Development and Validation of a Capillary Zone Electrophoresis Method with Indirect UV Detection for the Simultaneous Determination of Azelaic Acid and Salicylic Acid in Pharmaceutical and Cosmetic Preparations

Abstract

Azelaic acid (AZA) and salicylic acid (SA) are widely used active ingredients in pharmaceutical and cosmetic formulations for the treatment of acne, rosacea, hyperpigmentation, and other dermatological conditions. Despite their frequent co-administration, analytical methods for their simultaneous determination remain limited. In the present study, a capillary electrophoresis (CE) method with indirect UV detection was developed and validated for the simultaneous determination of AZA and SA in pharmaceutical and cosmetic preparations. Preliminary experiments demonstrated that direct UV detection was unsuitable because of the weak UV absorbance of AZA; therefore, indirect UV detection based on a sodium benzoate background electrolyte (BGE) was used. Following an initial one-factor-at-a-time (OFAT) screening, method optimization was performed using a face-centered central composite design (CCD) to evaluate the effects of BGE concentration, BGE pH, and separation voltage on the separation. The optimum separation was achieved using a 30 mM sodium benzoate BGE at pH 6.5 containing 5% (v/v) methanol, a separation voltage of +18 kV, a capillary temperature of 20 °C, and indirect UV detection at 230 nm. Baseline separation of both analytes was achieved within 5 min. The method was validated according to the ICH Q2(R2) guideline and demonstrated satisfactory accuracy, linearity, precision, selectivity, sensitivity, and robustness. The developed procedure was successfully applied to the analysis of commercial cosmetic formulations containing AZA, SA, or both active ingredients, providing assay results consistent with the declared contents. The proposed CE method provides a simple, rapid, cost-effective, and environmentally friendly alternative for the routine quality control of pharmaceutical and cosmetic formulations containing AZA and SA.

1. Introduction

Azelaic acid (AZA) and salicylic acid (SA) are among the most frequently used active ingredients in topical pharmaceutical and cosmetic formulations for the management of acne vulgaris, rosacea, seborrheic dermatitis, post-inflammatory hyperpigmentation and other skin disorders associated with abnormal keratinization. Due to their distinct pharmacological properties and favorable safety profiles, both substances are extensively incorporated into prescription medicines, over-the-counter products and cosmetic formulations [1,2].

AZA (nonanedioic acid) (Figure 1a) is a naturally occurring saturated straight-chain dicarboxylic acid (C₉H₁₆O₄, molecular weight 188.22 g mol⁻¹) produced by Malassezia species and present in cereals such as wheat, rye and barley. Structurally, the molecule consists of two terminal carboxyl groups linked by a seven-carbon aliphatic chain, resulting in weak diprotic acidity (pK_a1 = 4.5, pK_a2 = 5.5) [3]. Although only sparingly soluble in water at room temperature, its solubility increases upon ionization in alkaline media. AZA exhibits antimicrobial, anti-inflammatory, antioxidant and antiproliferative activities and inhibits mitochondrial oxidoreductases and tyrosinase [3,4]. These properties contribute to the reduction in Cutibacterium acnes proliferation, normalization of follicular keratinization and inhibition of melanogenesis, supporting its use in the treatment of acne vulgaris, rosacea, melasma and post-inflammatory hyperpigmentation. Topical formulations generally contain 10–20% AZA and are available as creams, gels, foams and emulsions [1,4].

SA (2-hydroxybenzoic acid) (Figure 1b) is an aromatic β-hydroxy acid (C₇H₆O₃, molecular weight 138.12 g mol⁻¹) containing both a carboxylic acid and a phenolic hydroxyl group, with pKa values of approximately 2.97 and 13.6, respectively [5]. SA possesses keratolytic, comedolytic, exfoliating and mild anti-inflammatory properties. By reducing intercellular cohesion within the stratum corneum, it promotes desquamation, facilitates follicular unclogging and decreases comedone formation. SA is widely used in the treatment of acne vulgaris, seborrheic dermatitis, psoriasis, dandruff and other hyperkeratotic skin disorders [5,6]. In cosmetic products, it is frequently incorporated into exfoliating formulations and chemical peels intended to improve skin texture and reduce post-inflammatory pigmentation. Conventional topical formulations contain 0.5–5% SA, whereas considerably higher concentrations are employed in professional peeling procedures [2,6].

The chemical structures of AZA and SA are presented in Figure 1.

Due to their complementary mechanisms of action, the two compounds are frequently used within the same therapeutic regimen and are increasingly incorporated into pharmaceutical and cosmetic formulations designed for acne-prone and hyperpigmented skin [7,8]. The growing availability of formulations containing both active ingredients has increased the need for reliable analytical methods for their simultaneous determination in routine quality control.

RP-HPLC is currently the most widely employed analytical technique for the determination of organic acids in pharmaceutical and cosmetic formulations because of its robustness, sensitivity and broad applicability [9]. However, HPLC methods generally require relatively large volumes of organic solvents, longer analysis times and higher operational costs. Consequently, alternative analytical techniques that provide comparable analytical performance while reducing solvent consumption and environmental impact remain of considerable interest.

Although several analytical procedures have been developed for the individual determination of AZA and SA, reports describing their simultaneous determination remain scarce and are limited to reversed-phase high-performance liquid chromatography (RP-HPLC) [10].

Capillary electrophoresis (CE) is a powerful analytical technique for the separation of ionic compounds and has been successfully applied to the analysis of pharmaceutical and cosmetic ingredients. Its high separation efficiency, short analysis time and minimal consumption of reagents make CE an attractive alternative to conventional chromatographic methods. Furthermore, the low volumes of background electrolyte (BGE) and samples required for analysis considerably reduce solvent consumption and waste generation, supporting the implementation of green analytical chemistry principles. These characteristics make CE suitable for routine quality control of active pharmaceutical and cosmetic ingredients and complex formulations [11,12].

Several CE methods have been reported for the determination of AZA, SA, or related compounds. Urbánek et al. developed a capillary zone electrophoresis (CZE) method with indirect UV detection for the simultaneous determination of AZA and sorbic acid in pharmaceutical cream using a benzoate BGE, demonstrating that indirect UV detection enables the direct analysis of AZA without derivatization [13]. Adler & Sirén described a CE method with indirect UV detection for the determination of homologous dicarboxylic acids, including AZA, in atmospheric aerosol samples after solid-phase extraction (SPE) [14]. Gómez et al. reported a CZE method for the simultaneous determination of SA, chloramphenicol, and resorcinol in anti-acne formulations [15], while Liu et al. developed a CD-modified CE method for seven cosmetic hydroxy acids, including SA, using chemometric optimization [16].

CE has also been successfully applied to cosmetic preservatives and related compounds. Micellar electrokinetic chromatography (MEKC) and CZE methods have been reported for the simultaneous determination of cosmetic preservatives, whitening agents, parabens, and related compounds, with SA included in all the reported methods [17,18,19,20]. More recently, Măgerușan et al. developed and validated a CZE method for the determination of SA in pharmaceutical and cosmetic formulations [21].

To provide a concise overview of the existing literature, the main characteristics of previously reported CE methods relevant to the determination of AZA, SA, and related compounds are summarized in

Table 1. Overview of previously reported CE methods for the determination of AZA, SA, and related analytes.

Ref.	Analytes	CE Mode Detection	Separation Conditions	Matrix	Analysis Time	Remarks
[13]	AZA + sorbic acid	CZE, indirect UV	30 mM benzoate BGE, pH~6 (adjusted with TRIS), 7 mM β-CD, 5% MeOH	Pharmaceutical cream	<15 min	AZA determination by indirect UV
[14]	C2–C10 dicarboxylic acids (including AZA)	CE, indirect UV	4 mM 2,6-pyridinedicarboxylic acid, 0.5 mM myristyltrimethylammonium hydroxide, pH 11	Atmospheric aerosols	<5 min	Environmental analysis SPE
[15]	SA + chloramphenicol + resorcinol	CZE, UV detection	50 mM sodium tetraborate/50 mM sodium phosphate BGE, pH 9.0	Anti-acne formulations	<6 min	Pharmaceutical formulations
[16]	Seven hydroxy acids (including SA)	CD-modified CE, UV detection	150 mM phosphate BGE, 0.5 mM CTAB, 3 mM γ-CD, 25% MeOH, pH 7.0	Cosmetic products	<10 min	Chemometric optimization (FFD + CCD)
[17]	Preservatives (including SA)	MEKC, UV detection	5 mM sodium tetraborate, 60 mM boric acid, 100 mM SDS, pH 8.3	Cosmetic products	<14 min	Eleven analytes analyzed simultaneously
[18]	Preservatives (including SA)	DLLME-CZE, UV detection	30 mM sodium tetraborate BGE, 12% ACN, pH 9.3	Cosmetic products	~12 min	DLLME coupled with CZE
[19]	Whitening agents (including SA)	MEKC, amperometric detection	40 mM sodium tetraborate BGE, 10 mM SDS, pH 9.0	Cosmetic products	~16 min	Six phenolic analytes
[20]	Whitening agents + parabens (including SA)	Sweeping MEKC, UV detection	15 mM sodium tetraborate BGE, 40 mM SDS, 0.100% PEO, pH 8.5	Cosmetic products	<10 min	Online sweeping preconcentration
[21]	SA	CZE, UV detection	25 mM sodium tetraborate BGE, pH 9.3	Pharmaceutical & cosmetic formulations	~3.5 min	Determination of SA only

ACN—acetonitrile; AZA—azelaic acid; β-CD—beta-cyclodextrin; γ-CD—gamma-cyclodextrin; BGE—background electrolyte; CCD—central composite design; CTAB—cetyltrimethylammonium bromide; CZE—capillary zone electrophoresis; DLLME—dispersive liquid–liquid microextraction; FFD—fractional factorial design; MEKC—micellar electrokinetic chromatography; MeOH—methanol; PEO—poly(ethylene oxide); SA—salicylic acid; SDS—sodium dodecyl sulfate; SPE—solid-phase extraction; TRIS—tris(hydroxymethyl)aminomethane.

.

These studies demonstrate the applicability of CE for the analysis of pharmaceutical and cosmetic ingredients using different separation modes and BGE. To the best of our knowledge, no CE method has previously been reported for the simultaneous determination of AZA and SA. Existing CE methods address either AZA or SA individually or together with unrelated compounds, whereas simultaneous analysis of these two widely co-administered dermatological agents has not yet been described.

The aim of the present study was to develop and validate a new simple, rapid, cost-effective and environmentally friendly CE method with UV detection for the simultaneous determination of AZA and SA in pharmaceutical and cosmetic preparations.

2. Materials and Methods

2.1. Analytes and Reagents

Pharmaceutical-grade AZA and SA were purchased from Fagron (Athens, Greece). Analytical-grade reagents were used throughout the study, including sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium tetraborate and sodium dodecyl sulfate (SDS) (Merck, Darmstadt, Germany), sodium benzoate (Fagron, Athens, Greece), methanol (MeOH) and sodium hydroxide (NaOH) (Lach-Ner, Neratovice, Czech Republic). Double-distilled deionized water obtained from a Millipore purification system (Millipore, Burlington, MA, USA) was used throughout the experiments. Commercial pharmaceutical and cosmetic preparations were purchased from local pharmacies.

2.2. Instrumentation and Software

Electrophoretic analyses were performed using an Agilent 1600 CE system equipped with a diode-array detector (DAD) (Agilent Technologies, Waldbronn, Germany). Electropherograms were acquired and processed using ChemStation software (version 7.01, Agilent Technologies). Separations were carried out in uncoated fused-silica capillaries (50 μm i.d., 40 cm total length, 32 cm effective length; Agilent Technologies). The pH of the BGEs was measured using a Terminal 740 pH meter (Inolab, Dortmund, Germany).

Design-Expert software (version 7.0, Stat-Ease Inc., Minneapolis, MN, USA) was used for the experimental design, response surface modelling, statistical analysis, and optimization of the electrophoretic conditions. Analysis of variance (ANOVA), evaluation of factor interactions, and generation of response surface and diagnostic plots were performed using the same software. Microsoft Excel (Microsoft 365, Microsoft Corp., Redmond, WA, USA) was used for the statistical analysis of the method validation data.

2.3. Electrophoretic Conditions

New capillaries were conditioned by flushing with 1 M NaOH for 30 min, followed by 0.1 M NaOH for 15 min and purified water for 15 min. Before each run, the capillary was rinsed with 0.1 M NaOH for 2 min, purified water for 1 min and finally with the BGE for 1 min.

The investigated BGEs (25–50 mM sodium benzoate) were freshly prepared by dissolving the appropriate amount of sodium benzoate in purified water. The pH was adjusted to the desired value (5.5–7.5) using 1 M NaOH, and MeOH (0–20%, v/v) was added where required.

Individual stock solutions of AZA and SA (1.0 mg mL⁻¹) were prepared separately in a MeOH-water (1:1, v/v) mixture and stored at 4 °C. Mixed working standard solutions containing both analytes were freshly prepared by combining equal volumes of the individual stock solutions and subsequently diluting the mixture with MeOH-water (1:1, v/v) to obtain the desired concentrations for calibration and validation experiments.

Prior to analysis, all the samples and BGEs were sonicated for 3 min. and filtered through 0.45 μm PTFE membrane filters (Millipore, Bedford, MA, USA). Preliminary experiments were performed under the following electrophoretic conditions: 25 mM BGE, capillary temperature 20 °C, separation voltage +20 kV, and hydrodynamic injection at the anodic end (50 mbar × 1 s).

During preliminary experiments, direct UV detection was evaluated at 210, 230 and 300 nm. However, because AZA showed weak UV absorption, indirect UV detection was selected for further development. UV detection was performed in indirect mode at 230 nm, using a UV-absorbing benzoate BGE. The detection wavelength was selected according to the absorption characteristics of the benzoate co-ion while ensuring adequate signal-to-noise ratio and baseline stability.

2.4. Sample Preparation

The applicability of the proposed method was evaluated by analyzing four commercially available topical cosmetic formulations containing AZA and/or SA. The investigated products included: (i) a 10% salicylic acid topical solution (Sample 1); (ii) Radiance Peel, a multi-acid chemical peel containing glycolic acid (34%), citric acid (10%), lactic acid (10%), kojic acid (5%), and salicylic acid (3%) (Sample 2); (iii) Purifying Peel, a topical solution containing azelaic acid (14%) and salicylic acid (12%) (Sample 3); and (iv) Night Peel with Azelaic Acid, containing azelaic acid (10%), mandelic acid (5%), and glycolic acid (3.5%) (Sample 4).

The concentrations of the active ingredients declared by the manufacturers were used solely to estimate the dilution factors required to prepare sample solutions within the validated concentration range. Because the manufacturers did not specify whether the declared percentages were expressed as w/w or w/v, the values were accepted as provided for this purpose only. The commercial products were analyzed to demonstrate the applicability of the developed method to real pharmaceutical and cosmetic formulations rather than to assess the agreement between the declared and experimentally determined concentrations.

An aliquot of 1.0 mL of each sample was transferred into a 100 mL volumetric flask, diluted to volume with a MeOH-water (1:1, v/v) mixture, and sonicated for 5 min. to ensure complete homogenization. Appropriate aliquots of the resulting solutions were further diluted to obtain analyte concentrations within the validated linear range. Prior to electrophoretic analysis, all the sample solutions were filtered through 0.45 μm PTFE membrane filters. All the analyses were performed in triplicate.

3. Results and Discussion

3.1. Preliminary Analysis

Preliminary experiments were carried out to evaluate the feasibility of the simultaneous determination of AZA and SA and to establish suitable electrophoretic conditions.

Initially, direct UV detection was evaluated. Although SA exhibited strong UV absorption and could be readily detected, AZA showed only weak intrinsic UV absorbance under the investigated conditions, resulting in poor sensitivity and inadequate detectability. Consequently, direct UV detection was considered unsuitable for the simultaneous determination of the two analytes, and indirect UV detection was investigated as an alternative approach.

Several BGEs were evaluated during preliminary method development, including phosphate, borate, phosphate-borate, MEKC systems based on sodium tetraborate and SDS, and benzoate-based electrolytes. Among the tested electrolytes, benzoate BGE was selected because it provides sufficient UV absorbance, enabling indirect detection of weakly absorbing compounds without derivatization. In addition, the benzoate-based BGE produced a stable baseline, satisfactory peak shapes, good migration-time reproducibility, and adequate separation of both analytes, and was therefore selected for further optimization.

Although several chromophoric co-ions have been reported for indirect UV detection, sodium benzoate was selected because its electrophoretic mobility is comparable to that of the investigated analytes, which is an important prerequisite for efficient indirect UV detection and minimized electromigration dispersion [22].

Indirect UV detection is particularly suitable for analytes that exhibit little or no intrinsic UV absorbance. In this detection mode, the BGE contains a UV-absorbing chromophoric co-ion that generates a stable absorbance signal. As the analytes migrate through the detection window, they temporarily displace the chromophoric co-ion, producing a transient decrease in absorbance that is recorded as a negative peak. The magnitude of the signal depends on the analyte concentration, the difference in molar absorptivity between the analyte and the chromophoric co-ion, and the compatibility of their electrophoretic mobilities [23].

In the benzoate BGE, the electrophoretic behavior of AZA and SA is governed by their acid-base properties and effective electrophoretic mobilities. Under alkaline conditions, AZA is predominantly present as a dianion, whereas SA migrates as the salicylate anion. Differences in molecular structure, charge, and charge-to-size ratio result in distinct effective electrophoretic mobilities, allowing their separation under the selected electrophoretic conditions. The benzoate BGE therefore fulfills a dual role, acting both as the UV-absorbing co-ion required for indirect detection and as the BGE providing suitable selectivity for the simultaneous separation of AZA and SA.

Under normal polarity, both analytes migrated toward the detector with the electroosmotic flow (EOF), while their own electrophoretic mobility was directed toward the anode. SA migrated before AZA, most likely because AZA, being predominantly dianionic at pH 5.5–7.0, exhibited a higher effective anionic mobility opposing the EOF and therefore a longer migration time.

3.2. Method Optimization

For method optimization a sequential one factor at a time (OFAT)—central composite design (CCD) strategy was adopted, whereby OFAT was first used to identify the critical experimental variables, followed by face-centered CCD to optimize these factors simultaneously and evaluate their interactions and quadratic effects.

OFAT screening was performed to identify the most influential variables before multivariate optimization. The effects of BGE composition and pH, MeOH content, separation voltage, capillary temperature, and injection parameters on the separation performance were investigated.

For each experimental parameter, three representative levels were evaluated during method optimization. The investigated variables included BGE concentration (25, 37.5, 50 mM), BGE pH (5.5, 6.5, 7.5), MeOH content (0, 10, 20%), separation voltage (15, 20, 25 kV), capillary temperature (15, 20, 25 °C), and hydrodynamic injection conditions (30, 40, 50 mbar/1, 2, 3 s).

Increasing the BGE concentration from 25 to 50 mM resulted in longer migration times for both analytes. This behavior can be attributed primarily to the higher ionic strength of the BGE, which reduces the EOF. Although higher BGE concentrations improved peak symmetry and resolution, they also increased the analysis time and generated higher current.

Increasing the BGE pH from 5.5 to 7.5 resulted in shorter migration times for both analytes due to the increase in EOF. Among the investigated conditions, pH 6.5 provided the best compromise between separation efficiency and analysis time. At this pH, SA is fully ionized, whereas AZA is predominantly present in its dianionic form, resulting in sufficiently different electrophoretic mobilities to achieve baseline separation. At lower pH values, the reduced EOF prolonged the migration times, whereas at higher pH values, the increased EOF shortened the analysis but slightly reduced the resolution.

The influence of MeOH concentration (0–20%, v/v) as BGE additive on the electrophoretic separation was investigated. Increasing the MeOH content resulted in longer migration times for both analytes owing to the increased viscosity and reduced dielectric constant of the BGE, which decreased both the EOF and the electrophoretic mobilities of the analytes. MeOH concentrations above 10% (v/v) considerably prolonged the analysis time without providing a significant improvement in resolution.

The effect of the separation voltage was evaluated in the range of +15–+25 kV. Increasing the voltage resulted in shorter migration times for both analytes due to the higher electric field strength. However, higher voltages also generated increased current and potential Joule heating, which may negatively affect peak shape and separation efficiency.

The influence of capillary temperature was investigated over the range of 15–25 °C. Increasing the temperature resulted in shorter migration times for both analytes owing to the lower viscosity of the BGE, which increased both the EOF and the electrophoretic mobilities of the analytes. However, higher temperatures also led to a minor decrease in separation efficiency.

The influence of the hydrodynamic injection parameters was investigated by varying the injection pressure (30–50 mbar) and injection time (1–3 s). Increasing either parameter increased the injected sample volume, resulting in higher detector response and improved sensitivity. However, excessive sample loading caused peak broadening, deterioration of peak symmetry, and a reduction in separation efficiency and resolution. Lower injection pressures and shorter injection times produced sharper peaks and better separation but lower signal intensity.

Following the preliminary OFAT screening experiments, BGE pH, BGE concentration, and separation voltage were identified as the critical method parameters and were therefore selected for subsequent design of experiments (DoE) optimization. Capillary temperature (20 °C), MeOH content (5%, v/v), and hydrodynamic injection conditions (50 mbar × 1 s) were kept constant throughout the optimization, as preliminary experiments demonstrated that their effects were mainly limited to analysis time and detector response, with only a minor influence on separation selectivity.

The selected factors were subsequently optimized using a face-centered CCD to establish the optimum electrophoretic conditions for the simultaneous determination of AZA and SA. The analytical responses selected for the DoE optimization were resolution between AZA and SA (R_s) and migration time of the last-migrating analyte (t_m). Resolution was selected as the critical response related to separation selectivity, whereas migration time was used to evaluate analysis speed.

CCD was selected because it allows simultaneous evaluation of the effects of the selected factors, their interactions, and quadratic terms, while providing efficient response surface modelling and optimization of the electrophoretic conditions with a limited number of experimental runs [24].

A three-factor, three-level DoE was generated, with BGE concentration (30–50 mM), BGE pH (5.5–7.5), and separation voltage (+15–+25 kV) selected as the independent variables. The experimental matrix consisted of 15 runs, including five replicated center points (40 mM BGE, pH 6.5 and +20 kV), allowing estimation of the experimental error and evaluation of the adequacy of the quadratic response surface model. The experimental design matrix is presented in

Table 2. Experimental design matrix and corresponding analytical responses obtained for the face-centered CCD.

Run	BGE Conc [mM]	BGE pH	Voltage [kV]	Rs	tm [min]
1	40.00	5.50	20.00	1.86	5.92
2	40.00	6.50	25.00	1.77	5.04
3	30.00	6.50	20.00	2.04	5.38
4	40.00	6.50	20.00	1.92	5.61
5	50.00	7.50	15.00	1.85	7.05
6	50.00	6.50	20.00	1.88	5.98
7	30.00	5.50	15.00	1.79	6.22
8	40.00	6.50	20.00	1.90	5.63
9	40.00	6.50	20.00	1.94	5.65
10	30.00	7.50	25.00	1.68	4.58
11	40.00	6.50	20.00	1.94	5.68
12	40.00	7.50	20.00	1.80	5.22
13	40.00	6.50	20.00	1.96	5.66
14	40.00	6.50	15.00	1.88	6.22
15	50.00	5.50	25.00	1.75	5.12

.

Based on the face-centered CCD, second-order polynomial models were established to describe the relationships between the investigated experimental factors and the selected analytical responses. For model construction, the independent variables were coded as A (BGE concentration), B (BGE pH), and C (separation voltage). The fitted quadratic models describing the effects of the investigated factors on R_s and t_m are presented in Equations (1) and (2), respectively.

R_s = 1.93 − 0.080A − 0.053C − 0.11BC + 0.034A² − 0.096B² − 0.10C²

(1)

t_m = 5.63 + 0.33A − 0.35B − 0.59C + 0.30AB − 0.42AC + 0.096A²

(2)

For R_s, the model indicated that BGE concentration, voltage, the interaction between pH and voltage, and the quadratic effects of all three factors contributed to the separation performance. In contrast, t_m was primarily affected by the linear effects of BGE concentration, BGE pH, and voltage, together with the interactions between BGE concentration and pH, and between BGE concentration and voltage.

The quadratic models developed for both analytical responses exhibited excellent goodness-of-fit. For R_s, the model generated an R² value of 0.9671; the adjusted R² (0.9424) was in good agreement with the predicted R² (0.9136), confirming satisfactory predictive ability and the absence of significant overfitting. An even better fit was obtained for t_m, with R², adjusted R², and predicted R² values of 0.9961, 0.9932, and 0.9690, respectively. The close agreement between the adjusted and predicted R² values for both responses (difference < 0.20) demonstrates the robustness and high predictive capability of the models. These results confirm that the quadratic polynomial equations adequately describe the relationships between the investigated electrophoretic factors and the analytical responses within the studied design space.

For each analytical response, three-dimensional response surface plots were generated (Figure 2 for R_s, Figure 3 for t_m). These plots illustrate the interactions between two experimental factors and their combined effect on the monitored response, while the third factor was maintained at its central level.

Numerical optimization was performed using the desirability function with the objective of minimizing migration time while maximizing resolution. Both responses were assigned equal importance. The optimal electrophoretic conditions predicted by the model were a BGE concentration of 30 mM, a BGE pH of 6.5, and a voltage of +18 kV. Experimental verification performed under these conditions confirmed the validity of the optimization, as the experimentally obtained migration time and resolution were in close agreement with the predicted values, demonstrating the adequacy and predictive capability of the developed models.

The optimized electrophoretic conditions consisted of a 30 mM sodium benzoate BGE (pH 6.5) containing 5% (v/v) MeOH, a separation voltage of +18 kV, a capillary temperature of 20 °C, and hydrodynamic injection at the anodic end (50 mbar × 1 s). Indirect UV detection was performed at 230 nm. Under these conditions, baseline separation of SA and AZA was achieved within 5 min, with SA migrating before AZA. A representative electropherogram obtained under the optimized conditions is presented in Figure 4. Negative peaks arise because analyte ions temporarily displace the UV-absorbing benzoate co-ion from the detection zone, producing a transient decrease in absorbance characteristic of indirect UV detection.

The environmental performance of the developed CE method was evaluated using the AGREE metric [25], which assesses analytical procedures according to the twelve principles of Green Analytical Chemistry. The method achieved an overall AGREE score of 0.71, indicating a favorable greenness profile. The AGREE assessment, including the individual evaluation of each of the twelve principles, is provided in Figure 5, allowing independent verification of the greenness assessment. The obtained score reflects the low solvent consumption, minimal waste generation, and high analytical efficiency of the proposed CE method and is comparable to or higher than those generally reported for conventional RP-HPLC methods.

3.3. Method Validation

The analytical performance of the optimized CE method was evaluated in terms of precision (repeatability and intermediate precision), linearity, limits of detection (LOD) and quantification (LOQ), accuracy, and robustness, following the recommendations of the ICH Q2(R2) guideline.

Repeatability (intra-day precision) was assessed by performing six consecutive injections of mixed standard solutions containing AZA and SA at three concentration levels (0.05, 0.25, and 0.50 mg mL⁻¹) within a single day. Intermediate precision (inter-day precision) was evaluated using the same concentration levels by performing six replicate injections per day over three consecutive days. Precision was expressed as the relative standard deviation (RSD, %) of migration times and peak areas.

Method linearity was evaluated over the concentration range of 0.025–0.50 mg mL⁻¹ for both analytes using eight concentration levels, each analyzed in triplicate. Calibration curves were constructed by plotting the corrected peak area against analyte concentration, and the corresponding regression equations and correlation coefficients were calculated.

The LOD and LOQ were estimated from the standard deviation of the intercept and the slope of the calibration curve, using multiplication factors of 3.3 and 10, respectively.

Representative electropherograms recorded at the validated LOQ concentrations are provided in the Supplementary Materials (Figure S1), demonstrating adequate signal intensity and baseline resolution for reliable quantification.

Accuracy was evaluated by the standard addition method using a previously analyzed cosmetic sample. Recovery studies were performed using Sample 3, the only commercial formulation containing both AZA and SA, as it represented the most complex sample matrix included in the study. Known amounts of both reference standards were added to the sample solution at three concentration levels (80, 100, and 120% of the nominal analyte concentration), and each level was analyzed in triplicate. The results were expressed as mean percentage recovery ± SD.

Selectivity was demonstrated by the analysis of cosmetic sample solutions containing both substances, which showed no interfering peaks at the migration times of AZA and SA. Baseline separation of both analytes was achieved under the optimized electrophoretic conditions, confirming the suitability of the method for their simultaneous determination.

The validation results are summarized in

Table 3. Validation parameters of the CE method developed for the simultaneous determination of AZA and SA.

Parameter	AZA			SA
	Conc. (mg mL−1)	RSD (%) tm	RSD (%) Area	Conc. (mg mL−1)	RSD (%) tm	RSD (%) Area
Intra-day precision (n = 6)	0.05	0.07	0.74	0.05	0.04	0.62
	0.25	0.05	0.68	0.25	0.04	0.56
	0.5	0.05	0.56	0.5	0.05	0.52
Inter-day precision (n = 18)	0.05	0.18	0.98	0.05	0.15	0.72
	0.25	0.16	0.83	0.25	0.12	0.57
	0.5	0.12	0.76	0.5	0.13	0.54
Accuracy (n = 3) (concentration 0.25 mg mL−1)	Conc. (mg mL−1)	Recovery (%)		Conc. (mg mL−1)	Recovery (%)
	0.20	96.38 ± 0.18		0.20	98.21 ± 0.12
	0.25	97.33 ± 0.17		0.25	98.33 ± 0.21
	0.30	98.21 ± 0.23		0.30	98.72 ± 0.31
Linearity	AZA			SA
Range (mg mL−1)	0.025–0.50			0.025–0.50
Regression equation	y = 163.52x + 0.3075			y = 207.69x + 3.1708
Coefficient of determination (R2)	0.997			0.998
LOD (mg mL−1)	0.007			0.006
LOQ (mg mL−1)	0.021			0.018

. All the validation parameters fulfilled ICH acceptance criteria.

Robustness was evaluated by assessing the effect of small deliberate variations in experimental conditions on migration times and confirmed using a Plackett–Burman design (PBD) (

Table 4. PBD matrix used to evaluate the robustness of the developed CE method.

Run	BGE Conc. [mM]	MeOH [%]	Voltage [kV]	Temperature [°C]	Rs	tm [min]
1	30.00	5.00	18.00	20.00	2.46	5.18
2	28.00	4.00	16.00	18.00	2.41	5.12
3	28.00	6.00	20.00	18.00	2.38	5.08
4	28.00	6.00	20.00	22.00	2.36	5.04
5	32.00	6.00	16.00	18.00	2.44	5.31
6	28.00	4.00	16.00	22.00	2.39	5.24
7	28.00	6.00	16.00	22.00	2.35	5.20
8	30.00	5.00	18.00	20.00	2.46	5.19
9	32.00	4.00	20.00	22.00	2.43	5.20
10	32.00	4.00	16.00	18.00	2.44	5.54
11	30.00	5.00	18.00	20.00	2.44	5.18
12	32.00	4.00	20.00	22.00	2.42	5.16
13	32.00	6.00	16.00	22.00	2.40	5.35
14	32.00	6.00	20.00	18.00	2.41	5.26
15	28.00	4.00	20.00	18.00	2.40	5.16

). PBD was used because it enables simultaneous evaluation of multiple small variations with a limited number of experiments [24]. The examined factors included BGE concentration (30 ± 2 mM), MeOH content (5 ± 1%, v/v), separation voltage (18 ± 2 kV), and capillary temperature (20 ± 2 °C). The pH of the BGE was considered a controlled preparation parameter and was therefore not included in the PBD robustness study. Its effect had already been systematically investigated during CCD optimization, whereas robustness testing focused on routine operational variables. Although small deliberate variations in the selected parameters affected migration time to some extent, baseline separation was maintained in all the experimental runs, confirming the practical robustness of the method.

The developed method was successfully applied to the determination of AZA and SA in commercially available cosmetic formulations containing either a single active ingredient or both compounds. A representative electropherogram obtained from Sample 3, containing both AZA and SA, is presented in Figure 6. No interfering peaks originating from the sample matrix were observed at the migration times of the analytes, demonstrating the adequate selectivity of the method. The assay results obtained for the analyzed samples are summarized in

Table 5. Assay results for AZA and SA in commercial formulations.

Sample	Declared Content (mg mL−1)		Found Content (mg mL−1) (±SD, n = 3)		Assay (% of Label Claim) (±SD, n = 3)
	AZA	SA	AZA	SA	AZA	SA
Sample 1		100		98.6 ± 0.81		98.6 ± 0.81
Sample 2		30		30.2 ± 0.19		100.7 ± 0.63
Sample 3	140	120	139.2 ± 0.32	119.5 ± 0.40	99.4 ± 0.23	99.6 ± 0.33
Sample 4	100		98.8 ± 0.69		98.8 ± 0.69

Declared contents were converted to mg mL⁻¹ assuming % (w/v) labeling for the liquid topical formulations, as the manufacturers did not specify whether the declared percentages were expressed as w/v or w/w. Values are expressed as mean ± standard deviation (SD, n = 3).

.

The determined contents of AZA and SA were in good agreement with the label claims, with satisfactory recoveries and acceptable repeatability, confirming the applicability of the proposed method for routine quality control of cosmetic formulations.

4. Conclusions

A CE method with indirect UV detection was successfully developed and validated for the simultaneous determination of AZA and SA in cosmetic preparations. Because AZA exhibits only weak intrinsic UV absorbance, indirect UV detection using a sodium benzoate BGE enabled its reliable determination without derivatization. Experimental conditions were optimized using a combination of OFAT screening and a face-centered CCD, resulting in baseline separation of both analytes within 5 min.

The method fulfilled the validation requirements of the ICH Q2(R2) guideline, demonstrating satisfactory accuracy, linearity, precision, selectivity, sensitivity, and robustness. Its applicability was confirmed through the analysis of commercial cosmetic formulations containing either one or both active ingredients, with the assay results in good agreement with the declared contents. In addition, the low consumption of reagents and favorable AGREE score support the environmental sustainability of the proposed procedure.

To the best of our knowledge, this is the first CE method developed and validated for the simultaneous determination of AZA and SA in pharmaceutical and/or cosmetic preparations. Unlike previously reported CE methods addressing either AZA or SA individually, the proposed procedure enables simultaneous determination of both active ingredients using a single indirect UV method suitable for routine quality control.

A limitation of the present study is that the method was evaluated only using commercial cosmetic formulations. Further studies involving a broader range of pharmaceutical dosage forms and more complex matrices would provide additional evidence of its applicability.

The developed method provides a simple, rapid, cost-effective, and environmentally friendly alternative for the routine quality control of cosmetic formulations containing AZA and SA.