1. INTRODUCTION:

Donepezil hydrochloride (Fig.1) 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-one hydrochloride. Donepezil hydrochloride is commonly referred to in the pharmacological literature as E2020. It has an empirical formula of C₂₄H₂₉NO₃HCl and a molecular weight of 415.96. Donepezil hydrochloride is a white crystalline powder and is freely soluble in chloroform, soluble in water and in glacial acetic acid, slightly soluble in ethanol and in acetonitrile and practically insoluble in ethyl acetate and in n-hexane^1-2.

A Through literature survey has revealed the following reported methods for the estimation of Donepezil in bulk, formulation and biological fluids using different analytical techniques. The reported methods include estimation of Donepezil in biological samples using LC-UV and LC-MS, HPLC, and Spectrophotometry for the estimation of Donepezil in bulk and pharmaceutical formulations^3-16. In the present study attempts were made to develop a rapid, economical, precise and accurate liquid chromatographic method for the estimation of donepezil in tablet formulations.

2.0 EXPERIMENTAL:

2.1 Chemicals and reagents:

All the reagents were of analytical and HPLC grade unless stated otherwise. Milli-Q-Water was used throughout the study. Hydrochloric acid, Sodium Hydroxide, Acetonitrile, Hydrogen Peroxide, trifluoro acetic acid (Merck, Mumbai, India) were used. Donepezil Standard was obtained as a gift sample from Dr. Reddy’s Laboratories, Hyderabad, India and the donepezil tablets were purchased from Local Pharmacy.

2.2 Instrumentation:

The HPLC system used was a Waters 2695 separation module with an auto injector and waters 2996 PDA Detector. The output signal was monitored and integrated using Empower software. Zorbax SB C18, 150 x 4.6mm, 5µm column was used.

2.3 Optimized Chromatographic conditions:

Mobile phase : A filtered and degassed mixture of water and Acetonitrile (32:68), pH adjusted to 4.5 with Trifluoro acetic acid.

Detection wavelength : 229 nm

Column : Zorbax SB C18, 150 x 4.6mm, 5µm

Flow rate : 1.0 mL min^-1

Injection volume : 20µL

Fig.1: Chemical structure of Donepezil

2.4 Standard Preparations:

Standard stock solution was prepared by dissolving 25 mg of Donepezil working standard in 25 mL of mobile phase. From the above stock solution, a series of solutions were prepared at concentration levels ranging from 5 µg mL^-1 to 25 µg mL^-1 concentration. Measured the peak area responses of solutions at all levels in duplicate.

2.5 Sample solution Preparation:

20 tablets were accurately weighed and grounded to fine powder. Donepezil tablet powder equivalent to the label claim was accurately weighed and transferred into a 100 mL volumetric flask, few mL of mobile phase was added and sonicated to dissolve and made up the volume with mobile phase. The above solution was filtered and diluted to get a final concentration of 15 µg mL^-1.

2.6 Stress degradation study:

To determine whether the analytical method was stability indicating, Donepezil API was stressed under various conditions includes photolytic degradation (Exposed to 1, 00,000 Lux.), acid hydrolysis (1N Hydrochloric acid), base hydrolysis (1N sodium hydroxide) and Oxidative degradation (10% Hydrogen peroxide). All the stress degradation studies were performed with a drug concentration of 1 mg mL^-1.

2.7 Method Validation:

The method was validated according to International Conference on Harmonisation (ICH) guidelines for validation of analytical procedures. The validated parameters were system suitability, specificity, range and linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, ruggedness and robustness^17-18.

3.0 RESULTS AND DISCUSSION:

To optimize the method, initially a 10% Acetonitrile was used. However, this 10% Acetonitrile was unable to elute Donepezil. To elute Donepezil peak, the mobile phase composition of Acetonitrile: Water (50:50 V/V), pH adjusted to 3.2 with Trifluoro acetic acid was tried and the peak elution time is 2.4min (Fig 1a). The λ-max of Donepezil was observed at 229nm (Fig 1b). To increase the retention time of Donepezil, the mobile Phase composition and pH is optimised to Acetonitrile: Water (32:68 V/V), pH adjusted to 4.5 with Trifluoro acetic acid. The peak elution time is 7.3min (Fig 1c). A successful isocratic run (mentioned in the experimental section) was employed to resolve all the known degradation products which were formed during degradation. The optimized injection volume and detection wavelength were 20 μL and 229 nm, respectively. To achieve the separation of degradation products, stationary phase of C18 and a combination of water and acetonitrile were used. The chromatographic behaviour of Donepezil in different mobile phases and stationary phases were shown Table 1&2. The separation of degradation products and Donepezil was achieved on a Zorbax SB C18, 150 x 4.6mm, 5µm column and a water and acetonitrile (68:32), pH adjusted to 4.5 with Trifluoro acetic acid was used as mobile phase (Fig 2). The developed was found to be specific and validated as per ICH guide lines.

Fig.1a: A typical chromatogram of Donepezil (Mobile Phase composition is a mixture of Acetonitrile: Water (50:50 V/V), pH adjusted to 3.2 with Trifluoro acetic acid)

Fig.1b: A typical Spectrum of Donepezil λ-Max

Fig.1c: A typical chromatogram of Donepezil.

Table 1: The chromatographic behaviour of Donepezil in different mobile phases.

Mobile phase	Retention time (min)
Acetonitrile: Water (50:50 V/V), pH adjusted to 3.2 with Trifluoro acetic acid	2.4
Acetonitrile: Water (45:55 V/V), pH adjusted to 3.2 with Trifluoro acetic acid	3.2
Acetonitrile: Water (40:60 V/V), pH adjusted to 3.2 with Trifluoro acetic acid	4.5
Acetonitrile: Water (32:68 V/V), pH adjusted to 4.5 with Trifluoro acetic acid	7.3

Table 2: The chromatographic behaviour of Donepezil in different Columns.

Column	Retention time (min)
Lichrospher RP-18; 15cm × 4.6 mm i.d., 5 μm particle	6.0
Zorbax SB C18; 15cm × 4.6 mm i.d., 5 μm particle	7.3
YMC ODS-A; 15cm × 4.6 mm i.d., 5 μm particle	8.4
Hypersil ODS C18; 15cm × 4.6 mm i.d., 5 μm particle	5.2

3.1 Degradation behavior:

The developed LC method was used to study degradation behaviour of Donepezil in its tablet formulations under various stress conditions such as thermal, humidity, photolytic, acidic, and alkaline, oxidation conditions. The control sample of Donepezil showed only one major peak, a typical chromatogram of control tablet sample was shown in Fig.1c. The separation of Donepezil from its degradation products was shown in Fig.2 and it can ensure that the separation was achieved within 15min.The degradation behaviour of donepezil in different conditions was shown in Table 3.

3.2 Method Validation:

The optimized chromatographic conditions were validated by evaluating Linearity, Precision, Accuracy, LOD and LOQ, robustness in accordance with ICH guidelines.

3.2.1 Linearity

The curve proved to be Linear over a concentration range of 5 µg mL^-1 to 25 µg mL^-1 (Fig 3). Standard solutions were prepared at five concentrations (5, 10,15,20 and 25 µg mL^-1) were injected in triplicate. The linear regression of concentration vs peak area resulted in an average coefficient of determination (R²) is 0.999992(Table 4).

Fig. 3: A Typical Linearity Plot for Donepezil.

3.2.2 Precision:

The Precision of the method was evaluated by carrying out 6 independent Assay Preparations of Donepezil. The Intermediate precision of the method was also evaluated using Inter day and Intraday with different instruments. The results obtained were indicates that the method is reproducible (Table 5a&5b).

Table 3: Results for Degradation behaviour of Donepezil.

Degradation mechanism / condition	% Assay	% Degradation	Purity Angle	Purity Threshold	Remarks
Un-degraded sample	99.9	-	0.022	0.553	Passed
photolytic at 254 nm for 168 Hrs	99.8	-	0.155	0.411	Passed
Acid /1 N HCl Heat on water bath for 8 Hrs	94.6	4.4	0.260	0.373	Passed
Base /1 N NaOH Heat on water bath for 16 Hrs	97.2	2.8	0.211	0.322	Passed
Peroxide /10.0% H₂O₂Heat on water bath for 12 Hrs	98.7	1.3	0.180	0.388	Passed

Table 4: Results for Linearity Plot for Donepezil.

Linearity Level	Concentration (µg/mL)	Response	Statistical Analysis
25%	5	532749	Slope	111027
50%	10	1086293	y-Intercept	-22508
100%	15	1646426	% of y- Intercept	-1.37
150%	20	2194893	Correlation Coefficient	0.999996
200%	25	2754124	Regression Coefficient-r²	0.999992

Table 5a: Results of System Precision.

	Injection	Peak Area	Theoretical Plates	Tailing factor
	1	1646466	20113	1.02
Concentration	2	1646211	20154	1.11
100%	3	1646343	20221	1.05
	4	1646231	20213	1.04
	5	1646416	20188	1.06
Statistical	Mean	1646333	20178	1.06
Analysis	SD	111.76
	% RSD	0.68%

Table 5b: Results for Inter and Intraday of Donepezil.

Preparation No.	Inter Day	Intraday
Preparation No.	% Assay	% Assay
1	100.04	100.01
2	99.77	99.97
3	100.22	100.11
4	100.02	100.05
5	99.88	99.89
6	99.89	99.84
Average	99.97	99.98
Std.Dev.	0.16	0.10
%RSD	0.16	0.10

3.3.3 Accuracy:

The accuracy of the method is determined by recovery experiments. The recovery was performed by Donepezil sample at 50%, 100% and 150% levels against Donepezil working standard. At each level, three sample preparations were used. The average % recovery on three preparations at each level, as 100.26%, 99.70% and 99.78% respectively (Table 6).

Table 6: Results for Accuracy (% recovery) of Donepezil.

% Spiked to the label amount	Amount added (mg)	Amount found (mg)	% Recovery	Mean % Recovery
% Spiked to the label amount	Amount added (mg)	Amount found (mg)	% Recovery	N=3,+SEM
50% 50% 50%	5.11	5.13	100.39	100.26+0.01
	5.05	5.06	100.20
	5.07	5.08	100.20
100% 100% 100%	10.08	10.05	99.70	99.70+0.04
	10.09	10.04	99.50
	10.05	10.04	99.90
150% 150% 150%	15.06	15.04	99.87	99.78+0.01
	15.05	15.01	99.73
	15.08	15.04	99.73

3.3.4 LOD and LOQ:

The limit of detection (LOD) is defined as the lowest concentration of an analyte that can be readily detected but not necessarily quantified. It is usually regarded as the amount for which the signal-to-noise ratio (SNR) is 3:1. The limit of quantitation (LOQ) is defined as the lowest concentration of an analyte that can be quantified with acceptable precision and accuracy. It is usually regarded as the amount for which the SNR is 10:1. Two types of solution, blank solution and solutions containing known, progressively decreasing concentrations of the analyte were prepared and analyzed. LOD and LOQ were 0.5 μg mL^-1 and 5 μg mL^-1 respectively.

3.3.5 Robustness:

To determine the robustness of developed method, experimental conditions were purposely altered. The flow rate of the mobile phase was 1.0 mL min-1. To study the effect of flow rate on the retention, flow was changed by 0.2 units from 0.8 to 1.2 mL min^-1. The effect of the percent organic strength on the retention and peak area was studied by varying Acetonitrile by +3 %. The pH of the mobile phase was altered by +0.2 units. The % RSD was Less than 1.0% (Table 7).

Table 7: Results for Robustness of Donepezil.

Robustness Parameter	Retention Time	Tailing factor	RSD (%)
Mobile phase Composition (v/v)
30:70	7.4	1.07	0.24
32:68 (Optimized)	7.3	1.03	0.11
34:66	7.2	1.03	0.09
Variation in flow rate (mL min-¹)
0.24	7.5	1.13	0.33
1.0 (Optimized)	7.3	1.04	0.12
1.2	7.1	1.01	0.21
Buffer pH
4.3	7.2	1.22	0.22
4.5(Optimized)	7.3	1.02	0.06
4.7	7.4	1.13	0.16

3.3 Stability of analytical solution and mobile phase

The % RSD for retention times of different intervals of 0hr, 24hrs, 30hrs and 48hrs was found to be 0.13% and 0.46% for mobile phase and analyte solution respectively, (Table 8).

Table 8: Results for Stability of analytical solution and mobile phase.

Time Interval	Retention time	Mobile phase stability	Analyte solution stability
Time Interval	( in minutes)	Mobile phase stability	Analyte solution stability
0 Hour (Initial)	7.31	100.02	100.11
12 Hours	7.31	99.93	100.03
24 Hours	7.32	100.03	99.98
30 Hours	7.31	99.98	99.91
48 Hours	7.31	99.91	99.94
Mean ± SEM (n= 4)	7.31±0.00	99.97±0.00	99.99±0.01
%RSD	0.06	0.05	0.08

4.0 CONCLUSION:

Forced degradation study on Donepezil in tablet formulation was carried out under the conditions of Photolysis, hydrolysis, Oxidation and thermal. Based on the information generated by forced degradation, stability indicating LC method was developed and validated. The Assay method adopted for Donepezil is specific, precise, linear and accurate. The Analyte solution was found to be stable up to 48 hours under ambient conditions and mobile phase was found to be stable up to 48 hours. Hence this method can be used for routine analysis and stability study.

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Received on 22.04.2011 Modified on 05.05.2011

Asian J. Research Chem. 4(10): Oct., 2011; Page 1508-1512