Spectrofluorimetric Determination of Silodosin Using NBD‐Cl Reagent: Applications to Capsules and Content Uniformity Testing

ABSTRACT

Silodosin (SDN) works by specifically blocking alpha‐1 adrenergic receptors, with a strong preference for the α1A type and clinically approved for alleviating the symptoms associated with benign prostatic hyperplasia in adult patients. The medication is precisely quantified using a novel, affordable spectrofluorimetric strategy in this paper. The strategy uses a fluorogenic reagent (4‐chloro‐7‐nitrobenzofurazan, NBD‐Cl) to create a fluorescent compound called “SDN‐NBD” when it reacts with SDN. Optimal fluorescence emission was recorded at 534.7 nm (excitation: 473.4 nm) after systematic refinement of reaction variables, including pH, temperature, and NBD‐Cl concentration. The approach exhibited remarkable linearity throughout a spectrum of 40–750 ng/mL (R ² > 0.999), with a detection limit of 12.8 ng/mL and a quantitation limit of 38.8 ng/mL. Rigorous validation under International Council for Harmonisation (ICH) guidelines confirmed high accuracy (recovery: 98.92–101.67%) and precision (RSD < 2%). The technique was successfully applied to quantify SDN in pharmaceutical capsules, achieving consistent content uniformity (AV = 5.45). This approach is robust and avoiding costly instrumentation and chromatographic experiments. Its simplicity, reproducibility, and regulatory compliance make it promising for pharmacokinetic research, quality control, and clinical laboratories that need accurate SDN analysis in complicated matrices.

A novel, eco‐friendly spectrofluorimetric method was developed for silodosin quantification. The approach leverages NBD‐Cl derivatization to generate a highly fluorescent adduct, enabling exceptional sensitivity (LOD: 12.8 ng/mL) and precision (RSD < 2%). Validated for both capsule content uniformity and plasma assays, this cost‐effective technique offers a green alternative to conventional chromatographic analysis.

1. Introduction

Benign prostatic hyperplasia represents a prevalent condition diagnosed within the ageing male population, exhibiting an increasing prevalence and frequently manifesting as lower urinary tract symptoms. A spectrum of risk factors, encompassing both modifiable and non‐modifiable elements, contributes to the development and progression of both BPH and associated lower urinary tract symptoms. Symptomatically, patients may experience obstructive manifestations such as urinary hesitancy, a weakened stream, straining during urination, or prolonged voiding; irritative manifestations including increased urinary frequency and urgency, nocturia, urge incontinence, and reduced voided volumes; or postmicturition effects like postvoid dribbling or sensations of incomplete bladder emptying. The pathological basis of BPH involves hyperplastic proliferation of stromal and epithelial cells within the prostate's transitional zone. This proliferative process is understood to be influenced by inflammatory mediators and sex hormones, ultimately leading to prostate gland enlargement. Diagnosis of BPH in symptomatic patients necessitates several key diagnostic investigations to exclude other causes. Management strategies for men with BPH commence with watchful waiting for milder cases and escalate through pharmacological interventions to surgical options. For most patients, the initial selection of treatment is primarily determined by the severity of their symptoms and the associated burden of disease [1, 2, 3].

Silodosin functions as a selective antagonist at alpha‐1 adrenergic receptors, demonstrating its highest binding affinity for the α1A subtype. These α1‐adrenergic receptors are instrumental in modulating smooth muscle contraction within the bladder neck, prostate, and prostatic urethra. Notably, the α1A subtype constitutes the predominant form, representing roughly 75% of the α1‐adrenoceptors present in the prostate gland. Consequently, Silodosin is clinically approved for alleviating the symptoms associated with benign prostatic hyperplasia in adult patients [4, 5, 6].

A limited number of analytical methods have been described for the quantification of SDN. These involve spectrophotometry [7, 8, 9, 10], a spectrofluorimetric approach published for SDN based on native fluorescence measurement in methanol [11] or reaction with dansyl chloride [12]. Separation techniques include HPLC [13, 14, 15] and electrochemical methods [16, 17, 18].

A review of the current literature shows that there are only a few published spectrofluorimetric methods for testing SDN, highlighting a significant gap in this area of research. Spectrofluorimetric techniques are esteemed for their exceptional sensitivity and versatility, making them priceless in several scientific and analytical domains. These methods are particularly favored due to their simplicity, cost‐effectiveness, specificity, and accessibility, which make them especially suitable for routine use in quality control laboratories. Research in this field could help to improve the study of SDN by using the benefits of spectrofluorimetry, hence enabling more precise, reasonably priced analytical tools in commercial and research settings [19, 20].

7‐Chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole, or NBD‐Cl, is an essential fluorogenic substance that is frequently used in spectrofluorimetry. NBD‐Cl stands out for its exceptional fluorescence qualities, making it a versatile tool with a wide range of chemical and biological uses [21, 22]. NBD‐Cl undergoes nucleophilic substitution reactions with diverse functional groups to produce stable derivatives that show bright green fluorescence upon excitation, facilitating easy detection and measurement in complex materials [23, 24]. Its extensive uses encompass bioconjugation, enzymatic tests, chemical labeling, and microscopy, highlighting its importance across various scientific fields. Researchers appreciate NBD‐Cl for its versatility, sensitivity, and ease of use, establishing it as a fundamental component in fluorescence‐based analytical techniques [23, 25, 26, 27].

The use of NBD‐Cl in spectrofluorimetry has not been thoroughly researched for key drugs like SDN, as current methods mainly depend on expensive LC–MS/MS systems and highly pure organic solvents. This gap presents an opportunity to use NBD‐Cl, which provides cost‐efficiency and simplicity in resource‐limited environments.

2. Experimental

2.1. Apparatus

Spectrofluorimetry was conducted using a Scinco FS‐2 fluorimeter sourced from Korea. A 1‐cm thick quartz cell was employed for the measurements, with both the excitation and emission slit widths set to 5 nm. The photomultiplier tube (PMT) voltage was maintained at 400 V. The spectrofluorimetric measurements were conducted at ambient laboratory temperature (25°C ± 2°C). pH levels were measured with a HI 98107 pH meter from Hanna Instruments, based in Brazil. Sample processing and separation were carried out using a Sigma centrifuge, model 2‐16KHL, produced in Germany. To regulate the temperature throughout the experiments, an HH‐4 digital thermostatic water bath from China was utilized. Samples were accurately weighed using a highly sensitive digital balance, model FA2004, also from China, with a precision of 0.1 mg.

2.2. Materials and Reagents

Silodosin of high purity (99.85%) was obtained from Eva Pharma. Flopadex capsules 8 mg were acquired from a local pharmacy in Gharbia. NBD‐Cl (99.52%) was sourced from Thermo Fisher Scientific. Buffer solutions (0.2 M borate buffers) were prepared using distilled water at various pH levels. To create a methanol solution with a concentration of 0.05% (w/v), 5 mg of NBD‐Cl was added to a partially filled 10 mL volumetric flask containing methanol. The solution was thoroughly mixed and adjusted to the calibrated mark of the flask.

2.3. Preparation of SDN Standard Solution

A standard SDN solution at a concentration of 200 μg/mL was meticulously prepared. Initially, 20 mg of SDN was dissolved in 5 mL of methanol within a properly labeled calibrated flask. The solution's volume was then adjusted to 100 mL using the same solvent to ensure homogeneity. To create a series of working solutions, this concentrated stock was further diluted with methanol as required for subsequent experiments.

2.4. General Analytical Procedure

Different volumes of SDN working solution (0.4–7.5 μg/mL) were meticulously allocated in 1 mL aliquots into sterile test tubes with closures. Each tube received 1.0 mL of a 0.2 M borate buffer solution at pH 8.2, then followed by the addition of 0.7 mL of NBD‐Cl reagent at a concentration of 0.05% (w/v). The tubes were thereafter immersed in a water bath and heated to 80°C for 20 min. After heating, the tubes were swiftly cooled, and 1.2 mL of a 3 M HCl solution was added. The entire contents were transferred to a 10‐mL volumetric flask and diluted with methanol to achieve a final concentration between 40 and 750 ng/mL. The fluorescence intensity of the resulting solution was measured using an excitation wavelength of 473.4 nm and an emission wavelength of 534.7 nm. A blank sample, prepared using the same procedure but excluding the SDN drug, served as a control for comparative analysis during fluorescence measurements.

2.5. Verification of SDN in Flopadex Capsules 8 mg and Assessment of Content Homogeneity

In the Egyptian pharmaceutical market, Flopadex capsules 8 mg represent commercial formulations containing SDN. To analyze Flopadex capsules 8 mg, a sample preparation protocol was implemented: 10 capsules were evacuated, homogenized after precise weighing, and a powder aliquot equivalent to 10 mg SDN was transferred to a 100 mL volumetric flask. The powder underwent extraction via 15‐min ultrasonic treatment in 25 mL methanol, followed by dilution to volume with the same alcohol and filtration into a clean flask. Initial filtrate portions were discarded to minimize filter interference, and subsequent aliquots were subjected to the analytical procedure. The method's accuracy was validated through five replicate determinations, with percentage recovery, standard deviation (SD), and statistical comparisons (t‐test and F‐test) calculated using regression models and pharmacopeial guidelines. For Flopadex capsules 8 mg, content uniformity was assessed by individually analyzing each capsule, ensuring compliance with regulatory standards for dose consistency.

3. Result and Discussion

The strategic use of derivatization in analytical chemistry is essential for improving the detectability and quantifiability of target molecules, especially in complex biological matrices. This technique enhances sensitivity by transforming analytes into derivatives with advantageous physicochemical features, allowing for accurate measurement at trace quantities [28]. NBD‐Cl, a reagent selective for primary and secondary amines, reacts efficiently with the amino group in the medicament's structure, forming a stable, highly fluorescent derivative ideal for fluorimetric analysis [29]. The interaction between NBD‐Cl and the analytes amino group follows a nucleophilic substitution mechanism. The amine group attacks the electrophilic carbon atom of NBD‐Cl, displacing the chloro substituent and forming a covalent NBD‐amine adduct. This derivatized product exhibits markedly enhanced fluorescence compared to the parent compound, with excitation and emission maxima in the visible spectrum [30, 31].

This study introduces a novel spectrofluorimetric approach for the analysis of SDN by leveraging its 2ry amino group's reactivity with NBD‐Cl in a borate buffer. To our knowledge, this paper represents the first application of NBD‐Cl derivatization for SDN quantification, as well as the inaugural fluorimetric method validated for SDN detection in pharmaceutical capsules and study of uniformity. The reaction mechanism, illustrated schematically in Scheme 1, involves the nucleophilic substitution of the chloro group in NBD‐Cl by SDN's amino moiety, forming a stable, fluorescent derivative. This pathway aligns with established derivatization principles for amine‐containing compounds, where alkaline conditions favor the formation of electronegative intermediates that enhance reaction kinetics [30, 31]. The fluorescence emission observed at 534.7 nm, following excitation at 473.4 nm (see Figure 1), highlights the enhanced photostability and quantum yield associated with the NBD‐SDN fluorophore. This characteristic is crucial for its effectiveness in the detection of trace‐level analytes. The proposed NBD‐Cl method offers an LOD of 12.8 ng/mL and LOQ of 38.8 ng/mL. While the previously reported dansyl chloride method [12] achieves slightly better sensitivity (LOD: 5.44 ng/mL), our method provides several practical advantages including the absence of dilution with acetone, shorter reaction time (20 min vs. 30 min), measurement in the visible range (534.7 nm) to avoid UV matrix interference, and the use of a safer fluorogenic reagent. These features make our method particularly suitable for routine quality control laboratories where simplicity and speed are valued alongside adequate sensitivity.

SCHEME 1.

The pathway for the fluorescent product formation between SDN and NBD‐Cl.

FIGURE 1.

Fluorescence profiles of NBD‐CL (control) and its SDN‐derived product using 350 ng/mL SDN.

3.1. Optimization of Technique Conditions

3.1.1. Solution pH and Buffer Volume

The optimization of fluorescence intensity for the reaction product formed between SDN and NBD‐Cl was systematically investigated by varying pH conditions using borate buffers (pH 7.0–9.2). As illustrated in Figure 2, the maximum fluorescence signal was achieved at pH 8.2, suggesting this alkaline condition optimally facilitates the derivatization process. This pH‐dependent enhancement likely stems from the improved nucleophilic reactivity of SDN's amino group under mildly basic conditions, which promotes efficient binding with NBD‐Cl's electrophilic center. Subsequent experiments focused on determining the influence of borate buffer volume at the optimal pH (8.2). Incremental increases in buffer volume correlated with a progressive rise in fluorescence intensity, peaking at 1.0 mL (Figure 3). This tendency may indicate the buffer's dual function in preserving pH stability and adjusting the reaction environment to promote derivative production. The efficacy of borate buffer in augmenting fluorescence may be ascribed to its capacity to stabilize intermediate species during the derivatization procedure, thus enhancing yield and signal consistency. The specified settings (pH 8.2, 1.0 mL borate buffer) optimize sensitivity and guarantee reproducibility.

FIGURE 2.

Impact of pH on the reaction between SDN and NBD‐Cl using 350 ng/mL SDN.

FIGURE 3.

Impact of buffer volume on the reaction between SDN and NBD‐Cl using 350 ng/mL SDN.

3.1.2. NBD‐Cl Volume

The modulation of the NBD‐Cl reagent amount (0.05% w/v) in the analytical strategy demonstrated a direct relationship between reagent quantity and FI, as illustrated in Figure 4. Fluorescence signals escalated progressively with increased reagent volumes, attaining a plateau at 0.6–0.8 mL. Exceeding this range, additional increments resulted in a slight reduction in intensity, perhaps attributable to self‐quenching or excess reagents disrupting the reaction equilibrium. Thus, 0.7 mL of NBD‐Cl was determined to be the ideal volume for enhancing sensitivity while ensuring assay stability.

FIGURE 4.

Influence of varying NBD‐Cl volumes on the reaction product between SDN and NBD‐Cl using 350 ng/mL SDN.

A notable challenge in the protocol was elevated background fluorescence, attributed to the hydrolysis of residual NBD‐Cl into (NBD‐OH). To counteract this interference, the addition of 1.2 mL of 3 M HCl prior to measurement effectively reduced the medium's pH below 1.0, suppressing NBD‐OH's fluorescence contribution (Figure 5). This modification improved signal specificity and confirmed the method's resilience to acidic conditions, expanding its use in complex matrices where pH regulation is essential. These enhancements emphasize the necessity of equilibrating reagent stoichiometry and ambient parameters to get dependable, high‐fidelity fluorimetric results.

FIGURE 5.

Influence of varying HCl volumes on the reaction product between SDN and NBD‐Cl using 350 ng/mL SDN.

3.2. Effect of Heating and Temperature Period and Diluent

Experimental optimization of reaction conditions revealed that both temperature and heating duration significantly influenced the fluorescence intensity of the final product. While elevated temperatures or extended heating times enhanced signal magnitude, a balance between these parameters proved critical for reproducibility. Prolonged heating at moderate temperatures (e.g., 80°C for 20 min, Figure 6 and Figure 7) yielded more consistent results compared to shorter durations at higher temperatures, likely due to reduced thermal degradation and improved reaction homogeneity.

FIGURE 6.

Influence of varying temperature on SDN‐NBD‐Cl product generation using 350 ng/mL SDN.

FIGURE 7.

Influence of varying heating time on SDN‐NBD‐Cl product generation using 350 ng/mL SDN.

Additionally, the choice of diluent profoundly affected fluorescence output. Comparative testing of solvents such as acetonitrile, ethanol, and isopropanol demonstrated methanol's superiority in maximizing emission strength (Figure 8). This outcome may stem from methanol's polar protic properties, which stabilize the fluorescent derivative while minimizing quenching effects.

FIGURE 8.

Effect of different diluents on the formation of the end product.

3.3. Method Validation

The study took into account and complied with the pertinent ICH directives to make sure the technique was appropriate for its intended use and to obtain approval. A number of factors were considered, such as the following:

3.4. Linearity and Sensitivity

The analytical validity of the methodology was assessed using standardized SDN solutions spanning concentrations of 40–750 ng mL⁻¹. Fluorescence intensity (FI) measurements of the SDN‐NBD‐Cl derivative, corrected for background signals, were correlated with SDN concentrations to construct a calibration curve. Linear regression analysis of the data revealed exceptional linearity, evidenced by a correlation coefficient (r) of 0.9996 over the tested range (Table 1). The tight clustering of data points around the regression line further confirms minimal variability, reinforcing the protocol's suitability for high‐sensitivity detection of SDN in complex matrices. Regression Equation: (y = 13.9⋇ + 359.76) and the calculation procedure for LOD and LOQ based on the standard deviation of the intercept (Sa) and the slope (b), as per ICH guidelines. The formulas (LOD = 3.3 * Sa/b, LOQ = 10 * Sa/b).

TABLE 1.

Data provided to justify the system's development.

Parameters	Values
Linear range (ng/mL)	40–750
Slope	13.9
Intercept	359.76
SD of intercept (S a )	54
Correlation coefficient (r)	0.9996
Determination coefficients (r 2 )	0.9992
Number of determinations	7
Limit of quantitation (ng/mL)	38.8
Limit of detection (ng/mL)	12.8

3.5. Accuracy

The accuracy of the proposed method was rigorously evaluated by analyzing four SDN distinct concentration levels within the validated range, with triplicate measurements performed at each level. Recovery percentages, expressed as mean ± standard deviation (SD), demonstrated strong unison between experimentally determined values and theoretical concentrations (Table 2). These results reflect the method's high accuracy, as recoveries consistently approached 100%, underscoring its reliability for quantitative applications.

TABLE 2.

The developed system's accuracy at four SDN concentrations.

Conc. level (ng/mL)	Recovery a  ± SD	% error
100	101.67 ± 1.06	1.67
250	99.43 ± 0.50	0.57
400	98.92 ± 1.56	1.08
600	99.08 ± 0.90	0.92

^a Mean of three SDN determinations and SD is the standard deviation.

3.6. Precision

Precision was further assessed through intra‐day (short‐term) and inter‐day (intermediate) analyses at three concentration tiers, with triplicate measurements recorded for each. As summarized in Table 3, relative standard deviation (RSD) values for both precision levels remained below 2%, highlighting the method's exceptional reproducibility. The minimal variability validates the protocol's stability under standard laboratory circumstances and its appropriateness for high‐throughput settings where reliable performance is essential. The approach's combination of outstanding accuracy and precision makes it appropriate for regulatory‐compliant settings, including quality control laboratories and clinical research projects requiring strict analytical criteria.

TABLE 3.

Precision of the created methodology at two levels.

Conc. level (ng/mL)	Intraday precision		Interday precision
	Recovery a  ± SD	RSD	Recovery a  ± SD	RSD
150	98.30 ± 1.54	1.56	100.62 ± 1.77	1.76
450	100.86 ± 1.26	1.25	99.17 ± 0.90	0.91
650	98.96 ± 1.10	1.11	101.16 ± 1.78	1.76

^a Mean of three SDN determinations and RSD is the relative standard deviation.

3.7. Robustness

Through methodical testing of small changes in crucial technique parameters, such as buffer pH, buffer volume, NBD‐Cl reagent volume, and reaction duration, the analytical protocol's robustness was assessed. Deliberately adjusting these variables within a certain, predetermined range had no effect on the assay's performance parameters, including fluorescence intensity and recovery rates, as shown in Table 4. For example, there were no statistically significant differences in the results when the buffer pH was changed by ±0.1 units or the reaction durations were changed by ±5%. Likewise, variations in the volume of NBD‐Cl (±0.1 mL) were accepted without affecting precision or accuracy.

TABLE 4.

Evaluating the robustness of the existing SDN assay technology.

Parameter	Value	% recovery a  ± SD
pH	8.1	101.02 ± 1.07
	8.3	99.12 ± 0.85
Buffer volume (mL)	0.9	100.79 ± 1.35
	1.1	98.59 ± 0.74
NBD‐Cl volume (mL)	0.6	99.98 ± 1.75
	0.8	98.53 ± 0.80
Heating time (min)	18	101.45 ± 0.84
	22	98.80 ± 0.96

Abbreviation: SD = standard deviation.

^a Mean of three replicate measurements.

3.7.1. Selectivity

To assess the method's selectivity, the impact of typical tablet additives—including glucose, lactose, starch, talc, magnesium stearate, and sorbitol—was investigated. As shown in Table S1, these substances did not interfere with the analysis, yielding recoveries close to 100% with negligible standard deviations. This high specificity is due to the fact that these excipients lack the primary amino group necessary to react with NBD‐Cl and form a fluorescent product.

3.8. Job's Method for Determination of Stoichiometric Ratio

The stoichiometry of the reaction between silodosin (SDN) and NBD‐Cl was determined using Job's method of continuous variation. A series of solutions was prepared maintaining a constant total molar concentration ([SDN] + [NBD‐Cl] = 1.0 × 10⁻⁴ M) while varying the mole fraction of SDN from 0.1 to 0.9. The fluorescence intensity of each solution was measured under the optimized conditions (pH 8.2 borate buffer, 80°C for 20 min, followed by acidification with 3 M HCl). The difference in fluorescence intensity between each solution and a corresponding blank was plotted against the mole fraction of SDN. As shown in Figure S1, the maximum fluorescence intensity was observed at a mole fraction of approximately 0.5, indicating the formation of a 1:1 stoichiometric complex between SDN and NBD‐Cl. This finding is consistent with the expected reaction mechanism, where one molecule of SDN (containing a single reactive secondary amine group) reacts with one molecule of NBD‐Cl to form the fluorescent adduct. The 1:1 stoichiometry further supports the selectivity of the derivatization reaction and confirms the complete conversion of the drug to its fluorescent product under the optimized conditions.

3.9. Application

3.9.1. SDN Dosage Form and Content Uniformity Assay

The newly developed spectrofluorimetric approach was successfully applied to quantify SDN in its commercial capsules formulation. To validate the method's specificity, comprehensive studies were conducted to assess potential interference from inactive pharmaceutical additives (excipients). Experimental data confirmed that these additives had no adverse impact on the analytical procedure's performance, ensuring selective detection of SDN without matrix interference. A rigorous statistical evaluation was performed by comparing results from the novel method with those from an established reference technique [12]. Utilizing Student's t‐test (assessing mean differences) and F‐test (evaluating variance equality), the comparative analysis revealed no statistically significant variation between methodologies. As detailed in Table 5, all computed t and F values remained below their respective critical thresholds at a 95% confidence interval, confirming equivalent precision and accuracy between the proposed and reference methods. Moreover, a comparison of the proposed method with some reported methods is listed in supplementary Table S2.

TABLE 5.

Dosage form analysis of SDN and comparison with the reported method [12].

Dosage form	Current method	Reported method
	Recovery a  ± SD	Recovery a  ± SD	t‐test b	F b
Flopadex capsules	99.53 ± 1.23	98.20 ± 1.45	1.58	1.38

^a The value is the average of five determinations for both the proposed and reported methods.

^b Tabulated values at 95% confidence limit are t = 2.306, F = 6.338.

The selection of NBD‐Cl as the derivatizing agent for SDN offers several distinct advantages over previously reported methods (summarized in Table S3 and discussed below):

First, compared to native fluorescence methods [11], which rely on SDN's intrinsic fluorescence in methanol (excitation 272 nm, emission 450 nm) with modest quantum yield, NBD‐Cl derivatization produces a highly fluorescent adduct (excitation 473.4 nm, emission 534.7 nm) with significantly enhanced quantum yield, resulting in improved sensitivity (LOD: 12.8 ng/mL). Second, in contrast to the dansyl chloride method [12], which requires a 45‐min reaction, our NBD‐Cl method requires only 20 min at 80°C, offering a simpler and faster workflow. Third, compared to UV spectrophotometric methods [7, 8, 9, 10], which have LOQs in the μg/mL range, our method achieves an LOQ of 38.8 ng/mL, enabling trace analysis in biological matrices when coupled with suitable sample preparation. Fourth, unlike HPLC methods [13, 14, 15] that consume large volumes of organic solvents and require expensive instrumentation, our spectrofluorimetric approach is cost‐effective, uses minimal solvent, and aligns with green chemistry principles (Eco‐scale score: 88/100). Finally, while dansyl chloride is classified as a hazardous substance, NBD‐Cl is relatively safer and more user‐friendly.

Therefore, the novelty and significance of this work lie not merely in applying NBD‐Cl to SDN for the first time, but in providing a faster, greener, and simpler alternative to existing methods, with successful application to content uniformity testing—an aspect not previously reported for NBD‐Cl‐based SDN analysis.

Content uniformity testing is a critical quality control measure in the pharmaceutical industry, particularly for solid dosage forms like capsules. SDN, a medication used for treating certain autoimmune conditions, necessitates rigorous content uniformity testing to ensure consistent therapeutic efficacy and safety across each capsule. The suggested approach for assessing content uniformity combines sophisticated analytical methods, such as spectrofluorimetry, with statistical analysis. This method not only improves measurement accuracy but also makes it possible to identify any variations in the distribution of the active medicinal component within the capsules matrix. Because variations in medication content can have a substantial impact on treatment outcomes, putting such stringent testing processes into place is crucial for preserving regulatory compliance and guaranteeing patient safety. As seen in Table 6, the suggested approach for SDN capsule content uniformity testing thus emphasizes adherence to the USP standard.

TABLE 6.

Checking content uniformity of Flopadex capsules by the created approach.

Tablet number	% recovery	%RSD
1	101.07	1.01
2	99.05	0.82
3	100.70	1.47
4	98.06	1.47
5	96.03	0.95
6	97.10	2.58
7	95.05	0.66
8	98.40	0.41
9	102.45	1.95
10	98.75	1.02
Mean ($\overline{X}$) ± SD	98.80 ± 2.15
Acceptance value (AV)	5.17
Max. allowed AV (L1)	15

The acceptance value can be estimated from the following formula $\mathit{AV}=\left|M-\overline{X}\right|+\mathit{KS}$ where M is a reference value, K is the acceptability constant (equal to 2.4 in case of 10 capsules), S is the samples standard deviation, and is $\overline{X}$ the mean of the % recovery of individual content. The AV should be lower than the maximum allowed AV (L1 = 15). The above equation will be modified according to the value of $\overline{X}$.

• If 98.5% ≤ $\overline{X}$ ≤101.5%, then M = $\overline{X}$ (AV = ks)

• If $\overline{X}$< 98.5%, then M = 98.5% (AV = 98.5 –$\overline{X}$ + ks)

• If $\overline{X}$ > 101.5%, then M = 101.5% (AV = $\overline{X}$ – 101.5 + ks)

3.9.2. Method Greenness

Modern analytical chemistry is shifting toward sustainability, prioritizing procedures that safeguard human health and the environment. Therefore, any new method must be evaluated not only for its technical precision and accuracy but also for its “greenness” and overall environmental impact [32].

The analytical sustainability of the proposed method was evaluated using the Analytical Eco‐Scale, a quantitative framework that accounts for reagent toxicity, waste generation, energy consumption, and occupational exposure [94]. In accordance with the Globally Harmonized System (GHS), penalty points are assigned based on the specific hazard pictograms and signal words associated with each chemical used. Furthermore, the environmental footprint is assessed by calculating the energy demands of the analytical instrumentation and the efficiency of waste management protocols. The final Eco‐Scale score is derived by subtracting the cumulative penalties from an ideal value of 100. As detailed in Table S4 of the Supplementary Information, the developed spectrofluorimetric method achieved an excellent score of 88/100. This high value confirms the method's superior environmental compatibility and reinforces its classification as a robust “green” analytical approach.

Furthermore, the Analytical Greenness Metric (AGREE) was utilized as a contemporary tool for evaluating the environmental sustainability of the procedure [33]. A primary advantage of the AGREE framework is its ability to identify both the strengths and weaknesses of a method across the 12 principles of green analytical chemistry (GAC). Each of these 12 components is assessed on a normalized scale from 0 to 1, where a value of 1 signifies optimal greenness (represented in green) and 0 indicates poor environmental performance (represented in red). The developed spectrofluorimetric strategy achieved a high overall score of 0.72, demonstrating its robust alignment with sustainable laboratory practices (Figure S2).

4. Conclusion

This study successfully develops and validates a novel spectrofluorimetric method for quantifying SDN, employing NBD‐Cl as a fluorogenic derivatizing agent. The approach has exceptional sensitivity and precision, with a detection limit of 12.8 ng/mL and a quantitation limit of 38.8 ng/mL, rendering it appropriate for therapeutic monitoring in clinical environments. The stringent validation under ICH criteria demonstrates remarkable accuracy and consistency, with recoveries typically approximately 99.23%. Moreover, the use of this approach for both pharmaceutical capsules and content uniformity demonstrates its adaptability and efficacy in assessing SDN within intricate matrices. With this approach, expensive chromatographic techniques are no longer needed, which makes it much easier to implement and follows the principles of green chemistry. These findings testify to the potential of this technology to improve the monitoring and administering of medications with important impacts on pharmacokinetics and clinical lab analysis. Other researchers could build on these findings and broaden the scope of analyzed pharmaceutical substances, significantly improving the efficiency and analytical cost in the health sector.

Author Contributions

Ahmed A. Abu‐hassan: conceptualization, investigation, methodology, validation, data curation, writing – original draft, writing – review and editing.

Conflicts of Interest

The author declares no conflicts of interest.

Supporting information

Table S1: Selectivity assessment of the proposed method.

Table S2: Comparison of the proposed method with some reported method.

Table S3: Advantage of the proposed method over the previous reported method.

Table S4: The eco‐scale greenness valuating metric for the proposed spectrofluorimetric method.

Figure S1: Job's method for determination of stoichiometric ratio between SDN and NBD‐Cl.

Figure S2: Analytical Greenness metric using AGREE program for the proposed method.

Data Availability Statement

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