Abstract
Alzheimer’s dementia is often treated with a combination of memantine and donepezil, which is more effective than monotherapy. Despite its clinical relevance, simultaneous determination is challenging due to the absence of a chromophore in memantine. Herein, a simple, accurate, and sensitive High-Performance Liquid Chromatography–refractive index method is reported for the first time for the simultaneous quantification of memantine and donepezil for routine analysis. The method employed a C18 column with a mobile phase of 1% diethylamine in water: methanol (50:50, v/v), adjusted to pH 6.0 using dilute glacial acetic acid. Optimized conditions (flow rate 0.8 mL/min, injection volume 20 µL at 40 °C) yielded a linearity ranging between 2.5 and 30 µg/mL (R² = 1) for both the drugs. Limits of detection and quantification were 0.194 and 0.589 µg/mL for Dnp and 0.237 and 0.718 µg/mL for Mem. Validation confirmed precision, robustness, and stability. The method was successfully applied to determine the amount of drug release from the intranasal nano-colloidal preparations, along with optimized sample preparations giving analyte recoveries within range without any interference from the formulation excipients. The derivatization exhibited acceptable selectivity with no interfering peaks from excipients, demonstrating its suitability for routine analysis. The sustainability of the developed method was assessed using different metrics, namely the Analytical GREEnness Metric Approach and Software, Green Analytical Procedure Index, and Blue Applicability Grade Index.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13065-026-01835-9.
Keywords: Chromatographic method, Donepezil, Memantine, Alzheimer’s disease, Greenness evaluation
Introduction
Dementia due to Alzheimer’s is the most common in elderly patients, accelerating with time [1]. The management of Alzheimer’s disease (AD) includes medications that can improve the symptoms and slow down the decline in cognitive functioning of the brain. The FDA-approved first-line therapy for AD is cholinesterase inhibitors such as donepezil, galantamine, and rivastigmine, and the N-methyl-D-aspartate (NMDA) receptor blocker memantine [2].
The combination therapy of memantine (Mem) and donepezil (Dnp) is commonly prescribed in moderate to severe AD and has been proven to be effective [3, 4]. The current treatment for AD primarily involves the use of a combination of Mem and Dnp (more effective and superior to monotherapy [5]) in oral dosage forms. However, these oral formulations have gastrointestinal side effects, especially in elderly patients [6]. Therefore, intranasal formulations could provide an alternative route offering targeted brain delivery and other advantages, including rapid absorption, ease of administration, and enhanced bioavailability [7]. The intranasal drug carriers should allow for controlled drug release profiles and prolonged retention time at the mucosal site, which mucoadhesive-based hydrogels could achieve [8]. These bases undergo in-situ gelling via a phase transition triggered by physiological environments, such as pH and temperature.
Mem (3,5-dimethyl-1-aminoadamantane) is a small molecule shown in Fig. 1A that acts as an NMDA receptor antagonist by modulating glutamate levels, thereby reducing excessive neuronal excitability [9]. The absence of a chromophore in its structure results in less effective detection due to negligible ultraviolet (UV) absorbance; therefore, refractive index (RI) detectors [10] can be used in the HPLC analysis of memantine [11, 12]. Dnp (2-[(1-benzylpiperidin-4-yl)methyl]-5,6-dimethoxy-2,3-dihydroinden-1-one) is a piperidine derivative, as shown in Fig. 1B, that exerts its therapeutic effect by inhibiting the acetylcholinesterase (AChE) enzyme and preventing the breakdown of the neurotransmitter acetylcholine [13]. Unlike Mem, Dnp has a chromophore, which allows for detection with UV-based methods [14].
Fig. 1.
The chemical structure of memantine (A) [15] and donepezil (B) [16]
Several analytical methods have been reported in the literature for the quantification of Mem and Dnp, either individually or in combination. Analytical techniques such as HPLC–UV/DAD [14, 17, 18], LC-MS/MS [19–21] Fluorometric methods [22] have been reported for the estimation of Dnp, leveraging its inherent UV-absorbing chromophore. In contrast, Mem, which lacks a chromophore, has been quantified using different derivatizing agents such as 9-fluorenylmethyl chloroformate (FMOC) [12, 23], 1-fluoro-2,4-dinitrobenzene (FDNB) [24], 1-chloro-2,4-dinitrobenzene [25], dansyl chloride [26, 27] derivatization-based UV methods [12, 28], or by HPLC-RID [11]. However, the derivatization adds complexity, time consumption, and variability. Although UV-based methods are suitable for Dnp, they are not reliable for simultaneous detection, including Mem, due to negligible UV absorbance. While LC-MS/MS offers high sensitivity and requires sophisticated instrumentation less suitable for routine simultaneous analysis of both the drugs.
Therefore, A simple, cost-effective, and derivatization-free method for the simultaneous estimation of both drugs was developed and validated according to ICH Q14 guidelines and validated as per ICH Q2(R1) [29]. This study addresses the analytical gap by presenting a direct and sensitive method with multiple advantages, including multi-analyte quantification, enhanced sensitivity, elimination of derivatization, short analysis time (20 min runtime), and low injection volume (20 µL). The developed method was used to determine Mem and Dnp contents in the formulated intranasal hydrogel and release samples. The greenness and applicability of the developed HPLC method were comprehensively evaluated using different tools, including Analytical GREEnness Metric Approach and Software (AGREE), Green Analytical Procedure Index (GAPI), and Blue Applicability Grade Index (BAGI).
Experimental
Materials
Memantine and donepezil were purchased from Combi Blocks USA. HPLC-grade methanol was purchased from Sigma-Aldrich (NSW, Australia). Chitosan, Poloxamer 407, and Poloxamer 188 were purchased from Sigma-Aldrich (Australia). Ultrapure Milli-Q water (18.2 mΩ) was obtained from a Millipore Milli-Q system (Sartorius, Germany). Diethylamine, glacial acetic acid, and absolute hydrochloric acid were purchased from Merck Ltd, Australia.
Instrumentation
An HPLC instrument (Shimadzu Corporation, Kyoto, Japan) equipped with a binary pump (LC-20AD), a degasser (DGU-20A3), an autosampler (SIL-20 A HT), and a Refractive index detector (RID-10 A) was utilized for the evaluation of Mem and Dnp. For interpretation and data collection, LabSolutions software (version 5.92) was used. A pH meter (Thermo Scientific, ORION STAR A111) was used for all pH measurements. LabSolutions software (version 5.132) was used for data processing and collection.
Chromatographic parameters
The mobile phase was composed of a 50:50 (v/v) mixture of methanol and ultrapure water containing 1% diethylamine, adjusted to pH 6.0 with 1 M glacial acetic acid. The flow rate was 0.8 mL/min, with a run time of 20 min, and an injection volume of 20 µL. The column used was a Kinetex 100 Å, C18 (250.0 × 4.6 mm; 5 μm), which was maintained at 40 °C. An RI detector was used for the analysis. The detector temperature was set at 40 °C with a sensitivity of 512. The sample and reference cells were purged with the mobile phase for 60 min each.
Preparation of standard solution
Accurately weighed amounts of both Mem and Dnp (10 mg) were transferred to a 10 mL volumetric flask containing 6 mL of the mobile phase as a diluent, and the mixture was sonicated for 10 min to obtain complete dissolution. The volume was then made up to 10 mL with the mobile phase. The stock solution was stored at 4°Ctill further use.
Method validation
Linearity and range
From the working stock solution containing both Mem and Dnp (1 mg/mL), further dilutions in a concentration range of 2.5 to 30 µg/mL were prepared using the mobile phase as a diluent. The prepared concentrations were analyzed using optimized HPLC-RID chromatographic conditions, and the obtained peak areas were plotted against the corresponding concentrations of each drug. The coefficient of determination (R2) and percentage recoveries were calculated.
Specificity
The specificity of the method was evaluated to confirm the absence of any co-eluting peaks with Dnp and Mem in in-vitro release media (Phosphate-Buffered Saline pH 7.4) and the mobile phase.
Precision and accuracy
Three standard solutions were prepared at three concentration levels: low (5 µg/mL), intermediate (15 µg/mL), and high (30 µg/mL), followed by injection into the HPLC system for analysis, and the percentage recoveries were determined (n = 6).
For precision, the same concentrations (5, 15, 30 µg/mL) were injected at different time points on the same day (intraday precision) and over three consecutive days (inter-day precision) for analysis. The relative SD was calculated for all data points.
Limit of detection (LOD) and limit of quantification (LOQ)
LOD is the lowest concentration of an analyte that can be detected but not necessarily quantified, and LOQ is the lowest concentration of an analyte that can be quantitatively detected with accuracy and precision. From the linearity concentration range (2.5–30 µg/mL), the standard deviation and slope were calculated using the LOD and LOQ equation from Shrivastava et al. [30].
Robustness
Method robustness was evaluated by slightly changing chromatographic conditions like temperature, pH, and mobile phase composition. The results were presented as % recoveries and relative standard deviation % (RSD).
Stability
To assess the stability of the drugs at different temperatures, a solution containing 30 µg/ml of both drugs was prepared and stored at refrigerated temp, room temperature, and 37 ˚C for 28 days, and samples were analyzed, and recovery was calculated.
Hydrogel formulation preparation and method application
Preparation of hydrogel
The poloxamer-chitosan-based hydrogel formulation shown in Table 1 was prepared using the cold method [31]. Firstly, the required amounts of poloxamer 407 and poloxamer 188 were added to pre-refrigerated triple-distilled water and kept overnight for complete dissolution and form a transparent solution. Separately, in another beaker, the weighed amount of chitosan (0.1% w/v) and the drug were added to the required amount of 1% acetic acid solution (v/v) and kept overnight at 4˚C. Finally, both the prepared solutions were mixed under continuous stirring to obtain a clear gel, which was stored in the refrigerator before use.
Table 1.
Intranasal in situ gel composition containing Dnp and Mem
| Components | % w/w | Function |
|---|---|---|
| Poloxamer 407 | 15 | Thermo-reversible in situ Gelling agent |
| Poloxamer 188 | 10 | Stabilizer and surfactant |
| Chitosan | 0.1 (in 1% acetic acid) | Permeation enhancer |
| Deionized water | q.s | Vehicle |
| Donepezil | 0.5 | Active ingredient |
| Memantine | 0.5 | Active ingredient |
Drug content determination
Accurately 500 µL of the intranasal formulation was transferred into a volumetric flask and was added with mobile phase up to the 10 ml mark. The solution was sonicated for 15 min to ascertain complete dissolution of the formulation in the mobile phase, which was filtered through a syringe filter of 0.22 micron, and analyzed using the method.
Drug release studies in an in vitro model
Approximately 200 mg of the formulation was weighed into a presoaked dialysis bag, then properly sealed. The formulation containing the dialysis bag (MWCO 12,000 KDa) was placed into 50 ml PBS pH 7.4, placed in an incubator at 37 ˚C at 100 rpm for 9 h [32]. At different time points, 10 ml volume was replaced with fresh media, and the samples collected were analyzed using the developed HPLC method.
Results and discussion
Analytical method development
Mem and Dnp are used in combination for the management of AD, necessitating a combination detection method capable of analyzing both drugs simultaneously.
To develop an optimal chromatographic method for the simultaneous detection of both drugs, various mobile phase compositions and parameters were systematically evaluated. Given the relatively high pKa values of both drugs, mobile phases in a buffer pH range of 5.0–7.0, containing different ratios of methanol and diethylamine (DEA) buffer, were tested. The pH of the mobile phase plays a significant role in the protonation of basic drugs and leads to better peak resolution and less tailing in the pH range 5 to 7 (Supplementary Fig. 6–10). Furthermore, diethylamine (DEA) was incorporated in concentrations ranging from 0.5% to 1.0% (v/v) to improve peak shape and minimize tailing, particularly due to the basic nature of the analytes. Among the tested conditions, a mobile phase consisting of methanol and 1% diethylamine buffer in a 50:50 (v/v) ratio at pH 6.0 provided the best results in terms of peak resolution, symmetry, and baseline stability when using a refractive index detector. Additionally, different flow rates from 0.5 to 1.0 mL/min were evaluated. The optimization data is provided in the supplementary sheet. Final flow rate (0.8 mL/min) was found to offer the optimal balance between analysis time, peak shape, and resolution, as shown in Fig. 3, and was therefore selected for the final method. A C18 Kinetex column was used to achieve isocratic elution, with average retention times (RT) of 6.7 min for Dnp and 9.8 min for Mem. The chromatogram of the blank mobile phase has been provided in Supplementary Fig. 16.
Fig. 3.
Chromatogram showing the detected peaks of Dnp (RT-6.7 min) and Mem (RT-9.8 min) eluted from a lab-prepared solution using the developed HPLC method
Analytical method validation
This developed method was validated following ICH Q2 (R1) guidelines [29].
Linearity
The calibration curves in the concentration range of 2.5–30 µg/mL for both Dnp and Mem showed linearity with a correlation coefficient (R²) of 1.000, and validation parameters are shown in Table 2; Fig. 2.
Table 2.
Table showing regression and validation parameters for the DNP and Mem developed method
| Compound | Linear range (µg/mL) | Slope | Intercept | R value | SD of Y-intercepts | LOD (µg/mL) | LOQ (µg/mL) |
|---|---|---|---|---|---|---|---|
| Donepezil | 2.5–30 | 326.01 ± 0.84 | –119.09 ± 13.98 | 1.000 | 19.22 | 0.19 | 0.59 |
| Memantine | 2.5–30 | 266.82 ± 0.84 | –116.91 ± 13.95 | 1.000 | 19.18 | 0.24 | 0.72 |
Fig. 2.
The calibration curve of Dnp and Mem in the range 2.5–30 µg/mL in mobile phase % (v/v) 1% v/v diethylamine in water: methanol (50:50, v/v), pH 6.0
Specificity
There are no additional co-eluted peaks from excipients included in the release media and mobile phase, as shown in Figs. 3 and 4.
Fig. 4.
Combination in release media PBS (7.4)
Selectivity
As indicated in Table 3, the selectivity test showed acceptable recoveries (within 98–102%) of both Mem and Dnp from the release media and mobile phase.
Table 3.
The selectivity of Dnp and Mem is presented in % recovery with standard deviations
| Selectivity | |
|---|---|
| Dilute | Recovery (%) ± SD |
| Mobile phase | 98.99 ± 0.09 |
| PBS (pH 7.4) | 98.19 ± 0.19 |
| Mobile phase | 101.46 ± 0.28 |
| PBS (pH 7.4) | 100.47 ± 0.52 |
(n = 3)
Accuracy
The method showed high accuracy, where drug recoveries were within the acceptable range and RSD less than 2.00% for both Dnp and Mem at low, medium, and high concentrations. The results are presented in Table 4.
Table 4.
The accuracy of Dnp and Mem is presented in % recoveries and relative standard deviation
| Drugs concentration (µg/mL) |
Accuracy | |
|---|---|---|
| Recovery (%) ± RSD | ||
| Dnp | 5.00 | 100.58 ± 0.73 |
| 15.00 | 98.53 ± 0.51 | |
| 30.00 | 99.22 ± 0.33 | |
| Mem | 5.00 | 100.7 ± 0.58 |
| 15.00 | 100.21 ± 0.93 | |
| 30.00 | 101.30 ± 0.46 | |
(n = 6)
LOD and LOQ
LOD and LOQ were determined by using linear regression analysis in Microsoft Excel. The standard deviation (σ), obtained from the standard error of the regression for donepezil (Dnp) and memantine (Mem), was 19.22 and 19.18, respectively. These σ values were applied in Eq. 2 to calculate the LOD and LOQ (µg/mL). The resulting LOD and LOQ values were 0.19 µg/mL and 0.59 µg/mL for Dnp, and 0.24 µg/mL and 0.72 µg/mL for Mem, respectively.
Robustness
The robustness of the analytical method was evaluated by varying the mobile phase conditions, including changes in pH (± 0.2), mobile phase composition methanol: diethylamine buffer (52:48 v/v), and a temperature decrease of 5 ˚C, and the results are presented in Table 5.
Table 5.
The recovery of Dnp and Mem was analyzed at varying chromatographic conditions
| Assay conditions | Recovery (%) ± SD | |
|---|---|---|
| Dnp | Mem | |
| Temperature 35 ˚C | 98.67 ± 0.17 | 99.16 ± 0.46 |
| Mobile phase ratio (52:48) | 99.21 ± 0.25 | 99.09 ± 0.35 |
| pH of mobile phase (6.2) | 98.55 ± 0.35 | 98.92 ± 0.34 |
(n = 3)
System suitability
A standard solution containing 30 µg/mL of Dnp and Mem was analyzed to evaluate system suitability. The resulting chromatographic peaks were assessed against established system suitability parameters, including theoretical plate count (N), retention time, resolution (Rs), height equivalent to a theoretical plate (HETP), tailing factor, and %RSD of peak area. The observed values, as shown in Table 6, met the predefined specification limits, confirming the usefulness of the optimized chromatographic method for subsequent analysis.
Table 6.
System suitability of the proposed HPLC analytical method for Mem and Dnp
| Parameter | Specification | Dnp | Mem |
|---|---|---|---|
| Retention Time (min) | 6.649 | 9.789 | |
| Retention Time SD | ≤ 2 | 0.003 | 0.003 |
| Plates | > 2000 | 7,085 | 12,494 |
| HETP | 21.143 | 12.019 | |
| Area %RSD | ≤ 2 | 0.40 | 0.57 |
| Tailing | ≤ 2 | 1.375 | 1.338 |
| Resolution (Rs1) | > 2 | 9.42 | |
(n = 6)
Precision
The developed method showed good precision, with %RSD values within an acceptable range (2.00%) as shown in Table 7. Both intra- and inter-day precision analysis confirmed reproducibility of the method.
Table 7.
Intra-day and inter-day precision of the developed HPLC method for Mem and Dnp expressed as %RSD
| Precision | ||||
|---|---|---|---|---|
| Intra-day precision | Inter-day precision | |||
| Drug | Drug concentration (µg/mL) | RSD | Drug concentration (µg/mL) | RSD |
| Dnp | 5 | 0.73 | 5 | 0.82 |
| 15 | 0.52 | 15 | 0.55 | |
| 30 | 0.33 | 30 | 0.72 | |
| Mem | 5 | 0.57 | 5 | 0.93 |
| 15 | 0.93 | 15 | 0.66 | |
| 30 | 0.45 | 30 | 0.57 | |
(n = 6)
Stability of the analytes
The stability test results for standard solutions of both drugs were evaluated over 28 days at different temperatures and are presented in Table 8. The data showed that both drugs are stable in the mobile phase and PBS 7.4 at different temperatures.
Table 8.
Stability studies of memantine and donepezil in the mobile phase and PBS 7.4
| Drug | Testing conditions | R % ±SD | ||||
|---|---|---|---|---|---|---|
| Diluent | Temp | Day 1 | Day 7 | Day 14 | Day 28 | |
| Dnp | Mobile phase | 4˚C | 100.45 ± 0.71 | 98.4 ± 0.16 | 100.29 ± 0.64 | 100.5 ± 0.37 |
| 25˚C | 100.11 ± 0.26 | 99.52 ± 0.67 | 100.15 ± 0.06 | 100.54 ± 0.27 | ||
| 37˚C | 99.86 ± 0.54 | 99.08 ± 0.80 | 100.18 ± 0.18 | 100.8 ± 0.28 | ||
| PBS (pH-7.4) | 4˚C | 98.95 ± 0.29 | 98.47 ± 0.67 | 98.65 ± 0.19 | 98.65 ± 0.19 | |
| 25˚C | 99.42 ± 0.86 | 99.28 ± 0.63 | 99.46 ± 0.43 | 100 ± 0.64 | ||
| 37˚C | 98.53 ± 0.48 | 98.28 ± 0.25 | 100.56 ± 0.61 | 99.84 ± 0.77 | ||
| Mem | Mobile phase | 4˚C | 98.90 ± 0.33 | 100.48 ± 0.40 | 100.7 ± 0.31 | 101.14 ± 0.53 |
| 25˚C | 98.49 ± 0.31 | 101.82 ± 0.35 | 100.55 ± 0.44 | 101.38 ± 0.38 | ||
| 37˚C | 98.88 ± 0.24 | 101.39 ± 0.57 | 101.41 ± 0.29 | 101.71 ± 0.35 | ||
| PBS (pH-7.4) | 4˚C | 98.76 ± 0.63 | 100 ± 0.57 | 98.65 ± 0.19 | 99.86 ± 0.25 | |
| 25˚C | 98.97 ± 0.63 | 101.28 ± 0.15 | 101.92 ± 0.08 | 100.87 ± 1.29 | ||
| 37˚C | 99.97 ± 0.1 | 100.75 ± 0.35 | 100.43 ± 0.63 | 101.42 ± 1.05 | ||
(n = 3)
Intranasal formulation preparation and application of the developed method
Formulation preparation of hydrogel
The hydrogel formulation of composition shown in Table 9 was prepared using the cold method. The final gel formulation was a transparent solution and free from particulate matter, as shown in Fig. 5.
Table 9.
Comparative Overview of Published Methods and the Developed Analytical Method for Donepezil and Memantine Analysis
| Method | Analyte(s) | Principle/Detection | Advantages | Limitations |
|---|---|---|---|---|
| HPLC–UV/DAD [14,17,18] | Donepezil | UV/Diode Array Detection | Simple, widely available, suitable for donepezil quantification | Limited to analytes with chromophores; not suitable for reliable memantine detection |
| LC–MS/MS [19–21] | Donepezil / Memantine / Combination | Mass Spectrometric Detection | Highly sensitive and selective; suitable for trace analysis | Expensive instrumentation; less suitable for routine QC |
| Fluorometric Methods [22] | Donepezil | Fluorescence Detection | High sensitivity for donepezil | Limited applicability to fluorescent/chromophoric analytes; not suitable for memantine |
| Derivatization-Based HPLC–UV (FMOC, FDNB, Dansyl Chloride) [12,23–28] | Memantine | Pre-column derivatization + UV Detection | Enables UV detection of memantine; improved sensitivity | Additional sample preparation, time-consuming, derivatization variability, and reagent cost |
| HPLC–RID [11] | Memantine | Refractive Index Detection | Derivatization-free detection of non-chromophoric analytes | Lower sensitivity; limited compatibility with gradient methods; baseline instability |
| Developed HPLC Method (Present Study) | Donepezil + Memantine | Simultaneous HPLC Method (Derivatization-Free) | Simultaneous multi-analyte quantification; derivatization-free; simple and cost-effective; short runtime (20 min); low injection volume (20 µL); suitable for routine analysis; applicable to formulation and release samples; sustainability evaluated (AGREE/GAPI/BAGI) | Lower sensitivity than LC–MS/MS; requires optimized detector setup for non-chromophoric analyte detection |
Fig. 5.
Intranasal gel formulation
Drug content analysis
The drug content of the formulated intranasal hydrogel was successfully determined using the developed analytical method. The results showed % recoveries of the Dnp and Mem as 98.15 ± 0.16 and 100.09 ± 0.59, respectively, presented in Table 10. The chromatogram showed no additional peaks in the Supplementary Fig. 17, and recoveries were within the acceptable range.
Table 10.
The drug assay data from the intranasal formulation
| Theoretical conc (mg/mL) | Observed conc (mg/mL) | Assay recovery | ||
|---|---|---|---|---|
| Formulation | Dnp | 49.93 | 49.01 | 98.15 ± 0.16 |
| Mem | 49.55 | 49.59 | 100.09 ± 0.59 |
(n = 3)
Release studies
Cumulative in vitro release studies, as shown in Fig. 6, revealed an initial release of approximately 30% for both drugs within the first hour, followed by a sustained release phase, with cumulative release reaching about 80% at 9 h. The chromatogram illustrating no additional peaks in the application of the method in formulation evaluation has been provided in Supplementary Fig. 15.
Fig. 6.
Drug release of Dnp and Mem from intranasal in situ gel formulation
Sustainable analytical performance profile of the developed method
The analytical method was developed with environmental impact in mind, aiming to replace and/or minimize the use of hazardous chemicals, reduce waste, and conserve energy, while maintaining analytical performance [33–35]. Unlike conventional studies that rely solely on greenness metrics, this work adopts a multidimensional sustainability assessment by integrating environmental impact (AGREE, GAPI) with method practicality and applicability (BAGI), providing a more comprehensive evaluation of analytical performance.
Methanol was used as the main solvent, in addition to water, for drug extraction from intranasal formulations, and the mobile phase, glacial acetic acid (pH adjustment), and dimethylamine (1% of the mobile phase) were also used in minimal quantities to optimise peak resolution and system suitability parameters. Based on the GlaxoSmithKline solvent guide, methanol and acetic acid received environmental impact scores of 9 (green) and 8 (green), out of 10, respectively, with water (10) representing the benchmark for the least environmental impact [36]. The use of non-green diethylamine was inevitable; however, only the smallest volume was employed, thereby limiting its environmental burden.
Descriptive evaluations of analytical method greenness, while informative, remain subjective and often insufficient for comparison across methodologies. Consequently, several quantitative greenness assessment tools have been introduced to provide structured, criterion-based evaluation frameworks. Many of these tools provide numerical scores and colour-coded charts, which facilitate thorough evaluation and easily highlight strengths and weaknesses of the assessed methods [37, 38]. In the present study, the greenness was assessed by AGREE and GAPI [39] tools, and the practicality of the developed method was evaluated using BAGI [40] tools. Collectively, these complementary tools integrate environmental impact, operational efficiency, and practical applicability, extending beyond greenness evaluation alone.
The AGREE evaluation, which quantifies compliance with the twelve principles of Green Analytical Chemistry (GAC) [39], produced a score of 0.57 (Fig. 6). This result reflects considerable greenness compliance, consistent with the use of greener solvents such as methanol, energy-efficient operation, the avoidance of derivatisation steps, and a good throughput, quantifying 3 binary mixtures per hour. The reduced score is primarily attributed to the method being off-line analysis with a few steps of sample preparation, the extraction method not being miniaturized, and the generation of more than 10 ml of waste. However, the waste is safely treated for disposal. The overall AGREE score suggests that the method successfully integrates several green chemistry concepts while maintaining analytical robustness.
The GAPI pictogram provided a visual overview of the environmental impact across all procedural stages, from sample preparation to waste management. The predominance of green and yellow fields, with only one red segment, indicates that most steps comply well with GAC principles. The red zones reflect off-line analysis as opposed to a greener in-line analysis and waste generation of more than 10 ml. The overall pattern demonstrated a well-optimized and environmentally conscious workflow, minimizing reagent consumption, waste, and exposure potential.
The BAGI scored 75 (Fig. 7), which is well above the recommended threshold of 60, indicating a high degree of practicality and user-friendliness. The BAGI pictogram showed a predominance of darker blue shades, indicating strong compliance with criteria related to method simplicity, time efficiency, reproducibility, and instrument accessibility. This emphasizes that the method not only aligns with sustainable analytical principles but is also realistic for routine laboratory use, supporting the blue component of the white analytical chemistry concept. The detailed breakdown of the scores for AGREE, GAPI, and BAGI is shown in supplementary Tables 1, 2, and Fig. 18, respectively.
Fig. 7.
AGREE, BAGI, and GAPI pictograms represent the greenness and practicality of the developed analytical method
By combining greenness (AGREE, GAPI) with practical applicability (BAGI), the method is not only environmentally conscious but also operationally feasible for routine use. This approach provides a holistic assessment confirming that the method achieves a balanced profile of sustainability and analytical performance.
Conclusion and future directions
This current analytical method is the first validated simultaneous RID method for donepezil and memantine. By avoiding derivatization and excipient interference, it provides a practical alternative to UV methods for routine formulation analysis. The developed analytical method is selective, sensitive, simple, rapid, and reproducible for the simultaneous detection of Dnp and Mem. This method can be utilized for formulation testing and quality purposes, containing both the drugs without the derivatization of memantine. In this paper, we applied the developed method to an intranasal in situ gel release study, and the results showed successful quantification in the samples. In addition to this, the method can be used to analyze drug content in marketed and experimental formulations. The use of a refractive index detector, combined with a relatively short run time, the use of the most common solvents, and good analyte separation at low concentration, offers a clear analytical advantage in this method. The moderate AGREE score, high BAGI score, and predominantly green/yellow GAPI profile collectively demonstrate that the method is environmentally sustainable and practically applicable.
Supplementary Material
Below is the link to the electronic supplementary material.
Acknowledgements
We sincerely thank Adelaide University for providing the facilities, resources, and academic support necessary to conduct this research.
Author contributions
Conceptualization, B.S., and S.G.; writing—original draft preparation, B.S. and S.H.Y; writing—review and editing, B.S., S.H.Y, A.M.U, Y.S., and S.G.; supervision, S.G. All authors reviewed the manuscript.
Funding
The authors declare that they have no known competing financial interests that could have appeared to influence the work reported in this paper.
Data availability
The data generated and/or analyzed during this study are included in this published article and its supplementary information files. Additional data is available from the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Informed consent
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Data Availability Statement
The data generated and/or analyzed during this study are included in this published article and its supplementary information files. Additional data is available from the corresponding author on reasonable request.