Development and Validation of Stability Indicating HPTLC Method for Determination of Donepezil Hydrochloride and its related substances in Bulk Drug and Pharmaceutical Dosage Form
Lawanya Lata Pandey *, Nirmal Dongre
Department of Pharmacy, SAGE University, Indore, Madhya Pradesh, India.
*Corresponding Author E-mail: pandeylawanyalata@gmail.com
ABSTRACT:
Context: A new, simple, selective, and affordable HPTLC technique for the analysis of donepezil hydrochloride (DH) and its associated compounds was developed and verified in accordance with ICH guidelines. Method: Donepezil Hydrochloride and Related Substances were densitometrically analyzed in the absorbance mode at 320 nm. The stationary phase was 60 F—254 silica gel pre-coated on aluminium TLC plates. Butanol, water, and glacial acetic acid were the components of the mobile phase (4:5:1, v/v/v). Result: It was discovered that this method provides compact places for DH (RF 0.53± 0.03) .Testing for stability indicators (force degradation) was done in acidic and alkaline conditions, as well as with dry heat and photodegradation. The degradation products were clearly distinguished from the pure drug by their markedly differing RF values. For Validation of method linearity, precision, robustness, limit of detection (LOD), limit of quantification (LOQ), ruggedness, and accuracy parameters are verified. LOD and LOQ were found to be 80.85 and 245 ng per spot, respectively, and linearity was found in the range 400-1200 ng per spot with a considerably high value of the correlation coefficient r2 = 0.999 is found. A 0.43% coefficient of variance is discovered. Conclusion: The approach was repeatable and specific for estimating donepezil hydrochloride and related compounds, according to statistical analysis Donepezil Hydrochloride can be quantified even when there are degraded products and related compounds present. It is commercially available for the estimation of related compounds and Donepezil hydrochloride.
KEYWORDS: Donepezil hydrochloride, HPTLC, Method Development, and Validation, Assay.
INTRODUCTION:
A piperidine derivative known as donepezil hydrochloride (DH) is a potent and specific inhibitor of the acetylcholinesterase (AChE) enzyme. 2,3 dihydro-5,6 dimethoxy-2-[1-(phenylmethyl)-4-piperidinyl] methyl1H-inden-1-one hydrochloride is the chemical name for DH 1, 2. Chemically, it differs from other AChE inhibitors3,4. DH is a white, crystalline powder that rapidly dissolves in n-hexane, glacial acetic acid, water, methanol, and chloroform but very slightly in ethanol and acetonitrile. DH, a drug used to treat mild to severe Alzheimer's-related dementia, reversibly inhibits AChE5. A review of the literature reveals that there aren't many HPTLC methods available for estimating donepezil and similar compounds. In Figure 1, the chemical composition of medications is displayed.
Fig 1. Chemical structure of donepezil hydrochloride (DH)
MATERIALS AND METHODS:
Materials
Unichem Research Laboratory (Goa, India) gave DH of pharmaceutical quality (99.98% m/m) as a gift sample. The other chemicals were purchased from Merck Chemicals (India), and all reagents are of analytical quality. The use of distillate water in analysis.
Instrumentation and Chromatographic conditions
Samples were spotted as 6 mm-long bands on a precoated silica gel aluminium plate 60 F—254 (20 x 10 cm) of 20 m thickness using a Camag Linomat V sample applicator (Muttenz, Switzerland). The plates were activated at 60°C for 10 minutes after being prewashed with methanol. Two bands were applied at a constant rate of 0.1 L s -1, 10 mm apart. The scanning speed was 20 mm s-1, and the slit size was maintained at 6 mm × 0.45 mm. The mobile phase was a mixture of butanol, water, and glacial acetic acid (4:5:1, v/v/v). A 20 mL mobile phase was included in each sample to create the chromatogram. Linear ascending development was conducted up to an 8 cm depth in a 20 cm × 10 cm twin-trough glass chamber (Camag, Muttenz, Switzerland) saturated with the mobile phase. The ideal chamber saturation time for the mobile phase was 20 minutes at room temperature (25°C 2°C) and relative humidity of 60 5%. The chromatogram was generated, and then the TLC plates were air dried. Under computer-controlled circumstances, densitometric scanning at 260 nm was performed using a Camag TLC scanner III in the absorbance mode.
Preparation of standard solutions and calibration graphs:
A stock solution of DH containing 1.0 mg/ml was created in methanol (Solution S1). To generate a standard solution of DH with a final concentration of 0.1 mg/ml, 100 g/ml, or 100 nanogram/ul, an appropriate volume of this solution was further diluted with 10 ml methanol.
Preparation of sample Solution
The weight and powder of twenty pills were done. 10 mg of Donepezil HCl-equivalent powder was put to a 50 mL calibrated volumetric flask. A 0.2 mg/mL-1 solution is produced. The final concentration is further diluted to (0.2 mg/ml), 200 g/ml-1, or 200 nanogram/ul.
A TLC plate was used to apply aliquots of 2, 4, 6, 8, 10, 12, and 14 L of the standard solution (donepezil hydrochloride), which has a concentration of 100ng/ul, and 3 and 5 ul of the sample solution (donepezil hydrochloride tablet), which has a concentration of 200ug/ml, for the purpose of charting the calibration curve. The DH concentrations that were obtained varied from 400 to 1200 ng mL-1. A chromatogram was created, allowed to dry, then was scanned. Calibration was performed using a plot of the peak regions against the relevant DH concentrations.. The DH concentrations that were obtained varied from 400 to 1200 ng mL-1. A chromatogram was created, allowed to dry, then was scanned. Calibration was performed using a plot of the peak regions against the relevant DH concentrations.
A new mobile phase for Donepezil Hydrochloride was developed to optimize TLC Procedure. On the TLC plates, samples were spotted and chromatograms were developed in different solvent systems. Dichloromethane and Methanol was selected as mobile phase for drug but the RF value of DH was too low so Butanol was taken as one of the components of the mobile phase for creating good resolution. However, polar solvents were added to increase the mobile phase strength. These were water and glacial acetic acid. The final compositions of the butanol: water: glacial acetic mobile phase were 4:5:1 (v/v/v) composition produced good resolution of the standard drug and its degradation products with sharp and symmetrical peak of DH at RF = 0.45 ± 0.03. When TLC plates were prewashed with methanol (after drying and activation) and the TLC chamber was saturated with the mobile phase for 20 min., the spot of DH was found to be highly compact. This procedure ensured good repeatability and good peak shapes. At a 320 nm wavelength, the produced plates were scanned.
RESULT AND DISCUSSION :
HPTLC Method validation of Donepezil Hydrochloride
Methods validation is performed and below characteristics tested during methods validation such as Linearity, Recovery, Specificity, Precision, Reproducibility, LOD and LOQ, Robustness and Ruggedness.
Linearity:
An analytical process must be linear in order to produce test results that are directly proportional to the analyte concentration in the sample. It can be shown directly on the analyte or on samples that have been spiked with at least five different drug concentrations spread across the whole working range. A proper statistical calculation called linear regression is also necessary, in addition to visually evaluating the analyte signal as a function of concentration. Reporting should include the slope, intercept, residual sum of squares, and correlation coefficient.
Table 1: Recovery Study of Donepezil Hydrochloride
|
Type of Recovery, % |
Standard taken |
Sample taken (Donep) |
Expected Value |
Amount Recovered (mg) |
Recovered*, % |
Mean Percentage
|
|
80 |
0.8 ul |
0.5 ul |
0.0046 |
0.004672903 |
96.12% |
97.1 %
|
|
100 |
1 ul |
0.5 ul |
0.0051 |
0.005171564 |
97.15% |
|
|
120 |
1.2 ul |
0.5 ul |
0.0056 |
0.005672825 |
98.14 % |
For the standard medication, the linearity of the response for DH was evaluated in the 400 ng to 1200 ng per spot range. The calibration curve was drawn between peak area and drug concentration (in ng). Data with a polynomial curve were statistically analysed, and the coefficient of variation (CV) and correlation coefficient R were found to be 0.43 and 0.999, respectively.
Fig 2 : Linearity Graph of Donepezil
Recovery:
Multiple level recovery tests were used to test the recovery of the new approach using standard addition at three distinct levels. Donepezil HCl 200 ng/ul sample stock solution was made from tablet formulation, and the amount of API spiked with Donepezil Hydrochloride is equivalent to 80, 100, and 120% of standard solution. The recovery data are displayed in the table-1.
Specificity:
Donepezil HCl was discovered to have an Rf value of 0.48 0.02 in a mixture of butanol, water, and acetic acid. By comparing the spectra of donepezil HCl at the peak start (S), peak apex (A), and peak end (E) positions, the peak purity of donepezil HCl was determined. The same solvent system is used to detect the mobile phase as well. Based on the data gathered, it was established that the peak of the reference medication solution contained no contaminants or degradation products. The retention time for donepezil hydrochloride is 0.48.
Fig 3: Retention time of Donepezil Hydrochloride
Precision of the assay:
Reproducibility of the procedure allowed for the calculation of precision. It was determined whether the procedure was repeatable by looking for intraday fluctuations, which indicated method reproducibility. measurement of the peak area repeatability On a TLC plate, 6 uL of DH solution (100 ng per uL) was identified seven times. Without moving the plate, the chromatograms were developed, dried, and the spot was scanned seven times. The measurement of the peak area's percentage coefficient of variation (% CV) was estimated. For DH concentrations of 100 ng per spot, the intra-day variance of the measurement result was assessed three times per day. Over a three-day period, the inter-day variation was similarly assessed..
Limit of detection and limit of quantification.
Methanol was spotted three times as a blank on a TLC plate to be used in the determination of the limit of detection and limit of quantification. Additionally, DH solutions of 200, 400, 600, 1000, and 1200 ng ml-1 were produced and put to a TLC plate. The relationship between the average response (peak area) dependence and the amount of DH per spot was shown, and the related regression equation was found. Then, concentration was plotted versus the responses' standard deviation. An estimate of the target's standard deviation (Std) was obtained from the intersection of the resulting linear plot and the associated regression equation. The following detection and quantification limits were discovered:
LOD(ng) 80.85594722
LOQ(ng) 245.0180219
Robustness of the method.
The findings of examining the impact of slight variations in saturation time were conducted. A 10-minute saturation period was maintained. There has not been a big change.
Assay of DH in commercial formulation
Pharmaceutical pills with a label claiming 10 mg of DH per tablet were pulverised and methanol was used to extract an amount of powder containing 10 mg of DH. The medication was sonicated for 30 minutes and centrifuged at 3000 rpm for 5 minutes to ensure full extraction. 1 mL of the aforementioned solution was spotted onto the plate, followed by development and scanning, and 10 mL of the supernatant was diluted with methanol to get the final concentration of 100 ug mL-1. The analysis was carried out three times. It was investigated whether excipients might interfere with the analysis.
Analysis of Donepezil Hydrochloride Impurities
For the examination of Donepezil Hydrochloride impurities, 5 ul of the DNP standard, DNP Quaternary, DNP MTD, DNP Des-Bezyl, and DNP Deoxy impurity were spotted on a TLC plate as an 8 mm band. The mobile system used was butanol, water, and acetic acid (4:5:1), and the RF value is shown below.
Table2. Retention time of Donepezil Related Substances
|
Sr,No |
Name Of Impurity |
Rf Value |
|
1. |
DNP Des B |
0.301 |
|
2. |
DNP Deoxy |
0.584 |
|
3. |
DNP Sample |
0.456 |
|
4. |
DNP Standard |
0.463 |
|
5. |
DNP Quaternary Salt |
0.487 |
DNP QUATERNARY IMPURITY
Fig 4 : Retention time of Donepezil Hydrochloride Quaternary Salt Impurity
DNP MTD IMPURITY
Fig 5 : Retention time of Donepezil Hydrochloride Mithylidine Impurity
DONEPEZIL DESBENZYL IMPURITY
Fig 6 : Retention time of Donepezil Hydrochloride Desbenzyl Impurity
DONEPEZIL DESBENZYL IMPURITY
Fig 7 : Retention time of Donepezil Hydrochloride Desbenzyl Impurity
CONCLUSION:
A new HPTLC Method is developed and validates for Donepezil and related Substances which takes short run time and cost effective. Its a simple ,sensitive precise ,and accurate. The results obtained with proposed method confirm suitability of this method for Pharmaceutical uses. The other ingredients and excipients does not interfere in process, hence it can be used for commercial dosage form estimations. It can be used for Donepezil Hydrochloride and Impurity analysis. There is still scope of further development of this method for Accurate analysis of related substances.
CONFLICT OF INTEREST:
The authors declares they have no conflict of interests..
ACKNOWLEDGEMENT:
We are thankful to the Unichem Laboratory, Goa for providing sample for carrying out this research work .
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Received on 07.04.2023 Modified on 10.05.2023
Accepted on 18.06.2023 © RJPT All right reserved
Research J. Pharm. and Tech 2023; 16(6):2890-2894.
DOI: 10.52711/0974-360X.2023.00476