A Novel Green Spectrofluorimetric Approach for Memantine Quantification With Mechanistic Elucidation Through Quantum Mechanical Modeling and Green Analytical Chemistry Evaluation

ABSTRACT

A novel, sustainable spectrofluorimetric method was developed for memantine determination using erythrosin B as a fluorescent probe, addressing critical analytical gaps in pharmaceutical quality control and environmental monitoring. The method exploits static fluorescence quenching through ground‐state complex formation between protonated memantine and anionic erythrosin B. Initially, UV–Vis absorption spectroscopy revealed a significant hypochromic effect accompanied by a hypsochromic shift of 5 nm, with the absorption maximum shifting from 527 to 522 nm upon complex formation, while fluorescence studies demonstrated concentration‐dependent quenching at 527 nm excitation and 553 nm emission wavelengths. Subsequently, temperature‐dependent Stern–Volmer analysis yielded quenching constants decreasing from 6.22 × 10⁵ to 4.47 × 10⁵ mol L⁻¹ (298–313 K), confirming the static quenching mechanism. Furthermore, Job's method established 2:1 memantine‐to‐erythrosin B stoichiometry, which was validated by quantum mechanical calculations revealing energetically favorable binding interactions. The developed method exhibited excellent analytical performance with a linear range of 0.02–2.0 μg/mL, a detection limit of 6.5 ng/mL, precision (%RSD < 2%), and accuracy (98.59%). Moreover, successful applications included pharmaceutical formulations and spiked environmental water samples, achieving quantitative recoveries ranging from 96% to 104% across different matrices. Finally, a comprehensive sustainability assessment demonstrated superior green credentials compared to conventional chromatographic approaches.

A novel derivatization‐free spectrofluorimetric method exploiting erythrosin B fluorescence quenching enables sensitive memantine determination in pharmaceutical formulations and environmental water samples.

1. Introduction

Memantine hydrochloride, a noncompetitive N‐methyl‐D‐aspartate (NMDA) receptor antagonist, represents a critical pharmaceutical intervention for moderate‐to‐severe Alzheimer's disease, with comprehensive meta‐analyses demonstrating its therapeutic efficacy in over 5004 patients across multiple clinical trials [1]. The global consumption of antidementia drugs, including memantine, has doubled in the past decade with widespread prescription patterns observed across different regions [2], contributing to an increasing environmental burden as pharmaceutical usage continues to expand globally. Importantly, pharmaceutical compounds have emerged as persistent environmental contaminants of emerging concern, with a comprehensive global database revealing that 631 different pharmaceutical substances have been detected above analytical detection limits in environmental matrices across 71 countries worldwide [3]. Among all, urban wastewater treatment plants serve as the dominant emission pathway for pharmaceuticals entering aquatic ecosystems, as conventional treatment systems are ineffective at removing these biologically active compounds, resulting in their continuous discharge into surface waters, groundwater, and marine environments [3, 4]. Hence, the environmental persistence and bioavailability of pharmaceuticals in aquatic systems pose significant ecological risks, as demonstrated by studies showing that pharmaceutical residues can promote toxic effects on nontarget organisms even at nanogram per liter concentrations [5]. Furthermore, research has established that aquatic model organisms, including zebrafish, express functional NMDA receptors and demonstrate high sensitivity to NMDA receptor antagonists, indicating potential neurological impacts on aquatic fauna exposed to memantine residues [6]. Consequently, the development of sensitive and selective analytical methodologies for environmental monitoring of memantine is therefore essential to assess its ecological fate, establish environmental exposure levels, and implement appropriate risk management strategies for protecting aquatic ecosystems from this emerging pharmaceutical contaminant.

A literature review revealed that the analytical determination of memantine presents significant challenges due to its lack of intrinsic chromophoric properties [7]. High‐performance liquid chromatography (HPLC) coupled with ultraviolet (UV) and fluorescence detection represents the most commonly employed approach for memantine analysis, consistently requiring pre‐column derivatization to enable detection due to memantine's lack of natural UV‐absorbing chromophores, which presents significant analytical challenges and high environmental concerns [8, 9, 10, 11]. Specifically, Suckow et al. developed an HPLC method utilizing dansyl chloride derivatization for memantine quantification in plasma, employing a C18 reversed‐phase column with phosphate buffer‐acetonitrile mobile phase and achieving a detection limit of 3 ng/mL [8]. Furthermore, Jalalizadeh et al. reported a stability‐indicating HPLC method for memantine hydrochloride using 1‐fluoro‐2,4‐dinitrobenzene (FDNB) derivatization, demonstrating the versatility of derivatization approaches for this compound [9]. Similarly, derivatization with o‐phthaldialdehyde (OPA) has been successfully applied for memantine determination using HPLC coupled with fluorescence detection at λ_ex 335 and λ_em 440 nm, utilizing mobile phases of acetonitrile‐phosphate buffer (50:50 v/v) [10]. Despite these developments, these derivatization procedures significantly extend analysis time and necessitate the use of organic solvents, raising concerns about environmental impact (EI) and operational complexity. Moreover, HPLC‐UV methods suffer from inherently low sensitivity, consequently requiring large sample volumes and extended analysis times. To address sensitivity limitations, liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods have been developed for memantine analysis, offering superior detection limits of 0.1–3 ng/mL using mobile phases of acetonitrile‐water containing 0.1% formic acid [12, 13]. However, these LC–MS/MS approaches necessitate expensive instrumentation and complex sample preparation involving liquid–liquid or solid‐phase extraction procedures. Gas chromatography–mass spectrometry (GC–MS) has also been applied for memantine determination using pentafluorobenzoyl chloride derivatization, providing good sensitivity but demanding volatile derivative formation and extensive sample cleanup [14]. Collectively, these established chromatographic methods present significant limitations including mandatory complex derivatization procedures, high reagent consumption, extensive sample preparation requirements, and substantial operational costs, thereby necessitating the development of simpler and more cost‐effective analytical approaches.

Spectrofluorimetric methods have emerged as powerful analytical alternatives that offer significant advantages over traditional chromatographic approaches for pharmaceutical analysis [15, 16, 17, 18, 19, 20]. These methods provide high sensitivity, typically achieving detection limits one to three orders of magnitude lower than UV–Vis spectrophotometry, with the capability to analyze compounds at nanogram levels [21, 22]. Furthermore, spectrofluorimetric techniques demonstrate substantial operational advantages over HPLC and LC–MS systems, including reduced analysis time, simplified instrumentation requirements, and elimination of complex mobile phase preparation [23, 24].

Erythrosin B (2′,4′,5′,7′‐tetraiodofluorescein disodium salt), a xanthene‐based food colorant dye, has demonstrated remarkable potential as a spectrofluorimetric probe for pharmaceutical analysis due to its unique analytical properties [25, 26]. This FDA‐approved food additive exhibits excellent fluorescence characteristics in the green region of the visible spectrum, providing a convenient analytical platform for drug determination through fluorescence quenching mechanisms [27]. Importantly, this green wavelength region offers significant advantages over UV‐based detection methods as it experiences minimal matrix interference effects commonly encountered in complex environmental samples [28]. The availability, affordability, rapid labeling capability, and high sensitivity of erythrosin B render it particularly suitable for routine pharmaceutical quality control applications [29]. Moreover, erythrosin B‐based methods offer significant environmental advantages by utilizing water as the primary solvent system, eliminating the need for large volumes of organic solvents typically required in chromatographic methods [30]. Recent studies have successfully applied erythrosin B for the determination of various pharmaceutical compounds, including naftidrofuryl, octreotide, and tamoxifen, demonstrating its versatility for amine‐containing drugs through ion‐pair complex formation mechanisms [31, 32, 33].

Our previously published investigation utilizing BSA‐stabilized copper nanoclusters for memantine determination in pharmaceutical formulations demonstrated excellent analytical performance with a linear range of 25–600 ng/mL and high precision [34]. However, this nanocluster‐based approach presents several limitations for environmental applications, including complex synthesis procedures requiring controlled conditions, potential instability of protein‐based matrices in varying environmental pH conditions, susceptibility to interference from metal ions commonly present in environmental samples, and relatively high operational costs due to nanocluster preparation requirements. Additionally, the BSA‐Cu nanocluster system exhibits limited robustness in complex environmental matrices containing high ionic strength and diverse organic matter that can interfere with the fluorescence response. In contrast, the proposed erythrosin B methodology offers superior environmental stability, simplified sample preparation protocols, enhanced cost‐effectiveness for routine monitoring applications, and improved tolerance to matrix effects commonly encountered in environmental samples, thereby providing a more practical analytical solution for memantine determination in environmental matrices such as wastewater, surface water, and groundwater systems.

This study presents the first spectrofluorimetric method for memantine determination in environmental water samples using erythrosin B as a novel fluorescent probe. The research aims to (i) comprehensively characterize memantine‐erythrosin B interactions through UV–Vis and spectrofluorimetric analysis, elucidating quenching mechanisms via Stern–Volmer analysis, temperature‐dependent kinetics, and Job's method for stoichiometric determination, (ii) calculate thermodynamic parameters and perform quantum mechanical DFT calculations to provide mechanistic insights into binding interactions, (iii) optimize critical factors affecting quenching efficiency, (iv) validate the method following ICH Q2(R2) guidelines, (v) demonstrate applications in pharmaceutical formulations and environmental water matrices, and (vi) conduct comprehensive sustainability assessment using a multi‐metric approach. The novelty lies in applying erythrosin B's green‐region fluorescence for environmental memantine analysis, offering superior matrix tolerance compared to UV‐based methods. This environmentally friendly approach addresses the analytical gap in memantine environmental monitoring while providing a cost‐effective alternative to complex chromatographic methods for routine pharmaceutical analysis.

2. Experimental

2.1. Materials

The memantine hydrochloride reference standard (99.65%) was obtained from Sigma‐Aldrich (St. Louis, MO, USA). The erythrosin B fluorescent dye (dye content ≥ 95%) was also obtained from Sigma‐Aldrich (St. Louis, MO, USA). HPLC‐grade acetonitrile was procured from Fisher Scientific (Hampton, NH, USA) for sample preparation procedures. Analytical grade chemicals, including boric acid and phosphoric acid, were purchased from Merck (Darmstadt, Germany), while acetic acid, sodium hydroxide, and sodium chloride were obtained from Sigma‐Aldrich (St. Louis, MO, USA). Commercial memantine hydrochloride tablets (Alzmenda, labeled to contain 10 mg of memantine hydrochloride per tablet) were acquired from local pharmacies in Hafr AlBatin, Saudi Arabia, for pharmaceutical analysis. Environmental water samples, including tap water and underground water, were collected from different locations in Hafr AlBatin, Saudi Arabia. Tap water samples were collected after allowing water to run for 5 min. Underground water samples were collected, filtered through 0.45 μm membrane filters, and stored at 4°C in amber glass bottles until analysis within 24 h.

2.2. Instrumentation

Fluorescence spectroscopic measurements were performed using a Jasco FP‐6200 spectrofluorometer (Jasco International Co. Ltd., Tokyo, Japan) equipped with a 150 W xenon arc lamp and standard 1 cm path length quartz cuvettes. The instrument parameters were optimized with excitation and emission slit widths set at 5 nm, and spectral scanning rate adjusted to 4000 nm/min. Fluorescence intensity measurements at the analytical wavelengths of 527 nm (excitation) and 553 nm (emission) were conducted using Spectra Manager software (version 1.53). Ultraviolet–visible absorption spectra were recorded using a Shimadzu UV‐1800 dual‐beam spectrophotometer (Shimadzu Corporation, Kyoto, Japan) with matched 1 cm quartz cells and operated through UV Probe software (version 2.43). Spectral acquisition was performed with 1 nm spectral resolution over the 200–700 nm wavelength range. Solution pH measurements and adjustments were accomplished using a Jenway 3510 digital pH meter (Jenway, Staffordshire, UK) calibrated daily using certified reference buffers at pH 4.0, 7.0, and 10.0.

2.3. Preparation of Memantine Standard Solution

Memantine hydrochloride reference standard (10.0 mg, 99.65% purity) was accurately weighed and transferred into a 100 mL volumetric flask, dissolved in distilled water, and diluted to volume to obtain a stock solution of 100 μg/mL. Working standard solutions in the range of 0.02–2.0 μg/mL were prepared daily by appropriate serial dilution of the stock solution with distilled water.

2.4. Preparation of Erythrosin B Solution

Erythrosin B (10.0 mg, dye content ≥ 95%) was accurately weighed and transferred into a 100 mL volumetric flask, dissolved in distilled water, and diluted to volume to obtain a stock solution of 0.01% w/v. The solution was stored in amber glass vessels at 4°C.

2.5. Quantum Mechanical Calculations

Molecular modeling studies were conducted using the Gaussian 09 computational chemistry software (Gaussian Inc., Wallingford, CT, USA) to investigate the binding interactions between memantine and erythrosin B molecules. The initial three‐dimensional structures of memantine, erythrosin B, and their potential binding complex were constructed and visualized using GaussView 6.0 molecular graphics interface. Complete geometry optimization was performed employing density functional theory (DFT) calculations using the RB3LYP hybrid functional with the LANL2DZ basis set, which provides accurate results for organic molecular systems containing heavy atoms such as iodine in erythrosin B.

The interaction energy between memantine and erythrosin B molecules was determined using the supramolecular approach according to the equation:

$$\mathrm{\Delta E}=\mathrm{E}\left(\text{complex}\right)-\mathrm{nE}\left(\text{memantine}\right)-\mathrm{E}\left(\text{erythrosin}\mathrm{B}\right)$$

where E (complex), E (memantine), and E (erythrosin B) correspond to the total electronic energies of the optimized complex structure, individual memantine molecule, and individual erythrosin B molecule, respectively, and n represents the stoichiometric ratio determined experimentally. Negative ΔE values indicate energetically favorable complex formation with thermodynamically stable binding interactions. Molecular descriptors including dipole moments were computed to characterize the electronic properties and intermolecular forces governing the association process. The calculations were performed under standard computational conditions with tight optimization criteria to ensure accurate energy minimization and reliable molecular property predictions.

2.6. Optimization of Reaction Conditions

A systematic one‐variable optimization approach was employed to determine the optimal experimental conditions for maximum fluorescence quenching efficiency between erythrosin B and memantine. Each parameter was individually optimized while maintaining other variables constant. The effect of pH was investigated over the range 3.0–9.0 using a Britton–Robinson buffer solutions with volumes varied from 0.5 to 3.0 mL. Erythrosin B concentration was studied across the 6–24 μg/mL range. Reaction time optimization was performed by monitoring fluorescence response over 0–10 min. All optimization experiments were performed in 10 mL volumetric flasks with memantine at a fixed final concentration of 1.0 μg/mL. The procedure involved adding appropriate volumes of memantine stock solution, followed by the variable parameter being optimized, and diluting to 10 mL with distilled water. After mixing, solutions were equilibrated before fluorescence measurements at 527 nm excitation and 553 nm emission wavelengths. The fluorescence quenching efficiency was evaluated using the F0/F ratio, where F0 represents erythrosin B fluorescence alone and F represents fluorescence after memantine addition. All experiments were conducted at ambient temperature (25°C ± 2°C) with triplicate measurements. Optimal conditions providing maximum quenching efficiency were selected for subsequent studies.

2.7. General Analytical Procedure and Construction of the Calibration Curve

The optimized analytical procedure for memantine determination using erythrosin B fluorescence quenching was performed as follows: Into 10 mL volumetric flasks, appropriate volumes of memantine standard solution or sample solution were added to achieve final concentrations within the analytical range of 0.02–2.0 μg/mL. Subsequently, 1.0 mL of Britton–Robinson buffer (pH 5.5) and erythrosin B stock solution were added to obtain a final concentration of 16 μg/mL. The solutions were mixed thoroughly and allowed to stand for 3 min at ambient temperature (25°C ± 2°C) to ensure complete interaction between the fluorescent probe and memantine molecules. The reaction mixtures were then diluted to volume with distilled water and mixed again.

Fluorescence measurements were conducted immediately after dilution using the optimized instrumental parameters. A blank solution containing all reagents except memantine was prepared under identical conditions, and its fluorescence intensity (F0) was recorded. The fluorescence intensity (F) of each standard or sample solution was measured under the same experimental conditions. For calibration curve construction, memantine working standard solutions were prepared at seven concentration levels of 0.02, 0.05, 0.10, 0.25, 0.50, 1.00, and 2.00 μg/mL by appropriate serial dilution of the stock solution with distilled water. Calibration curves were constructed by plotting the fluorescence intensity ratio (F0/F) against the corresponding memantine concentration in μg/mL. All measurements were performed in triplicate, and the mean values were used for quantitative calculations.

2.8. Applications to Real Samples

2.8.1. Analysis of Pharmaceutical Formulations

Commercial memantine hydrochloride tablets (Alzmenda, labeled to contain 10 mg memantine hydrochloride per tablet) were selected for analysis based on their market availability, widespread clinical prescription in the study region, and suitability as a representative pharmaceutical dosage form for method validation purposes. Ten tablets were accurately weighed to determine the average tablet weight, then pulverized to a homogeneous fine powder using a clean mortar and pestle. A portion of the powdered sample equivalent to 10 mg memantine was accurately transferred to a 100 mL volumetric flask and dissolved in approximately 30 mL of distilled water with the assistance of ultrasonication for 15 min to ensure complete drug extraction from the tablet matrix. The resulting solution was filtered through Whatman No. 42 filter paper to remove insoluble excipients and tablet debris, and the clear filtrate was diluted to volume with distilled water to obtain a stock solution containing 100 μg/mL memantine. Working solutions within the analytical range were prepared by appropriate serial dilution with distilled water and subjected to analysis following the general analytical procedure described in Section 2.7. Blank tablet matrix solutions were prepared using the same extraction procedure without drug addition to account for potential fluorescence interference from pharmaceutical excipients.

2.8.2. Analysis of Environmental Water Samples

Environmental water samples, including tap water and underground water, were analyzed for memantine residues using the developed method. The water samples were filtered through 0.45 μm membrane filters and spiked with known amounts of memantine to achieve final concentrations of 0.1, 0.3, 0.5, and 1.0 μg/mL for recovery studies. For sample preparation, 3 mL of each water sample was transferred to 10 mL volumetric flasks, followed by 3 mL of acetonitrile and 0.5 g of sodium chloride for salting‐out assisted liquid–liquid extraction (SALLE). The mixture was vortexed vigorously for 2 min and centrifuged at 5000 rpm for 10 min to achieve phase separation. The clear organic layer was carefully collected and evaporated to dryness under a gentle nitrogen stream at 40°C. The dried residue was reconstituted with 1 mL of distilled water and filtered through a 0.22 μm syringe filter before analysis according to the optimized procedure. Blank environmental samples were processed identically to subtract background interference. Matrix‐matched calibration curves were prepared by spiking blank water samples with known concentrations of memantine and processing under identical conditions.

3. Results and Discussion

3.1. Spectroscopic Characterization

Erythrosin B, a xanthene‐based fluorescent dye containing four iodine substituents and carboxylate functional groups, exhibits characteristic spectroscopic properties due to its extended conjugated π‐electron system (Figure S1). The spectral properties following interaction with memantine were investigated using UV–Vis absorption spectroscopy, which provided initial evidence for the formation of a ground‐state complex. As illustrated in Figure 1A, the addition of memantine to erythrosin B solution resulted in a distinct hypochromic effect, characterized by a significant decrease in absorbance intensity at the characteristic absorption wavelength. Furthermore, a notable hypsochromic shift of 5 nm was observed, with the absorption maximum shifting from 527 nm for free erythrosin B to 522 nm upon complex formation. This blue shift indicates perturbation of the electronic environment around the erythrosin B chromophore, consistent with the formation of a ground‐state association complex. The observed spectral changes can be attributed to the interaction between the positively charged amino group of memantine and the anionic carboxylate groups of erythrosin B, leading to alterations in the π‐electron system of the xanthene dye structure and subsequent modifications in its electronic transitions.

FIGURE 1.

Spectroscopic characterization of memantine‐erythrosin B interaction. (A) UV–Vis absorption spectra showing hypochromic effect and 5 nm hypsochromic shift upon complex formation. (B) Fluorescence excitation and emission spectra of erythrosin B (λex = 527 nm, λem = 553 nm). (C) Concentration‐dependent fluorescence quenching with increasing memantine concentrations (0–2 μg/mL).

Regarding fluorescence properties of erythrosin B, its rigid xanthene backbone exhibits strong emission in the green region of the visible spectrum due to efficient π‐π* transitions within the conjugated ring system, enhanced by planar molecular geometry that restricts nonradiative decay pathways [26, 30]. As demonstrated in Figure 1B, the fluorescence excitation and emission spectra showed optimal wavelengths at 527 and 553 nm, respectively, confirming its suitability as a fluorescent probe for analytical applications. Additionally, the emission in the green region significantly minimizes potential matrix interference effects commonly encountered with UV‐based detection methods, as this wavelength range experiences less interference from organic compounds and background fluorescence in environmental samples. Upon interaction with memantine, fluorescence spectroscopic investigations revealed characteristic quenching behavior that provided further evidence for complex formation. Progressive addition of memantine to erythrosin B solution demonstrated concentration‐dependent fluorescence quenching (Figure 1C) where systematic decreases in emission intensity were observed with increasing memantine concentrations from 0 to 2 μg/mL. Such consistent quenching response across the concentration range established the basis for quantitative memantine determination in the present work.

3.2. Mechanistic Studies

To understand the interaction between memantine and erythrosin B, several potential quenching pathways were evaluated. Memantine's molecular structure lacks aromatic chromophores or extended conjugation, immediately excluding inner filter effects that would require competitive light absorption (Figure S2). The absence of suitable electronic transitions in memantine also eliminates Förster resonance energy transfer, which demands spectral overlap between donor emission and acceptor absorption bands for efficient energy migration.

The distinction between dynamic and static quenching mechanisms was examined through temperature variation studies. Stern–Volmer analysis at three temperatures revealed linear relationships (Figure 2A) with quenching constants decreasing from 6.22 × 10⁵ mol L⁻¹ at 298 K to 4.47 × 10⁵ mol L⁻¹ at 313 K (Table 1). This inverse temperature dependence strongly indicates static quenching through ground‐state complex formation, since thermal energy disrupts intermolecular binding. Conversely, dynamic quenching processes would show enhanced quenching at higher temperatures due to increased molecular mobility and collision frequency. Furthermore, quantitative assessment through the relationship kq = KSV/τ₀ produced bimolecular rate constants of 7.78 × 10¹⁴ M⁻¹ s⁻¹, substantially exceeding diffusion limits in aqueous media (~10¹⁰ M⁻¹ s⁻¹). This four‐order magnitude difference definitively confirms static quenching rather than dynamic collision‐based encounters.

FIGURE 2.

Comprehensive mechanistic investigation of memantine‐erythrosin B binding interactions through temperature‐dependent and stoichiometric studies. (A) Stern–Volmer plots at three different temperatures (298 K, 303 K, 313 K) showing linear relationships with decreasing quenching constants, confirming static quenching mechanism. (B) Van't Hoff plot (ln Ka vs. 1/T) for thermodynamic parameter determination, yielding enthalpy and entropy values for complex formation. (C) Job's method continuous variation plot establishing 2:1 memantine‐to‐erythrosin B binding stoichiometry with characteristic maximum at mole fraction 0.67.

TABLE 1.

Temperature‐dependent thermodynamic parameters for memantine‐erythrosin B complex formation.

Temperature (K)	Ksv (105L mol −1)	Kb (104L mol −1)	Ka (106L mol −1)	ΔG (kJ/mol)	ΔH (kJ/mol)	ΔS (J/(mol·K)
298	6.22	8.71	1.13	−34.56	−18.55	53.69
303	5.69	8.35	1.02	−34.87
313	4.47	6.48	0.79	−35.36

The binding affinity between memantine and erythrosin B was subsequently characterized through complementary analytical approaches. Initially, binding constants (K_b) were determined using the double logarithmic equation: log[(F0‐F)/F] = log K_b + n log[Q], yielding values of 8.71 × 10⁴, 8.35 × 10⁴, and 6.48 × 10⁴ mol L⁻¹ across the temperature range studied. Additionally, association constants (K_a) were calculated using the modified Stern–Volmer equation F0/(F0‐F) = 1/Ka[Q] + 1, providing consistently higher values of 1.13 × 10⁶, 1.02 × 10⁶, and 0.79 × 10⁶ mol L⁻¹ at 298, 303, and 313 K, respectively. The systematic decrease in both K_b and K_a values with increasing temperature further corroborates the static quenching mechanism and demonstrates the thermal sensitivity of the intermolecular association.

Thermodynamic evaluation revealed consistently negative Gibbs free energy values of −34.56, −34.87, and −35.36 kJ/mol at the three temperatures studied, confirming the spontaneous nature of complex formation under all experimental conditions (Table 1). Subsequently, van't Hoff analysis (Figure 2B) provided enthalpy and entropy parameters of ΔH = −18.55 kJ/mol and ΔS = 53.69 J/(mol·K), respectively. The negative enthalpy indicates an exothermic binding process driven by favorable intermolecular interactions, particularly electrostatic attraction between memantine's protonated amino group and erythrosin B's anionic carboxylate substituents. Meanwhile, the positive entropy change suggests that the association process involves desolvation effects and structural reorganization of the hydration sphere around both interacting species, contributing to the overall thermodynamic favorability of complex formation. This thermodynamic signature is characteristic of ionic interactions combined with hydrophobic contributions in aqueous media.

Stoichiometric analysis was conducted using Job's method of continuous variations to determine the binding ratio between memantine and erythrosin B. Equal volumes of stock solutions (2.00 × 10⁻⁵ M) were mixed in varying proportions while maintaining a constant total molar concentration of 2.00 × 10⁻⁶ M in the final analytical solution. The resulting Job's plot (Figure 2C) exhibited a characteristic maximum at a mole fraction of 0.67, unambiguously establishing a 2:1 memantine‐to‐erythrosin B binding stoichiometry. This finding indicates that two memantine molecules interact with each erythrosin B molecule, consistent with the dye's structure containing multiple anionic sites capable of accommodating positively charged ligands. Collectively, these mechanistic investigations confirm static quenching through ground‐state complex formation and provide the experimental basis for subsequent quantum mechanical modeling of the binding interactions.

3.3. Quantum Mechanical Analysis

DFT calculations were performed to provide theoretical insights into the memantine‐erythrosin B binding interactions and validate the experimental stoichiometry determined from Job's plot analysis. The calculations employed the RB3LYP hybrid functional with the LANL2DZ basis set, which was specifically selected to accommodate the heavy iodine atoms in erythrosin B through effective core potentials. While this basis set is primarily optimized for geometry optimization of large molecular systems rather than high‐precision energy calculations, it provides adequate computational treatment for initial structural and energetic assessments. The interaction energy was calculated using the supramolecular approach according to the equation:

$$\mathrm{\Delta E}=\mathrm{E}\left(\text{complex}\right)-\mathrm{E}\left(\text{erythrosin}\mathrm{B}\right)-\text{2E}\left(\text{memantine}\right)$$

where E (complex) = −2237.663156 Eh, E (erythrosin B) = −1187.293709 Eh, and E (memantine) = −525.004381 Eh. The resulting binding energy of −0.361 Eh (−226.3 kcal/mol) indicates exceptionally strong intermolecular associations that support the experimental observations of stable complex formation. This computational binding energy represents the intrinsic electronic interaction strength under vacuum conditions and differs substantially from the experimentally derived Gibbs free energy values (−34.56 to −35.36 kJ/mol), which incorporate solvation effects, temperature contributions, and entropic factors that are absent in the gas‐phase calculations. Hence, these calculations provide valuable qualitative insights into the binding mechanism and molecular interactions, while quantitative energetic comparisons should be interpreted with consideration of the methodological limitations and environmental differences between computational and experimental conditions.

Dipole moment calculations provided additional evidence for the nature of the binding interactions. Individual dipole moments of 9.58 D for erythrosin B and 8.99 D for memantine were determined, while the optimized complex exhibited a dipole moment of 18.23 D. This value represents a partial cancellation compared to the theoretical additive sum of 27.56 D, indicating an organized molecular orientation and partial charge neutralization through electrostatic interactions between the protonated amino groups of memantine and the anionic sites of erythrosin B. The dipole moment reduction suggests that the binding involves specific geometric arrangements rather than random association, with the molecular orientations optimized to minimize electrostatic repulsion while maximizing attractive interactions.

To complement these dipole moment findings, the geometric analysis of the optimized structures provided detailed insights into the specific binding interactions. The optimized geometries of individual components and the 2:1 complex were obtained with all structures achieving convergence as evidenced by RMS gradient norms results (Figure 3). Initially, the individual erythrosin B structure (Figure 3A) displays the planar xanthene backbone with four iodine substituents and carboxylate functional groups that provide multiple anionic binding sites. Similarly, the optimized memantine geometry (Figure 3B) reveals the characteristic adamantane cage structure with the protonated amino group positioned to facilitate electrostatic interactions. Subsequently, analysis of the complex geometry (Figure 3C) revealed specific binding sites and interaction distances that explain the strong association observed experimentally. Each memantine molecule is stabilized through dual interactions with erythrosin B, creating a highly organized binding motif. The primary interactions involve electrostatic attraction between the protonated amino groups of memantine and the anionic functional groups of erythrosin B. Specifically, one memantine molecule forms a strong electrostatic interaction with the carboxylate group at a distance of 2.6 Å, while the second memantine molecule interacts with the hydroxyl substituent at 2.5 Å. These short distances are characteristic of strong ionic interactions and consequently explain the substantial binding energy observed.

FIGURE 3.

Quantum mechanical analysis of molecular binding interactions using DFT calculations at RB3LYP/LANL2DZ level. (A) Optimized geometry of erythrosin B displaying planar xanthene backbone with four iodine substituents and anionic carboxylate groups serving as binding sites. (B) Optimized memantine structure showing characteristic adamantane cage with protonated amino group positioned for electrostatic interactions. (C) Optimized 2:1 complex geometry revealing dual binding mechanisms: electrostatic interactions between amino groups and carboxylate/hydroxyl sites (2.5–2.6 Å) and halogen bonding with iodine substituents (3.9 Å).

Furthermore, each memantine molecule participates in halogen bonding interactions with the iodine substituents of erythrosin B, with both interactions occurring at identical distances of 3.9 Å. These halogen bonds provide secondary stabilization and contribute to the overall binding affinity and geometric organization of the complex. Therefore, the combination of primary electrostatic interactions and secondary halogen bonding creates a robust binding network where each memantine molecule is anchored through two distinct interaction modes, ultimately accounting for the high stability and specific 2:1 stoichiometry observed in the experimental investigations.

3.4. Optimization of Analytical Conditions

The analytical performance of the memantine–erythrosin B fluorescence quenching system was systematically optimized through one‐variable analysis to establish conditions that maximize sensitivity and reproducibility. Each experimental parameter was individually evaluated while maintaining other variables constant, guided by the mechanistic understanding established through quantum mechanical calculations.

Solution pH emerged as the most critical parameter affecting complex formation efficiency (Figure 4A). The quenching response increased progressively from pH 3.0 to a maximum at pH 5.5, followed by a plateau extending to pH 7.0, and then decreased at higher pH values. This behavior reflects the ionization states of both species. Erythrosin B possesses two ionizable sites with calculated pKa values of 3.52 (carboxylic acid) and 4.35 (phenolic hydroxyl) (Figure S1), while memantine's amino group has a pKa of approximately 10.45 (Figure S2). At pH values below 3.5, the carboxylic acid group remains largely protonated, reducing anionic binding sites. Moreover, between pH 3.5 and 4.3, progressive deprotonation occurs, increasing negative charge density and enhancing complex formation. At pH 5.5, both erythrosin B groups are fully deprotonated while memantine remains completely protonated, creating optimal electrostatic conditions. Furthermore, decreased efficiency above pH 7.0 may result from memantine deprotonation or altered fluorescence properties. Additionally, the influence of buffer volume on analytical response was examined from 0.5 to 3.0 mL (Figure 4B). The response increased from 0.5 mL to a maximum at 1.0 mL, then decreased at higher volumes. The initial increase reflects the necessity of adequate buffering capacity to maintain optimal pH conditions and provide sufficient ionic strength for complex stabilization. However, excessive buffer concentrations above 1.0 mL result in decreased quenching efficiency, likely due to competitive interactions between buffer components and the binding sites on erythrosin B.

FIGURE 4.

Systematic optimization of experimental conditions using one‐variable analysis approach for maximum analytical performance. (A) Effect of solution pH (3.0–9.0) on fluorescence quenching efficiency, showing optimal performance at pH 5.5 based on ionization equilibria of both interacting species. (B) Influence of Britton‐Robinson buffer volume (0.5–3.0 mL) on analytical response, demonstrating maximum efficiency at 1.0 mL. (C) Erythrosin B concentration optimization (5–25 μg/mL) revealing plateau region at 16–20 μg/mL corresponding to optimal stoichiometric conditions. (D) Reaction time studies (0–10 min) confirming rapid equilibration within 3 min and sustained stability.

Erythrosin B concentration optimization (Figure 4C) demonstrated the importance of reagent stoichiometry for maximum analytical response. The quenching response increased steadily from 5 μg/mL to a plateau at 16–20 μg/mL, followed by a slight decrease at 25 μg/mL. This concentration‐dependent behavior reflects the 2:1 binding stoichiometry established experimentally, where sufficient erythrosin B concentration is required to accommodate the binding of two memantine molecules per dye molecule. Moreover, the plateau region indicates saturation of binding sites and optimal stoichiometric conditions for maximum complex formation. The subsequent decrease at higher concentrations may result from inner filter effects, where excessive dye concentration causes reabsorption of emitted light. In addition, aggregation phenomena may reduce the effective fluorescence quantum yield and alter the binding microenvironment. Reaction time was investigated over the range 0 to 10 min to provide insights into complex formation kinetics (Figure 4D). The response increased rapidly within the first 3 min and maintained a stable plateau throughout the remaining time period. This rapid equilibration supports the static quenching mechanism, where ground‐state complex formation occurs immediately upon mixing. Moreover, the sustained stability indicates that the electrostatic associations are not subject to time‐dependent degradation or conformational rearrangements. Therefore, a reaction time of 3 min was selected to ensure complete equilibration while maintaining practical efficiency for routine analysis. Based on these studies, optimal conditions were pH 5.5, buffer volume 1.0 mL, erythrosin B concentration 16 μg/mL, and reaction time 3 min, providing maximum sensitivity with robust performance, and have been employed in further validation studies.

3.5. Method Validation and Analytical Performance

The developed spectrofluorimetric method was comprehensively validated following ICH Q2(R2) guidelines [35] to establish its reliability for quantitative memantine determination. Under optimized conditions, the method demonstrated excellent linearity over the concentration range 0.02–2.0 μg/mL with a correlation coefficient of 0.9996 (Table 2). The regression equation F0/F = 2.8977C + 1.1761 provided the basis for quantitative analysis, where C represents the memantine concentration in μg/mL. Furthermore, the limits of detection and quantification were determined as 6.5 and 19.4 ng/mL, respectively, based on the standard deviation of the blank response (σ) and the slope of the calibration curve (S) according to the ICH Q2(R2) guidelines, where LOD = 3.3σ/S and LOQ = 10σ/S [36, 37], indicating superior sensitivity compared to conventional UV‐based methods. These low detection limits are particularly advantageous for environmental monitoring applications where memantine residues are expected at trace levels.

TABLE 2.

Comprehensive analytical performance characteristics and method validation parameters following ICH Q2(R2) guidelines including linearity, sensitivity, precision, accuracy, and robustness evaluation.

Parameters		Memantine
Excitation wavelength (nm)		527
Emission wavelength (nm)		553
Linearity range (μg/mL)		0.02–2.0
Slope		2.8977
Intercept		1.1761
Correlation coefficient (r 2)		0.9996
LOD (ng/mL)		6.5
LOQ (ng/mL)		19.4
Accuracy (%R) a		98.59 ± 0.958
Repeatability precision (%RSD) b		0.971
Intermediate precision (%RSD) c		1.582
Robustness (%R)	Buffer (pH)	98.7 ± 0.969
	Erythrosine B (μg/mL)	100.88 ± 1.339
	Reaction time (min)	100.5 ± 1.295

^a Average of 9 determinations (3 concentrations repeated 3 times).

^b % RSD of 9 determinations (3 concentrations repeated 3 times) measured on the same day.

^c % RSD of 9 determinations (3 concentrations repeated 3 times) measured in the three consecutive days.

Accuracy assessment through recovery studies yielded 98.59% ± 0.958%, confirming the method's reliability for quantitative analysis across the working range (Table 2). Subsequently, precision studies revealed satisfactory reproducibility with repeatability precision (%RSD) of 0.971% and intermediate precision of 1.582%, both well within acceptable limits for pharmaceutical analysis. These precision values demonstrate the method's consistency under both intra‐ and inter‐day conditions. Moreover, robustness evaluation demonstrated acceptable tolerance to minor variations in critical parameters, with recovery values remaining close to 100%: pH variations (±0.1 units, 98.7% ± 0.969%), erythrosin B concentration changes (±1 μg/mL, 100.88% ± 1.339%), and reaction time modifications (±0.5 min, 100.5% ± 1.295%). These minimal deviations from theoretical values confirm the method's stability under routine analytical conditions.

Selectivity investigations examined potential interferences from common pharmaceutical excipients and environmental matrix components (Figure S3). The results revealed minimal interference from typical substances, with quenching efficiency remaining below 5% for most tested compounds, including common excipients and inorganic salts. However, like other fluorescence‐based methods, selectivity limitations exist compared to LC–MS/MS techniques, particularly regarding structurally similar compounds or highly concentrated matrix components. Nevertheless, these limitations can be effectively addressed through sample pretreatment procedures, such as the SALLE extraction employed for environmental samples, or by implementing matrix‐matched calibration approaches. Additionally, the green‐region emission of erythrosin B minimizes interference from organic compounds that typically absorb or fluoresce in the UV region, thereby providing inherent selectivity advantages over UV‐based detection methods.

Therefore, the validation parameters collectively demonstrate that the developed method meets international standards for pharmaceutical and environmental analysis, offering a reliable alternative to more complex chromatographic techniques while maintaining adequate analytical performance for intended applications.

3.6. Applications to Real Samples

The practical applicability of the developed spectrofluorimetric method was evaluated through the analysis of pharmaceutical formulations and environmental water samples to demonstrate its utility for routine analytical applications.

3.6.1. Pharmaceutical Analysis and Method Comparison

The method was successfully applied to the analysis of commercial memantine hydrochloride tablets using the optimized analytical procedure. The results were statistically compared with a reported HPLC‐UV method [8] to establish analytical equivalence (Table 3). The developed method yielded a mean recovery of 99.94 ± 0.697%, while the reference HPLC method provided 99.69 ± 0.948%. Statistical analysis using Student's t‐test revealed no significant difference between the methods (t‐calculated = 0.481 < t‐tabulated = 2.306 at p = 0.05), confirming comparable accuracy (Table 3). Furthermore, F‐test analysis demonstrated no significant difference in precision (F‐calculated = 1.854 < F‐tabulated = 6.338 at p = 0.05), indicating equivalent analytical performance (Table 3).

TABLE 3.

Statistical comparison between the developed spectrofluorimetric method and reported HPLC‐UV reference method for pharmaceutical formulation analysis using t‐test, F‐test, and bias assessment.

Method	Mean a	SD	t‐test (2.306) b	p	F‐value (6.338) b	p	θL c	θU c
Developed method	99.94	0.697	0.481	0.645	1.854	0.565	−0.960	1.466
Reported method	99.69	0.948

^a Average of five determinations.

^b The values in parenthesis are tabulated values of “t”and “F” at (p = 0.05).

^c Bias of ±2% is acceptable.

The equivalence assessment through calculation of bias limits (θ_L = −0.960, θ_U = 1.466) confirmed that both values fall within the acceptable range of ±2%, establishing analytical equivalence according to regulatory guidelines. These results validate the proposed method as a reliable alternative to chromatographic techniques, offering advantages of reduced analysis time, simplified instrumentation, and lower operational costs while maintaining comparable analytical performance.

3.6.2. Environmental Water Analysis

The method's environmental applicability was assessed through recovery studies using spiked tap water and underground water samples to evaluate performance under realistic monitoring conditions. Sample preparation was conducted using SALLE to eliminate matrix interferences and concentrate the analyte prior to analysis. Recovery studies were performed at four concentration levels (0.1, 0.3, 0.5, and 1.0 μg/mL) spanning the expected environmental concentration range for pharmaceutical contaminants.

Both water matrices demonstrated excellent analytical performance (Table 4). Underground water samples yielded recoveries ranging from 96.14% to 104.28% with relative standard deviations below 4%, while tap water analysis provided recoveries between 99.60% and 104.07% with %RSD values below 3% (Table 4). These results confirm minimal matrix effects and good precision across different aquatic environments. The SALLE pretreatment effectively eliminated potential interferences while maintaining quantitative recovery, demonstrating the method's robustness for environmental applications.

TABLE 4.

Recovery studies for memantine determination in environmental water matrices using salting‐out assisted liquid–liquid extraction pretreatment across multiple concentration levels.

Samples	Spiked (μg/mL)	Found (μg/mL)	Recovery (%)	RSD (n = 3, %)
Underground water	0.1	0.104	104.28	3.987
	0.3	0.309	102.92	2.179
	0.5	0.506	101.1	1.089
	1	1.012	101.17	1.092
Tap water	0.1	0.096	96.14	2.928
	0.3	0.312	104.07	2.453
	0.5	0.498	99.60	1.744
	1	1.026	102.62	1.419

To further enhance selectivity for complex environmental matrices, molecularly imprinted polymers (MIPs) could be integrated as advanced sample preparation techniques. MIP‐based solid phase extraction would provide selective recognition sites complementary to memantine's molecular structure, effectively addressing the inherent selectivity limitations of fluorescence‐based probes without requiring chromatographic separation. This approach would combine molecular recognition capabilities with the cost‐effectiveness and rapid analysis time advantages of spectrofluorimetric detection, offering enhanced specificity while maintaining operational simplicity. Overall, the successful environmental applications demonstrate the method's suitability for monitoring memantine residues in aquatic systems, addressing the critical need for accessible analytical tools in pharmaceutical environmental surveillance programs.

3.7. Green Analytical Chemistry Assessment

The environmental sustainability and practical applicability of the developed spectrofluorimetric method were comprehensively evaluated using the Environmental, Performance, and Practicality Index (EPPI) [38] alongside the White Analytical Chemistry/RGB12 model (WAC) [39]. This streamlined two‐metric assessment approach was selected in recognition of EPPI's comprehensive dual‐index framework, which simultaneously integrates ecological sustainability through its EI Index and real‐world analytical applicability through its Performance and Practicality Index (PPI), thereby providing a balanced and holistic evaluation of the method across all critical dimensions of sustainability and practicality within a single unified tool (Figure 5).

FIGURE 5.

Comprehensive sustainability assessment using a dual‐metric evaluation approach. (A) EPPI assessment showing EI Index (95.4/100, ideal green), PPI (63.0/100, acceptable practicality), and four core EI components across sample preparation, instrumentation, reagents, and waste, yielding a total EPPI score of 79.2/100. (B) WAC/RGB12 comprehensive assessment displaying analytical efficiency (92.8%, red), environmental friendliness (96.3%, green), and practical efficiency (71.7%, blue) with overall whiteness score of 86.9%.

3.7.1. EPPI Assessment

The developed spectrofluorimetric method was evaluated using the EPPI, a comprehensive dual‐index framework that simultaneously addresses ecological sustainability, analytical performance, and real‐world applicability through its EI Index and PPI, as shown in Figure 5A.

The EI Index achieved an outstanding score of 95.4/100, placing the method in the ideal green category (score range 85–100, dark green) according to the EPPI interpretation framework. This exceptional environmental performance reflects the method's strong compliance with the principles of green analytical chemistry (GAC) and green sample preparation (GSP) across all four core EI components. The sample preparation component scored excellently, benefiting from the absence of chemical pre‐synthesis requirements, minimal sample preparation involving simple dilution for pharmaceutical samples, and the use of distilled water as the primary solvent system, which is recognized as the most environmentally benign solvent with a reagent score of 100. The instrumentation component achieved high scoring through minimal energy consumption (≤ 0.1 kWh per sample) from the spectrofluorometer, consistent with energy‐efficient analytical instrumentation. The reagent component demonstrated superior environmental credentials through the exclusive use of erythrosin B in aqueous solution and Britton‐Robinson buffer components, all of which carry minimal GHS hazard classifications, eliminating the need for toxic organic reagents typically required in chromatographic methods. Furthermore, the waste generation component scored favorably due to the production of less than 10 mL of aqueous waste per sample, substantially lower than conventional HPLC and LC–MS/MS methods requiring large volumes of organic mobile phases. Collectively, these EI component scores confirm the method's minimal ecological footprint and its alignment with sustainable laboratory practices.

The PPI achieved a score of 63.0/100, falling within the acceptable practicality range (score 50–74, light purple), reflecting the method's solid real‐world applicability with identified opportunities for further improvement. The method scored maximally on the nature of method criterion as a fully quantitative analytical approach and achieved excellent scores for reagent availability through the commercial accessibility of erythrosin B at low cost, instrument accessibility and cost of analysis through the widespread availability of spectrofluorometers in standard analytical laboratories at a cost below $10 per sample, and instrument maintenance through the long operational lifespan and minimal maintenance requirements of spectrofluorometric instrumentation. Full ICH Q2(R2) validation further contributed positively to the PPI score. The method's nanogram‐level sensitivity (LOD: 6.5 ng/mL) secured a high sensitivity score consistent with nanogram‐range detection capabilities. However, the PPI score reflects certain limitations in the practicality domain, including the absence of Design of Experiments optimization, lack of AI integration in method development, manual operation requirements, and limited sample throughput compared to automated high‐throughput systems. These factors represent clear opportunities for future methodological enhancement.

The total EPPI score of 79.2/100 classifies the developed method as a “Highly Recommended—Green and Practical, Efficient Method” according to the EPPI significance framework (score 75–100). This total score validates the method's holistic excellence, demonstrating successful integration of outstanding environmental sustainability with acceptable analytical practicality, positioning it as a superior sustainable alternative to conventional chromatographic approaches for routine memantine determination in pharmaceutical quality control and environmental monitoring applications.

3.7.2. WAC Assessment

The WAC assessment revealed an overall whiteness score of 86.9%, demonstrating high holistic method performance across analytical, environmental, and practical dimensions (Figure 5B). The analytical efficiency (red) category achieved 92.8%, with outstanding precision and accuracy scores validating the method's reliability for quantitative memantine determination. The environmental friendliness (green) category scored 96.3%, reflecting perfect performance in energy consumption and direct environmental impacts, with excellent toxicity and waste management scores. This high green performance confirms successful implementation of sustainable analytical practices without compromising measurement quality. The practical efficiency (blue) category achieved 71.7%, showing good cost‐efficiency and time‐efficiency but limited operational simplicity due to manual procedures and laboratory restrictions. The balanced RGB performance demonstrates successful integration of all three analytical chemistry dimensions, avoiding the common trade‐offs between sustainability and performance. The high whiteness score validates the method's holistic excellence, positioning it as a sustainable analytical solution that maintains analytical integrity while reducing environmental impact. This comprehensive assessment confirms that the spectrofluorimetric approach represents a paradigm shift toward sustainable pharmaceutical analysis, offering a practical alternative to conventional methods while addressing recent demands for environmentally responsible analytical practices.

3.8. Comparison With Reported Spectrofluorimetric Methods for Memantine Determination

The developed spectrofluorimetric method was critically compared with reported spectrofluorimetric approaches for memantine determination. All existing spectrofluorimetric methods rely exclusively on chemical derivatization strategies to generate the measurable fluorescent signal. Atia et al. reported a method based on Hantzsch condensation between memantine's primary amino group and acetylacetone/formaldehyde in acetate buffer (pH 4.8), generating a yellow fluorescent derivative measured at λex/λem = 418/484.5 nm with a linear range of 0.2–1.0 μg/mL and LOD of 15.3 ng/mL [40]. While bioanalytically applicable, this method requires multi‐reagent derivatization with precise pH control, and its UV‐excitation wavelength renders it susceptible to matrix fluorescence interference. Similarly, Ravisankar et al. employed NQS in alkaline medium followed by potassium borohydride reduction, achieving a linear range of 0.25–1.25 μg/mL and LOD of 9.0 ng/mL [41]. Despite acceptable sensitivity, the mandatory two‐step reaction sequence substantially extends analysis time and increases procedural complexity. Kepekci Tekkeli and Erturk Toker developed an HPLC‐fluorimetric method using FMOC‐Cl precolumn derivatization achieving a high LOD of 0.3 ng/mL [42], however, at the expense of expensive instrumentation, toxic organic mobile phases, and complex derivatization procedures. Our previously reported BSA‐copper nanocluster probe demonstrated excellent sensitivity with a linear range of 25–600 ng/mL [34], however, the complex synthesis procedures, susceptibility to metal ion interference, and limited robustness in high ionic strength environmental matrices restrict its broader analytical applicability.

In contrast, the present erythrosin B‐based approach represents the first derivatization‐free spectrofluorimetric method applied to environmental water matrices for memantine determination. The elimination of derivatization reduces total preparation time to only 3 min, the green‐region emission at 553 nm minimizes matrix interference, and the exclusive use of water as the primary solvent eliminates organic solvent consumption entirely, collectively conferring superior green analytical credentials confirmed by the outstanding EI Index score of 95.4.

4. Conclusion

This study successfully developed and validated a novel spectrofluorimetric method for memantine determination using erythrosin B as a fluorescent probe, addressing critical analytical gaps in pharmaceutical quality control and environmental monitoring. The comprehensive investigation revealed static quenching through ground‐state complex formation with 2:1 memantine‐to‐erythrosin B stoichiometry, confirmed by quantum mechanical calculations showing energetically favorable binding interactions involving electrostatic and halogen bonding mechanisms. Subsequently, the validated method demonstrated excellent analytical performance with superior sensitivity (LOD: 6.5 ng/mL), satisfactory precision (%RSD < 2%), and accuracy (98.59%). Moreover, statistical comparison confirmed equivalence with reference HPLC methods, while successful applications to pharmaceutical formulations and environmental water samples using SALLE pretreatment achieved quantitative recoveries (96%–104%) across different matrices. Furthermore, comprehensive sustainability assessment using the (EPPI: total score 79.2/100; EI Index: 95.4; PPI: 63.0) and the White Analytical Chemistry model (WAC: 86.9%) demonstrated superior green credentials through minimal energy consumption, reduced waste generation, and elimination of toxic reagents compared to conventional chromatographic approaches. Therefore, the method represents a sustainable analytical solution that maintains measurement quality while reducing environmental impact, offering a practical alternative for routine memantine analysis in both pharmaceutical and environmental applications.

It is worth noting that several limitations require acknowledgment in the presented work. Among all, selectivity is the most significant constraint compared to LC–MS/MS techniques, particularly for complex matrices containing structurally similar compounds. Furthermore, manual operation requirements increase analysis time and operator dependency, while the 2:1 binding stoichiometry results in nonlinear reagent consumption at higher concentrations. Moreover, single‐analyte capability limits versatility compared to multicomponent chromatographic methods. Consequently, future research should focus on implementing sample preparation techniques, such as molecularly imprinted polymer technology, to increase method selectivity. Besides, developing portable instrumentation for field applications and integrating automation through flow injection or microfluidics systems are other emerging opportunities. Investigation of alternative fluorescent probes and spectral deconvolution techniques represents another avenue that could enable multi‐analyte determination capabilities. Overall, the presented work establishes a robust foundation for sustainable memantine analysis while identifying clear pathways for methodological enhancement and expanded analytical scope in pharmaceutical and environmental monitoring applications.

Author Contributions

Muneef M. Aldhafeeri: conceptualization, investigation, writing – original draft, methodology, validation, visualization, writing – review and editing, formal analysis, data curation.

Funding

The author has nothing to report.

Conflicts of Interest

The author declares no conflicts of interest.

Supporting information

Figure S1: Chemical structure of erythrosin B.

Figure S2: Chemical structure of memantine.

Figure S3: Selectivity studies showing interference effects of various compounds.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.