.924	0.367

Table 9: Results of LOD, LOQ for Memantine

S.No

LOD

LOQ

Memantine

HCl

3.3 × 0.3020/ 22.13

0.0450 µg/ml

10 × 0.3020/ 22.13

0.1364 µg/ml

Specificity

Specificity of an analytical method is its ability to measure the analyte accurately and specifically in the presence of component that may be expected to be present in the sample matrix. Chromatograms of standard and sample solutions were compared in order to provide an indication of specificity of the method.

Assay of pharmaceutical formulation:

The proposed validated method was successfully applied to determine Memantine HCl in their tablet dosage form (Namenda). And the % Assay results were shown in table 10.

Table 10: Results of % assay

S.No

Amount Found (mg/ tablet)

% Assay

20.105

20.061

20.054

100.5

100.3

100.2

Stability studies:

In order to demonstrate the stability of both standard and sample solutions during analysis, both solutions were analyzed over a period of 24hr at room temperature. The results show that for both solutions, the retention time and peak area of Memantine HCl remained almost similar (% R.S.D. less than 2.0) ands no significant degradation within the indicated period, thus indicated that both solutions were stable for at least 24hr, which was sufficient to complete the whole analytical process.

The results of Forced degradation studies¹⁹ were shown in table 11.

Acid degradation study:

Accurately weighed 10 mg of the drug was taken in a 10 ml volumetric flask and few drops mobile phase was added to dissolve the drug .The volume was made up with freshly prepared 0.1N HCl. Then this solution was kept inside a water bath maintained at a temperature of 60°C for 30 min, followed by analysis by the proposed method. The chromatogram for acid degradation study was shown in figure 4.

Figure 4: chromatogram for acid degradation study

Base degradation study:

Accurately weighed 10 mg of the drug was taken in a 10 ml volumetric flask and few drops mobile phase was added to dissolve the drug .The volume was made up with freshly prepared 0.1N NaOH. Then this solution was kept inside a water bath maintained at a temperature of 60°C for 30 min, followed by analysis by the proposed method. The chromatogram for base degradation study was shown in figure 5.

Figure 5: chromatogram for base degradation study

Peroxide degradation study:

Accurately weighed 10 mg of the drug was taken in a 10 ml volumetric flask and few drops mobile phase was added to dissolve the drug .The volume was made up with freshly prepared 30% H₂O₂. Then this solution was kept inside a water bath maintained at a temperature of 60°C for 30 min, followed by analysis by the proposed method. The chromatogram for peroxide degradation study was shown in figure 6.

Figure 6: Chromatogram for peroxide degradation study

Thermal degradation study:

Sample was subjected to thermal degradation by heating the sample at 105°C for 2 days, followed by analysis by the proposed method. The chromatogram for thermal degradation study was shown in figure 7.

Figure 7: Chromatogram for thermal degradation study

CONCLUSION:

A simple, rapid, accurate, and precise RP-HPLC method for the analysis of Memantine in pure and in tablet dosage forms had been developed and validated in accordance with ICH guidelines. The RP-HPLC method developed is cost-effective due to short retention time which enabled analysis of memantine samples with a small amount of mobile phase. From the % RSD values of precision and recovery studies the method was found to be precise and accurate. The low detection and quantification limits achieved indicate the method is very sensitive. The robustness data gathered during method validation showed that the method is not susceptible to small changes in chromatographic conditions. The proposed RP-HPLC method developed by the author is suitable for routine analysis and quality assessment of Memantine in pharmaceutical products.

Table 12: Summary of validated parameters for proposed method

Parameter Result

Linearity range 20 – 120 µg/ml

Regression equation Y = 22.13x + 13.04

Slope 22.13

Intercept 13.04

Correlation coefficient 0.999

System precision (% RSD, n=5) 0.3564

Method precision (% RSD, n=5) 0.4402

LOD (µg/ml) 0.362

LOQ (µg/ml) 1.096

% Recovery (Accuracy, n =3) 101.08%

% Assay (% Assay, n=3) 100.36%

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Received on 07.09.2013

Modified on 25.09.2013

Accepted on 02.10.2013

Research Journal of Pharmaceutical Dosage Forms and Technology. 5(6): November-December, 2013, 334-340

Stress condition (100µg/ml)	Duration of stress	Peak area (mV.sec)	% Degradation	% of Active ingredient
Standard	-	2374.018	-	-
Acid (0.1 N HCl)	30 min	2288.335	3.61	96.390
Base (0.1N NaOH)	30 min	2258.164	4.88	95.119
Peroxide (30% H₂O₂)	30 min	2237.030	5.771	94.229
Thermal	2days	2305.292	2.895	97.105