Abstract
A sensitive and environmentally friendly spectrofluorimetric method was developed for the determination of guanfacine in pharmaceutical formulations. The method is based on derivatization of guanfacine with 4-chloro-7-nitrobenzofurazan in alkaline medium to yield a highly fluorescent product measured at 535 nm after excitation at 466 nm. Experimental conditions affecting the reaction were systematically optimized to achieve maximum fluorescence intensity. The method exhibited excellent linearity over the concentration range of 50–500 ng/mL with a correlation coefficient of 0.9994. The limits of detection and quantitation were found to be 13.80 and 41.82 ng/mL, respectively, indicating high sensitivity. The method was validated in accordance with ICH guidelines and demonstrated satisfactory accuracy, precision, robustness, and selectivity. It was successfully applied to the analysis of guanfacine in commercial tablet formulations without interference from excipients. Greenness assessment using Analytical Eco-scale and AGREE tools confirmed the environmentally benign nature of the method, achieving a high Eco-scale score of 89 and an AGREE score of 0.68. Compared with a reported method, the proposed approach offers improved sensitivity and superior environmental benignity, making it suitable for routine quality control analysis.
Keywords: Guanfacine, Spectrofluorimetry, NBD-Cl derivatization, Green analytical chemistry.
Subject terms: Chemistry, Environmental sciences
Introduction
Guanfacine hydrochloride (GFN), Fig. 1, is a selective α₂A-adrenergic receptor agonist that has gained considerable clinical importance in the management of attention-deficit hyperactivity disorder (ADHD) and hypertension1. It is a non-stimulant agent that exerts its therapeutic effect primarily through modulation of noradrenergic signaling in the prefrontal cortex, a brain region responsible for attention, impulse control, and executive function2. By enhancing prefrontal cortical activity and reducing sympathetic nerve impulses, GFN contributes to improved behavioral regulation and decreased vascular resistance, ultimately lowering blood pressure and enhancing cerebral and peripheral blood flow3. The extended-release oral formulation of GFN has been approved for the management of ADHD in children and adolescents (6–17 years), offering the advantage of once-daily dosing and enhanced patient adherence4. Chemically, GFN (N-amidino-2-(2,6-dichlorophenyl) acetamide monohydrochloride) possesses a secondary amine functional group that renders it amenable to derivatization reactions with electrophilic reagents5. Given its increasing therapeutic utilization and pharmacological relevance, the development of sensitive, selective, and cost-effective analytical methods for its quantification in pharmaceutical formulations is of significant importance in quality control and routine analysis.
Fig. 1.
Chemical structure of GFN.
Several analytical techniques have been reported for the determination of GFN, including liquid chromatography6 –10,Gas chromatography11,12, Spectrofluorimetry13, and spectrophotometry14. For instance, LC-MS/MS methods developed for GFN in biological matrices report LLOQ values in the low picogram-per-milliliter range6–10, reflecting exceptional sensitivity suited to pharmacokinetic and bioanalytical studies; however, the associated instrumentation costs and sample preparation complexity limit their routine use for pharmaceutical quality control, where simpler and more economical techniques are often preferred. In contrast, spectrophotometric methods are simpler and more accessible but may suffer from limited sensitivity, particularly at low concentration levels. Spectrofluorimetric techniques, therefore, represent an attractive alternative, offering a favorable balance between sensitivity, selectivity, and operational simplicity for routine pharmaceutical analysis.
To the best of our knowledge, only one spectrofluorimetric method has been reported for the determination of guanfacine, based on its derivatization with benzoin under alkaline conditions13. Although this method demonstrated acceptable sensitivity, it involves relatively complex reaction conditions requiring the use of auxiliary reagents such as β-mercaptoethanol and sodium sulfite, in addition to extraction steps when applied to biological samples. Such requirements may limit its practicality and environmental compatibility in routine applications.
Fluorescence spectroscopy has gained increasing attention in pharmaceutical analysis due to its high sensitivity and selectivity. However, many pharmaceutical compounds, including GFN, exhibit weak or no native fluorescence, necessitating derivatization with suitable fluorogenic reagents. Among these, 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is widely recognized for its high reactivity toward nucleophilic functional groups, particularly primary and secondary amines, leading to the formation of highly fluorescent derivatives. The reaction of NBD-Cl with amine-containing compounds proceeds via nucleophilic substitution, where the chlorine atom is displaced by the amino group, yielding a stable fluorescent product. This strategy has been successfully applied in the determination of numerous pharmaceutical compounds, providing highly sensitive and selective methods under relatively mild experimental conditions15,16.
In addition to its analytical performance, NBD-Cl is considered a relatively environmentally benign reagent, aligning with the principles of green analytical chemistry. Methods based on NBD-Cl derivatization typically involve reduced reagent consumption, minimal use of hazardous solvents, and simplified procedures without the need for labor-intensive extraction steps, thereby enhancing their environmental benignity17.
Based on the above considerations, the present study aims to develop a novel, sensitive, and environmentally friendly spectrofluorimetric method for the determination of GFN in pharmaceutical formulations. The proposed method is based on derivatization of GFN with NBD-Cl to produce a highly fluorescent derivative, followed by optimization of reaction conditions and validation in accordance with International Council for Harmonization (ICH) guidelines. The applicability of the method for routine quality control analysis is also investigated.
Experimental
Materials and chemicals
Guanfacine hydrochloride (GFN) and 4-chloro-7-nitrobenzofurazan (NBD-Cl) were obtained from Merck KGaA (Darmstadt, Germany). Intuniv® extended-release tablets, labeled to contain 1 mg of GFN per tablet, were manufactured by Takeda Pharmaceutical Company Limited (Spain). HPLC-grade solvents, including methanol, ethanol, dichloromethane, acetonitrile, and acetone, were supplied by Sigma-Aldrich (USA). Analytical-grade chemicals were procured from local suppliers and used as received. All solutions were prepared using freshly double-distilled water. buffer solutions within the pH range of 7.0–10.0 were prepared following the procedures described in the United States Pharmacopeia. A fresh methanolic solution of NBD-Cl (0.2% w/v) was prepared and used throughout the experiments.
Instrumentation
Fluorescence measurements were performed using a Jasco FP-6200 spectrofluorometer (Japan) equipped with a 150 W xenon arc lamp. The excitation and emission slit widths were both set at 10 nm. Measurements were carried out using a 1 cm quartz cuvette.
Standard solutions
A primary stock solution of GFN (100 µg/mL) was prepared by dissolving an accurately weighed amount of the drug in distilled water. Working standard solutions were subsequently obtained by serial dilution of the stock solution to yield concentrations in the range of 1.5–15 µg/mL.
General fluorescence procedure
Aliquots (1.0 mL) of GFN working standard solutions were transferred into a series of 10 mL stoppered test tubes. To each tube, 1.25 mL of borate buffer (pH 9) and 1.5 mL of NBD-Cl solution (0.2% w/v) were added. The reaction mixtures were thoroughly mixed and heated in a thermostatically controlled water bath at 80 °C for 25 min to ensure complete derivatization. After heating, the tubes were immediately cooled in an ice bath to terminate the reaction. Subsequently, 1.0 mL of 2 M hydrochloric acid was added to each tube. The reaction mixtures were quantitatively transferred into 10 mL volumetric flasks and diluted to volume with distilled water. A reagent blank was prepared following the same procedure. The fluorescence intensity of the formed derivative was measured at an emission wavelength of 535 nm after excitation at 466 nm. A calibration curve was constructed by plotting relative fluorescence intensity versus the corresponding GFN concentration.
Analysis of pharmaceutical formulation
Five Intuniv® tablets, each containing 1 mg of GFN, were accurately weighed and finely powdered. The amount of the powder equivalent to one tablet was transferred into a 100 mL volumetric flask, dissolved in approximately 50 mL of distilled water, and sonicated for 10 min to ensure complete extraction. The resulting solution was filtered to remove insoluble excipients, and the filtrate was diluted to volume with distilled water. Suitable aliquots were further diluted to fall within the working concentration range and analyzed using the procedure described above.
Results and discussion
Spectral characteristics
Guanfacine hydrochloride (GFN) does not exhibit significant native fluorescence under the studied conditions, which necessitates derivatization for its spectrofluorimetric determination. Upon reaction with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in alkaline medium, a highly fluorescent derivative was formed. The emission spectrum of the reaction product showed a well-defined maximum at 535 nm when excited at 466 nm, whereas the reagent blank displayed negligible fluorescence under the same conditions (Fig. 2). This confirms the successful formation of a fluorescent NBD-derivative and demonstrates the selectivity of the proposed method. The observed fluorescence can be attributed to the formation of a highly conjugated NBD-GFN derivative following nucleophilic substitution of the chlorine atom in NBD-Cl by the amino group of guanfacine. The resulting product exhibits enhanced rigidity and extended π-electron delocalization, leading to a significant increase in fluorescence intensity. The excitation and emission wavelengths obtained are consistent with those reported for NBD-derivatives of other amine-containing compounds, further supporting the proposed reaction mechanism15,16.
Fig. 2.
Fluorescence spectra of the NBD-GFN derivative.
Reaction mechanism
The reaction between GFN and NBD-Cl proceeds via a nucleophilic substitution mechanism in alkaline medium. Under these conditions, the amino group of guanfacine becomes deprotonated, increasing its nucleophilicity. Nucleophilic nitrogen attacks the electron-deficient carbon atom bearing the chlorine substituent in NBD-Cl, resulting in the displacement of chloride ion and formation of a stable NBD-GFN derivative (Fig. 3). This derivatization step introduces a strong fluorophore into the molecular structure, thereby enabling sensitive spectrofluorimetric detection. Enhanced fluorescence is mainly due to the electron-withdrawing nitro group and the rigid benzofurazan moiety of NBD, which promote efficient radiative transitions.
Fig. 3.
Suggested reaction pathway of GFN with NBD-Cl.
Optimization of experimental conditions
The experimental variables affecting the formation and stability of the fluorescent product were systematically investigated to achieve maximum sensitivity.
Influence of buffer pH and buffer volume
The nucleophilic substitution reaction between NBD-Cl and amine-containing compounds is favored under alkaline conditions. Accordingly, the effect of pH on the fluorescence response of the GFN-NBD derivative was systematically examined using borate buffer solutions over the pH range of 7–10. The results indicated that maximum fluorescence intensity was achieved at pH 9 (Fig. 4a), which was therefore selected as the optimum pH for subsequent experiments. The effect of buffer volume was also evaluated by varying the volume of pH 9 borate buffer. An increase in fluorescence intensity was observed up to 1.25 mL, beyond which no further improvement was noted, indicating that this volume is sufficient to maintain the optimal reaction environment. It is well recognized that NBD-Cl undergoes hydrolysis under alkaline conditions, leading to the formation of NBD-OH, which contributes to background fluorescence. To minimize this interference, the reaction mixture was acidified after completion of the reaction and cooling, using 1 mL of 2 M hydrochloric acid. This step effectively reduced the contribution of NBD-OH and enhanced the selectivity of the measurement.
Fig. 4.
Optimization studies for GFN reaction with NBD-Cl.
Effect of NBD-Cl volume
The influence of the reagent concentration was investigated by varying the volume of a 0.2% (w/v) NBD-Cl solution. The fluorescence intensity of the formed derivative increased with increasing reagent volume, reaching a maximum at 1.5 mL (Fig. 4b). Further increases did not result in significant enhancement, and therefore 1.5 mL was selected as the optimal volume to ensure complete reaction without excess reagent interference.
Effect of reaction temperature and heating time
The derivatization reaction between NBD-Cl and amines is kinetically slow at room temperature and typically requires elevated temperatures to proceed efficiently. The effect of temperature was studied over the range of 50–100 °C. The fluorescence intensity of the GFN-NBD derivative increased with temperature, reaching a maximum at 80 °C (Fig. 4c), beyond which no significant improvement was observed. The influence of heating time was also investigated under the optimized temperature. The fluorescence signal increased progressively with time and reached its maximum after 25 min, indicating completion of the reaction. Prolonged heating did not improve the response, and thus 25 min was selected as the optimal reaction time.
Effect of diluting solvent
The choice of diluting solvent can significantly influence the fluorescence characteristics of the formed derivative. Therefore, different solvents, including water, dichloromethane, methanol, ethanol, acetone, and acetonitrile, were evaluated. Among the tested solvents, water provided the highest fluorescence intensity (Fig. 4d), which may be attributed to its compatibility with the reaction medium and reduced quenching effects. Consequently, water was selected as the diluting solvent for all subsequent measurements.
Method validation (ICH Q2-R1)
Linearity and range
The linearity of the proposed spectrofluorimetric method was evaluated by correlating the measured relative fluorescence intensity of the GFN-NBD derivative (after blank correction) with the corresponding drug concentrations. A direct proportional relationship was observed over the concentration interval of 50–500 ng/mL. The calibration plot exhibited excellent linear behavior, with a coefficient of determination (r²) of 0.9995, confirming the reliability of the method within the studied range. The corresponding regression parameters are summarized in Table 1.
Table 1.
Regression and validation data for the determination of GFN by the proposed method.
| Parameters | GFN |
|---|---|
| Emission wavelength | 535 nm |
| Excitation wavelength | 466 nm |
| Linearity range | 50–500 ng/mL |
| Slope | 1.5170 |
| Intercept | 4.3617 |
| Coefficient of determination (r2) | 0.9994 |
| LOD | 13.80 ng/mL |
| LOQ | 41.82 ng/mL |
Limits of detection and quantitation
The sensitivity of the method was assessed through the estimation of the limits of detection (LOD) and quantitation (LOQ). These parameters were calculated based on the standard deviation of the response (σ) and the slope (S) of the calibration curve, following the ICH-recommended equations:
 |
 |
The standard deviation (σ) was estimated as the standard deviation of the residuals of the calibration curve, constructed from n = 3 independent measurements. The calculated values, presented in Table 1, indicate the high sensitivity of the developed method, enabling reliable quantification of GFN at low concentration levels.
Accuracy and precision
The accuracy and precision of the method were investigated using three concentration levels within the working range (100, 300, and 400 ng/mL). Each level was analyzed in triplicate within the same day to evaluate repeatability, and the procedure was repeated over three consecutive days to assess intermediate precision. Accuracy was expressed in terms of percentage recovery (%R), while precision was evaluated as relative standard deviation (%RSD). The results obtained (Table 2) demonstrated that the method provides consistent and accurate measurements, with acceptable variability and recoveries close to 100%.
Table 2.
Accuracy and precision outcomes.
| Concentration (ng/mL) | Within-day assay | Between-days assay | ||||
|---|---|---|---|---|---|---|
| %R* | %Bias* | RSD* | %R* | %Bias* | RSD* | |
| 100 | 99.89 | -0.11 | 0.95 | 98.10 | -1.90 | 1.86 |
| 300 | 99.52 | -0.48 | 1.37 | 98.68 | -1.32 | 1.50 |
| 400 | 100.54 | 0.54 | 0.88 | 99.01 | -0.99 | 1.91 |
*Average of three determinations.
Robustness
The robustness of the proposed procedure was examined by introducing minor deliberate variations in key experimental conditions. A fixed concentration of GFN was analyzed while varying one parameter at a time, including slight changes in pH, reagent volume, and reaction time, while keeping other conditions constant. The recovery values obtained under these modified conditions remained within acceptable limits, as shown in Table 3, indicating that the method is sufficiently robust and not significantly affected by small operational changes.
Table 3.
Evaluation of robustness of the proposed spectrofluorimetric method at a GFN concentration of 400 ng/mL.
| Parameter | %R* ± SD |
|---|---|
| Optimium conditions | 99.33 ± 0.57 |
| Effect of buffer pH | |
| pH 8.8 | 98.81 ± 0.99 |
| pH 9.2 | 98.12 ± 0.73 |
| Volume of buffer | |
| 1 mL | 100.18 ± 1.25 |
| 1.5 mL | 98.98 ± 0.97 |
| Volume of NBD-Cl | |
| 1.25 mL | 99.70 ± 0.58 |
| 1.75 mL | 99.45 ± 0.81 |
| Reaction temperature | |
| 75 °C | 99.27 ± 0.93 |
| 85 °C | 100.63 ± 1.08 |
| Heating time | |
| 20 min | 98.62 ± 1.10 |
| 30 min | 99.05 ± 0.64 |
*Average of three determinations.
Selectivity and application to pharmaceutical formulation
The selectivity of the method was confirmed through its successful application to the determination of GFN in pharmaceutical dosage forms without interference from commonly encountered excipients. Specificity was further evaluated by examining the potential interference of common excipients present in the Intuniv® formulation, namely lactose monohydrate, microcrystalline cellulose, povidone, crospovidone, hypromellose, colloidal silica, and polysorbate 80. Each excipient was added in excess (10- to 50-fold relative to GFN) to a fixed GFN concentration of 300 ng/mL, and the fluorescence intensity was measured under the optimized conditions. None of the tested excipients produced a significant deviation in the fluorescence signal of GFN, with percent recoveries ranging from 98.87% to 100.69% and %RSD values not exceeding 1.05% (Table 4), confirming the absence of excipient interference and the suitability of the method for direct application to the tablet matrix without prior separation. The absence of matrix interference was further verified using the standard addition technique, which yielded satisfactory recovery values. In addition, the analytical performance of the proposed method was statistically compared with that of a previously reported spectrofluorimetric method13, based on the derivatization of guanfacine with benzoin in alkaline medium in the presence of β-mercaptoethanol and sodium sulfite, using Student’s t-test and F-test at a 95% confidence level. The calculated values (Table 5) were found to be lower than the corresponding theoretical values, indicating no significant difference between the two methods in terms of accuracy and precision.
Table 4.
Effect of common excipients on the determination of GFN (300 ng/mL) by the proposed method (n = 3).
| Excipient | Fold excess over GFN | Recovery (%) ± RSD |
|---|---|---|
| Lactose monohydrate | 50 | 100.10 ± 1.01 |
| Microcrystalline cellulose | 50 | 99.99 ± 0.88 |
| Povidone | 20 | 99.41 ± 0.79 |
| Crospovidone | 20 | 100.69 ± 1.05 |
| Hypromellose | 20 | 100.07 ± 0.89 |
| Colloidal silica | 10 | 99.66 ± 0.56 |
| Polysorbate 80 | 10 | 98.87 ± 0.97 |
Table 5.
Quantitative analysis of GFN in pharmaceutical tablets by the proposed method and statistical comparison with the reported method.
| Sample | Percent recovery ± RSD | t-test (2.306)* | F-test (6.388)* | |
|---|---|---|---|---|
| Proposed method | Reported method13 | |||
| Intuniv® Tablets** | 99.34 ± 0.74 | 99.36 ± 1.19 | 0.03 | 2.58 |
| Standard addition *** | 101.24 ± 1.37 | |||
*The values in parenthesis are tabulated values of “t “and “F” at (P = 0.05).
**Average of five determinations.
*** Average of three determinations.
Comparative evaluation with reported method
A critical comparison between the proposed method and the reported spectrofluorimetric method13 was conducted with respect to analytical sensitivity and environmental benignity (Table 6). From a sensitivity standpoint, the proposed method exhibits a clear analytical advantage, achieving LOD of 13.80 ng/mL compared to 20 ng/mL for the reported method. This represents an approximate 1.45-fold improvement in detection capability, which can be attributed to the superior fluorogenic behavior of NBD-Cl.
Table 6.
Comparative evaluation with the reported method.
| # | The described approach | The reported approach13 | ||
|---|---|---|---|---|
| LOD | 13.80 ng/mL | 20 ng/mL | ||
| Analytical eco-scale | Parameters | Penalty points | Parameters | Penalty points |
| Reagents | Reagents | |||
| Water | 0 | Water | 0 | |
| Borate buffer pH 9 | 2 | Potassium hydroxide | 4 | |
| NBD-Cl | 1 | Benzoin | 0 | |
| HCl | 1 | β-mercaptoethanol | 8 | |
| Sodium sulphite | 1 | |||
| Instrument spectrofluorometer | Instrument spectrofluorometer | |||
| Energy: <0.1kWh per sample | 0 | Energy: <0.1kWh per sample | 0 | |
| Heating energy (water bath, 80 °C/25 min) | 1 | |||
| Occupational hazards | 0 | Occupational hazards | 0 | |
| Waste | 6 | Waste | 6 | |
| ∑ Penalty points | 11 | ∑ Penalty points | 19 | |
| Total scores | 100 − 11= 89 | Total scores | 100 − 19= 81 | |
| AGREE |
|
|
||
In terms of green analytical performance, the proposed method demonstrates a significantly improved environmental profile. According to the Analytical Eco-scale, the proposed method achieved a score of 89, compared to 81 for the reported method13. This 10-point difference is substantial and reflects a meaningful reduction in environmental burden. The higher score of the proposed method is primarily attributed to the avoidance of hazardous reagents such as β-mercaptoethanol, which contributes significantly to the penalty points. Instead, the proposed method employs relatively safer reagents, resulting in lower overall environmental impact. Further support is provided by the AGREE assessment, where the proposed method achieved a score of 0.68, whereas the reported method13 showed a lower score of 0.63. This difference confirms the superior compliance of the proposed method with the principles of green analytical chemistry. The higher AGREE score is mainly associated with improved reagent safety, reduced chemical hazard, and better overall greenness metrics. It is noteworthy that both methods exhibit comparable energy consumption, instrumentation requirements, and waste generation; therefore, the observed improvement in greenness is directly attributable to rational reagent selection rather than procedural differences. Overall, the proposed method offers a dual advantage of enhanced sensitivity and superior environmental benignity, supported by both Eco-scale and AGREE metrics. These findings highlight the method as a more advanced, efficient, and greener alternative for the determination of guanfacine in pharmaceutical analysis.
While LC-MS/MS methods reported for GFN offer markedly superior sensitivity, for instance, LLOQ values in the low picogram-per-milliliter range have been achieved for GFN quantification in biological matrices9, such techniques require expensive triple-quadrupole instrumentation, skilled personnel, and labor-intensive sample preparation (e.g., liquid–liquid extraction), rendering them less accessible for routine pharmaceutical quality control settings. The proposed spectrofluorimetric method, by contrast, achieves nanogram-per-milliliter sensitivity (LOD 13.80 ng/mL) sufficient for the analysis of GFN in tablet formulations, using inexpensive, widely available instrumentation and a straightforward derivatization procedure without extraction or cleanup steps. This positions the proposed method as a practical, cost-effective, and environmentally friendly alternative for routine QC laboratories, where LC-MS/MS-level sensitivity is not required, while chromatographic and mass spectrometric methods remain the methods of choice for trace-level bioanalytical applications such as pharmacokinetic studies in plasma.
It should be noted that the proposed method was developed and validated solely for the quantification of GFN in pharmaceutical tablet formulations and has not been evaluated for application to biological matrices such as plasma or urine. Extension to bioanalysis would require additional validation steps, including a protein precipitation or extraction protocol to isolate GFN from the matrix, assessment of matrix effects and selectivity against endogenous plasma/urine components and metabolites, and re-evaluation of sensitivity, since therapeutic plasma concentrations of GFN are typically in the low nanogram- to picogram-per-milliliter range6–10, which may approach or fall below the LOD of the present method. Consequently, the method in its current form is recommended for routine pharmaceutical quality control applications rather than pharmacokinetic or bioanalytical studies, for which the LC-MS/MS methods discussed above remain more appropriate.
Conclusion
A simple, sensitive, and eco-friendly spectrofluorimetric method was successfully developed for the determination of GFN in pharmaceutical formulations. The method relies on derivatization with NBD-Cl to form a highly fluorescent product, enabling sensitive detection at low concentration levels. Validation studies confirmed that the method is accurate, precise, robust, and selective, complying with ICH requirements. The proposed method demonstrated superior analytical performance compared to the reported method, offering a lower limit of detection (13.80 ng/mL vs. 20 ng/mL) and enhanced sensitivity. In addition, greenness evaluation revealed a clear environmental advantage, with higher Analytical Eco-scale (89) and AGREE (0.68) scores, reflecting reduced use of hazardous reagents and improved environmental benignity. The method, in its current validated form, is intended for the analysis of GFN in pharmaceutical formulations rather than biological samples. Extension to bioanalytical applications would require further validation, including matrix-specific sample preparation and sensitivity assessment. Overall, the method provides a reliable, cost-effective, and environmentally responsible alternative for routine quality control analysis of GFN, combining high sensitivity with green analytical performance.
Acknowledgements
The authors gratefully acknowledge Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia for supporting thiswork through Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2026R466),Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. The authors extend their appreciation to the Deanship ofScientific Research at Northern Border University, Arar, KSA for funding this research work through the project number “NBU-FFR-2026-69-03“.
Author contributions
AAA, ASA: Data curation. SIA, MHA; Validation. FMA, MEA; Writing-review & editing. SR, AHA; Methodology, Writing original draft. All authors reviewed the manuscript.
Funding
This work was supported by the Princess Nourah bint Abdulrahman University Researchers Supporting Project number(PNURSP2026R466), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. The work was also supported by theDeanship of Scientific Research at Northern Border University, Arar, KSA for funding this research work through the projectnumber “NBU-FFR-2026-69-03“.
Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Declarations
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 datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.