Analytical Method Development and Validation of Donepezil HCl by

RP-HPLC

Rupanshi Sahu*, Rakhee Kapadia

Sage Institute of Research and Technology-Pharmacy, Sage, Bhopal, 462022, Madhya Pradesh, India.

*Corresponding Author E-mail: rupanshis21@gmail.com

ABSTRACT:

Hypothesis: The precise measurement of Donepezil HCl in intricate pharmaceutical and biological matrices is crucial for assessing its pharmacokinetic properties, therapeutic effectiveness, and formulation quality.  Current analytical techniques frequently exhibit restricted sensitivity, insufficient specificity in biological matrices, and inadequate reproducibility.  We proposed the establishment & verification of a high-performance liquid chromatography system that uses reverse phase separation method to ensure reliable, accurate, and sensitive quantification of Donepezil HCl in diverse matrices, including emulsomal formulations, in vitro release media, plasma, and brain tissue. Experiments: Acetonitrile and 0.01 M ammonium formate buffer with a pH of 6.4 was used as mobile phase to develop a RP-HPLC method, C18 column of a Waters Nova-Pak (3.9 × 150mm) and packing 4 μm was utilized in a 62:38 v/v ratio.  The methodology was confirmed in accordance with the requirements described in ICH Q2(R1), encompassing evaluations of specificity, linearity (0.05–5µg/mL), accuracy, reliability, precision, limit of detection (LOD), & limit of quantification (LOQ) are all important aspects to consider. Injection volume of 20µL and 1.0mL/min flow rate of resulted in detection at 268nm. The method's suitability was confirmed across several concentrations and matrices, including placebo, plasma, and brain homogenate. Results: The retention period of Donepezil HCl was around 6.0 minutes, exhibiting an excellent peak shape and negligible matrix interference. Within the range of 0.05–5µg/mL (R² = 0.9984) linearity was detected. The approach exhibited significant specificity without interference at the retention time of Donepezil HCl.  Recovery rates varied between 97.8% and 98.52%, while %RSD values for precision experiments were consistently below 0.05%. The detection limit was established to be 0.037 µg/mL and quantitation limit to be 0.113 µg/mL. Robustness testing validated the dependability of method and slight fluctuations in operational parameters. This validated RP -HPLC method is reproducible, specific, and sensitive for routinely quantifying donepezil HCl in biological and pharmaceutical applications.

KEYWORDS:  Donepezil HCl, RP-HPLC, Method Validation, Plasma, Brain Homogenate, Emulsomes, Analytical Chemistry.

INTRODUCTION:

Alzheimer's disease (AD) is a devastating neurological ailment predominantly impacting the elderly, marked by increasing memory loss, cognitive decline, and behavioural disturbances^1,2. Donepezil hydrochloride (Donepezil HCl) is a commonly utilized cholinesterase inhibitor authorized for Alzheimer's disease, which improves cholinergic neurotransmission by reducing acetylcholinesterase activity in the brain³. Because of its therapeutic significance, there is an increasing need for highly sensitive and specific analytical methods to accurately quantify donepezil HCl in drug delivery systems, pharmaceutical formulations, and biological matrices like brain and plasma homogenates^4,5.

Donepezil HCl demonstrates modest hydrophilicity and is primarily taken orally in most commercial formulations⁶. Nonetheless, difficulties related to its inconsistent bioavailability, hepatic metabolism, and crossing the blood-brain barrier have prompted the creation of innovative drug delivery technologies⁷, including emulsomes, nanoparticles, and transdermal carriers⁸. Precise measurement of the medication in these formulations and biological contexts is crucial for comprehending its pharmacokinetic properties, biodistribution, and formulation efficacy⁹.

One efficient technique is HPLC for the quantitative analysis of medicines due to its superior resolution, reliability, and capacity to evaluate materials with intricate matrices¹⁰. Although numerous analytical techniques for Donepezil HCl are available, a method that provides precision, accuracy, specificity, robustness, and sensitivity across diverse sample types remains critically important¹¹. Furthermore, the existence of matrix interferences in plasma or brain tissue extracts requires a strategy that reduces background noise while preserving linearity and recovery¹².

This work developed and validated a straightforward, fast and dependable (RP-HPLC) technique for quantifying Donepezil HCl¹³, in conjunction with a C18 column as well as a mobile phase (composed of ammonium formate & acetonitrile buffer at a pH of 6.4)^14,15. The technique was refined to guarantee distinct peak profiles, less tailing, and high resolution in a little duration. The method's validation was conducted in compliance with ICH Q2(R1) recommendations, encompassing all critical analytical performance parameters, including accuracy, linearity, precision, specificity, sensitivity, and robustness¹⁶.  This validated method is applicable for routine quality control, pharmacokinetic investigations, and formulation development for Donepezil HCl, especially in innovative drug delivery systems designed to improve brain targeting¹⁷.

MATERIALS AND METHOD:

Donepezil hydrochloride was obtained as a complimentary sample from Alkem Pharmaceuticals, Mumbai. HPLC-grade acetonitrile and analytical-grade ammonium formate were procured from Merck Ltd. (India)¹⁸. Double-distilled water was purified using a Milli-Q system. Every reagent used was of analytical grade or higher. The mobile phase was made with the composition of acetonitrile and 0.01M ammonium formate buffer in a ratio of 62:38 v/v, also the buffer was adjusted to pH 6.4 with formic acid, and then the mobile phase was filtered through a 0.22μm membrane filter and degassed before use^19,25.

The analytical measurement of Donepezil HCl was conducted utilizing a RP-HPLC system with a UV-PDA detector²⁰. Chromatographic separation was carried out using a Waters Nova-Pak C18 column (3.9 × 150mm, 4 μm particle size). A buffer consisting of acetonitrile and 0.01M ammonium formate were combined in a ratio of 62:38(v/v) to generate the mobile phase. After setting the detecting wavelength to 268nm, the rate of flow was maintained at 1.0mL/min throughout the experiment. A sample (Donepezil HCl) volume of 20µL was injected, and the total run time was ten minutes overall. There was an observation that the retention time of donepezil hydrochloride was around 6.0 minutes^21,22.

Preparation of Standard and Working Solutions:

In order to ensure that the drug was completely dissolved, 1mg/mL of donepezil hydrochloride was prepared in the mobile phase and then sonicated to obtain the stock solution. From this stock solution, working standards were freshly prepared by serial dilution to obtain concentrations between 0.05 and 5.0 µg/mL. During the process of constructing the calibration curve and determining linearity, these standard solutions were utilized.

Table 1: Chromatographic Parameters for Donepezil HCl

Parameter	Description
Chromatographic System	HPLC with PDA Detector (λ = 268 nm)
Column	C18, Waters Nova-Pak (3.9 × 150 mm, 4 μm particle size)
Mobile Phase	Acetonitrile: 0.01 M Ammonium Formate (62:38 v/v)
Flow Rate	1.0 mL/min
Injection Volume	20 μL
Retention Time	Approximately 6.0 min
Run Time	10 min

Validation of the Method (in line with ICH Q2          [R1])^19,20,27:

System Suitability:

A 0.5µg/mL standard solution of Donepezil HCl was injected six times to test system compatibility. Evaluations were performed on the peak area, tailing factor, theoretical plates, and retention time factors. Due to the fact that it had regular retention periods and peak characteristics, this method was perfect for routine analysis.

Specificity:

The method's specificity was tested using blank samples, placebos, and plasma and brain homogenate spiked with Donepezil HCl.  Chromatograms showed no interference peaks at Donepezil HCl retention time, making the technique very specific²⁶.

Linearity:

For linearity testing, a concentration range of 0.05 to 5.0 µg/mL was selected based on the anticipated therapeutic and analytical levels of Donepezil HCl in plasma, brain homogenate, and emulsomal formulations. Graphing peak area versus concentration developed a calibration curve²³.  

Accuracy:

The method's accuracy was assessed using recovery experiments at three concentrations such as low (0.1 µg/mL), medium (0.5µg/mL), and high (2.5µg/mL).   Donepezil HCl was incorporated into placebo matrices and subsequently quantified ²¹.

Precision:

The study evaluated precision by analyzing intra-day and inter-day variations at concentrations of range 0.1, 0.5, and 2.5µg/mL. Each sample underwent triple analysis. The precision and reproducibility of the approach were validated by the results of assays that exhibited percentage of the RSD values of less than 2% both within & between days²².

LOD and LOQ:

The calculation of the detection limit was calculated by using formula 3.3 × (σ/S) and quantification limit by 10 × (σ/S) was identified by calculating both the calibration curve slope as well as the standard deviation of the intercept²³.

Robustness:

In order to assess method durability, small adjustments were made to chromatographic parameters, comprising the method for the development of the mobile phase (with a tolerance of ±2% acetonitrile), flow rate (with a tolerance of ±0.1mL/min), the detection wavelength (with a tolerance of ±2nm), and the column temperature (with a tolerance of ±5). This demonstrates that the technique is able to withstand operational turbulence, as it was successful despite these alterations²⁴.

RESULTS AND DISCUSSION:

The method was developed and validated (RP-HPLC) for determination of Donepezil HCl by using a mobile phase composed of acetonitrile and 0.01M ammonium formate buffer, which was adjusted to pH 6.4. It was determined that the wavelength of maximum absorbance (λmax) for the ultraviolet spectrum of Donepezil HCl was 268nm. This was determined by scanning the spectrum ranging from 200 to 400nm. This wavelength was selected for further analysis as the drug exhibited sharp absorbance, good sensitivity, and linearity at this point.

By injecting 6 replicates of a 0.5µg/mL standard solution of Donepezil HCl, System suitability of the chromatographic method was assessed. The retention time (~6.0min), tailing factor, number of theoretical plates and peak area conformed to the permissible parameters outlined by ICH Q2(R1), indicating suitability of the system for routine analysis. The method's specificity was assessed by examining blank samples, placebo formulations, and biological matrices (plasma and brain homogenates) fortified with Donepezil HCl. During the retention period of the analyte, there were no peaks that interfered with the analysis, confirming that the method is specific and free from interference by excipients or biological components.

Linearity was established across 0.05 and 5.0µg/mL, the range of concentrations. A strong linear connection between peak area and drug concentration, with a correlation coefficient (R2) of 0.9984, the calibration curve was demonstrated and suggesting high linearity as well as suitability for quantitative estimations over a broad concentration range. Recovery tests were conducted at three different spiking concentrations (low, medium, and high, with concentrations ranging from 0.1, 0.5, and 2.5µg/mL) to identify the accuracy of the approach. The fact that the percentage recovery ranged between 97.80% and 98.52% is evidence that the procedure is accurate and capable of producing findings that can be relied upon.

Table 2: System Suitability Parameters

Injection No.	Retention Time (min)	Peak Area	Tailing Factor	Theoretical Plates
1	6.02	1,118,500	1.42	2100
2	6.00	1,118,900	1.43	2120
3	6.01	1,118,700	1.41	2095
4	6.03	1,118,600	1.44	2080
5	6.01	1,118,800	1.42	2110
6	6.02	1,118,750	1.43	2090
Mean ± SD	6.015 ± 0.012	1,118,708 ± 120	1.425 ± 0.012	2099.17 ± 15.5
%RSD	0.16%	0.012%	0.67%	0.62%

Table 3: Linearity Data of Donepezil HCl

Concentration (µg/mL)	Peak Area
0.05	11400
0.5	109875
1.0	219923
2.5	545,021
5.0	1,123,647
Slope	224889
Intercept	-2579.2
Correlation Coefficient (R²)	0.9984

Table 4: Accuracy and Recovery Studies

Level	Std. Amount (µg)	Spiked Amount (µg)	Total (µg)	Recovered (µg)	% Recovery
Low	100	100	200	195.6	97.80%
Medium	500	500	1000	985.2	98.52%
High	2500	2500	5000	4925.0	98.50%
Mean	–	–	–	–	98.27%

Fig. 1: HPLC Chromatograms for System Suitability

Fig 2: HPLC Chromatograms for Accuracy/Recovery

Fig 3: Calibration curve Donepezil HCl

Fig 4: HPLC Chromatograms for Linearity

Fig 6: HPLC Chromatograms for Specificity

Precision was identified via intra-day and inter-day analyses, employing replicate injections at three concentration levels. The peak area relative standard deviation (%RSD) was less than 2% in all instances, signifying that the approach is exact and reproducible.  Robustness was assessed by implementing minor, intentional modifications in chromatographic parameters, such as flow rate (±0.1 mL/min), detection wavelength (±2 nm), composition of mobile phase (±2% acetonitrile) and temperature of the column (±5 °C). The analytical results remained consistent, and %RSD values were within acceptable limits, confirming that the method is unaffected by minor variations and robust in analytical conditions.

The results demonstrated that the approach is robust and reliable under a wide range of operational settings, as evidenced by the fact that there was only a little amount of difference in the retention time and peak area. After careful consideration, it was observed that the newly designed RP-HPLC method is uncomplicated, accurate, specific, exact, robust, and resilient. It is suitable for the quantitative measurement of donepezil hydrochloride in bulk substances, pharmaceutical compositions, & biological matrices, such as plasma and brain tissue, and it satisfies the requirements prescribed by the ICH Q2(R1).

Table 5: Precision Study Data

Replicate	Intra-day Peak Area	Inter-day Peak Area
1	111,800	111,600
2	111,700	111,550
3	111,850	111,580
4	111,750	111,590
5	111,820	111,610
6	111,780	111,620
Mean	111,783.33	111,591.67
%RSD	0.043%	0.020%

Table 6: Robustness Study

Parameter Modified	Condition	Retention Time (min)	Tailing Factor	% RSD
Mobile Phase	±2% Acetonitrile	6.01	1.43	1.2
Flow Rate	±0.1 mL/min	6.04	1.44	1.5
Detection Wavelength	±2 nm	6.00	1.42	1.3
Column Temperature	±5°C	6.02	1.43	1.1

Fig 5: HPLC Chromatograms for Precision

Fig 7: HPLC Chromatograms for Robustness

CONCLUSIONS:

This study effectively established and validated a straightforward, quick, and dependable RP-HPLC method for quantifying Donepezil hydrochloride in pharmaceutical formulations and biological matrices, including plasma and brain homogenates. The approach was developed to guarantee excellent sensitivity, specificity, and repeatability, which are crucial for assessing innovative drug delivery methods aimed at improving the therapeutic efficacy of Donepezil in the management of Alzheimer’s disease.

The technique utilized a Waters Nova-Pak C18 column by using acetonitrile and 0.01M ammonium formate buffer (pH 6.4) as a mobile phase of ratio 62:38 v/v.  Detection was carried with PDA detector at 268nm, which produced a sharp and well-resolved peak with a retention time of approximately 6.0 minutes. The system suitability characteristics, such as theoretical plates, tailing factor, retention time, and peak area, conformed to the ICH Q2(R1) criteria, hence guaranteeing the reliable functioning of the chromatographic system.

The validation results affirmed the method's robustness under diverse analytical settings. Throughout the concentration ranging between 0.05 and 5.0µg/mL, the calibration curve exhibited remarkable linearity, demonstrating its superior linearity, with a correlation value (R²) of 0.9984.  The approach exhibited exceptional precision, with %RSD values much under 2% of intra-day and inter-day and remarkable accuracy, as indicated by recovery values between 97.80% and 98.52%. The determined LOD (0.037 µg/mL) and LOQ (0.113 µg/mL) validated the method's efficacy for identifying low levels of Donepezil HCl in intricate matrices. Furthermore, intentional alterations in technique parameters demonstrated no substantial effect on performance, affirming the method's robustness and resilience.

The established RP-HPLC method adheres to ICH Q2(R1) requirements and is appropriate for routine analysis, quality control, pharmacokinetic assessment, and formulation evaluation of Donepezil HCl. The successful validation endorses its use in the continued study and development of sophisticated delivery systems, such as emulsomes and nanoformulations, for the targeted treatment of neurological illnesses.

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Received on 30.06.2025      Revised on 22.11.2025

Accepted on 08.02.2026 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):3007-3012.

DOI: 10.52711/0974-360X.2026.00428

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