Development of a reverse-phase HPLC method for the simultaneous determination of curcumin and dexamethasone in polymeric micelles

Abstract

Background

A rapid, simple, accurate, and robust reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed to quantify curcumin and dexamethasone in polymeric micelle nanoparticle formulations simultaneously.

Methods

The optimized chromatographic conditions involved a Universal HS C18 column, isocratic elution with a methanol: acidic water (pH 3.5, 80:20, v/v) mobile phase, and detection wavelengths of 425 nm for curcumin and 254 nm for dexamethasone. The developed method was subsequently validated according to ICH guidelines, and demonstrated excellent linearity (R² > 0.999), precision (RSD% < 2%), and accuracy (mean recovery was 98.7% for curcumin and 101.7% for dexamethasone).

Results

The limits of detection (LOD) were 0.0035 mg/mL for curcumin and 0.0029 mg/mL for dexamethasone, while limits of quantification (LOQ) were 0.0106 mg/mL for curcumin and 0.0088 mg/mL for dexamethasone, respectively. The method was applied to evaluate the encapsulation efficiency (EE%) of curcumin and dexamethasone into polymeric micelle nanoparticles formulated using Soluplus^® and DOPE in a 1:10 molar ratio. EE% values were 78.84 ± 0.05% for curcumin and 54.33 ± 0.05% for dexamethasone.

Conclusions

the current developed method indicates suitability for the simultaneous determination of curcumin and dexamethasone, thereby facilitating the quality control and optimization of such advanced drug delivery systems.

PLAIN LANGUAGE SUMMARY

Cancer therapies frequently employ poorly water-soluble small molecules, thereby hampering their bioavailability and, consequently, therapeutic effectiveness. Both curcumin—an abundant polyphenol derived from turmeric—and dexamethasone, a widely antineoplastic anti-inflammatory glucocorticoid, fall within this class. To enhance their therapeutic window, we engineered sub-50-nanometer self-assembled amphiphilic polystyrene–b–poly (ethylene oxide) polymeric micelles capable of hosting curcumin and dexamethasone within their hydrophobic cores. An auxiliary high-performance liquid chromatography (HPLC) protocol was subsequently validated for simultaneous dual quantitation, thereby enabling rapid loading characterization. Analytical metrics, including calibration curves, limits of detection, and inter- and intraday accuracy, support a standard deviation of less than 5% at sub-micromolar concentrations. When this validated pipeline was directed at the pro-formulated micellar pools, quantification revealed a 79% drug loading capacity for curcumin and a 54% loading for dexamethasone. By delivering quantitation of both payloads in a single analytical scan, this approach obviates solvent macroscale extraction and reproducibly accelerates formulation refinement cycles. Consequently, the proposed HPLC analytical interface establishes a rapid and cost-effective platform amenable to the rational design and iterative optimization of multifunctional amphiphilic drug delivery systems, laying a preclinical groundwork for subsequent combination regimens that leverage curcumin and dexamethasone in oncologic applications.

ARTICLE HIGHLIGHTS

1. We report an efficient RP-HPLC procedure for determining curcumin and dexamethasone concentrations in polymeric micellar formulations.

2. An isocratic system employing an 80:20 methanol:acidic water mixture provides complete resolution in under 7 minutes of column occupancy.

3. Comprehensive validation, adhering to ICH Q2(R1) criteria, substantiates an excellent linear correlation (R² exceeds 0.999), repeatability, and intermediate precision (RSD < 2%), as well as recoveries of 98.7% for curcumin and 101.7% for dexamethasone.

4. Detectability and quantification limits were established at sub-nanomolar levels for each compound, confirming the method’s adequate sensitivity.

5. Analysis of Soluplus/DOPE micelles yielded encapsulation efficiencies of 78.8% for curcumin and 54.3% for dexamethasone, indicating the system’s potential for co-delivery.

6. By combining robustness, rapidity, and low solvent consumption, the proposed RP-HPLC strategy serves as a convenient platform for quality control and optimization of polymeric nanocarrier formulations in translational investigations.

1. Introduction

One of the major challenges in effective cancer therapy is the poor aqueous solubility of many potent chemotherapeutic and supportive agents, which limits their bioavailability, therapeutic efficacy, and clinical application. The design of drug delivery systems based on nanoparticles has emerged as a powerful strategy to address these limitations by enhancing solubility, improving pharmacokinetics, and enabling targeted delivery [1].

Polymeric micelles are nanoscale colloidal carriers formed by the self-assembly of amphiphilic block copolymers in aqueous environments. They typically exhibit a core-shell architecture, where the hydrophobic core serves as a reservoir for poorly water-soluble drugs, while the hydrophilic shell stabilizes the structure in biological fluids [2]. Mixed micelles, composed of two or more amphiphilic components—such as polymers and lipids—offer enhanced structural versatility and loading capacity compared to single-polymer micelles [3]. This hybrid architecture enables improved solubilization, enhanced encapsulation efficiency, and controlled release of hydrophobic drugs [3]. Due to their small size, biocompatibility, and tunable properties, mixed polymeric micelles have attracted considerable attention as effective nanocarriers for delivering poorly soluble chemotherapeutic agents in cancer treatment [3].

Curcumin or [(1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)- 1,6-heptadiene-3,5-dione] (Figure 1) is a polyphenolic compound extracted from the rhizomes of Curcuma longa species; a traditionally used plant known as turmeric [4,5]. Curcumin interacts with several molecular targets and pathways in the body, therefore exerting numerous biological activities [6]. For example, it significantly inhibits nuclear factor kappa-B (NF-κB), a transcriptional factor responsible for cell survival, cytokine production, and other cellular functions, thereby reducing inflammation and associated tissue damage [7–9]. It also suppresses some enzymes that play a role in inflammatory response, including cyclooxygenase-2 (COX-2) and 5-lipoxygenase (LOX) [6,10]. Curcumin has been shown to have powerful antitumor effects and is found to regulate multiple tumor-signaling pathways, including cell proliferation, inflammation, angiogenesis, and metastasis [11–13]. For example, it suppresses the Wnt/β-catenin pathway [14], modulates STAT transcriptional proteins [15], and induces apoptosis in cancer cells by targeting protein kinases and pathways such as the Akt/mTOR cascade [16]. Curcumin is also known for its antioxidant [6] and antimicrobial [17] effects.

Figure 1.

Chemical structure of curcumin and dexamethasone.

Dexamethasone, a synthetic glucocorticoid, is recognized for its potent anti-inflammatory and immunosuppressive actions, allowing it to play a significant role in cancer therapy. It inhibits pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β), and suppresses the activation of NFκB [18]. These effects contribute to its ability to enhance the efficacy of chemotherapeutic agents by modulating cytokine expression and facilitating increased drug accumulation within tumors [19,20]. At high doses, dexamethasone was shown to modulate the tumor microenvironment, inhibiting the accumulation of most immune cells and downregulating glucose and lipid consumption, thereby inducing apoptosis and inhibiting tumor growth in animal xenograft models [21]. Additionally, dexamethasone is employed in various oncological treatments to mitigate adverse effects such as chemotherapy-induced nausea and vomiting while acting as a chemosensitizer to potentiate antitumor responses [19,22].

Given their aforementioned health benefits, a combination of curcumin and dexamethasone appears to be a promising therapeutic strategy, particularly in cancer treatment, as dexamethasone reduces inflammation and modulates the tumor microenvironment, while curcumin suppresses tumor growth. However, one main challenge that faces such a type of application is the poor water solubility of both agents, which results in low oral bioavailability [23,24]. Several drug delivery systems have been introduced to improve solubility and enhance bioavailability for each individual drug. For example, curcumin-containing polymeric nanoparticles, made from biodegradable synthetic polymers such as poly(D, L-lactic-co-glycolic acid) (PLGA), showed a nine-fold increase in bioavailability compared to unprocessed curcumin and were able to enhance anti-inflammatory effects and anticancer efficacy [25]. Curcumin was also encapsulated in liposomes [26], polymeric micelles [27], polymeric nanogels [28], or self-assembled peptide systems [29]. Moreover, dexamethasone was encapsulated in nanomicelles [30] and liposomes [31], to improve its bioavailability and achieve targeted delivery to tumor sites [31]. In addition, nanodispersions containing a combination of curcumin and dexamethasone were attempted to enhance the corneal permeability of both drugs for the treatment of ocular diseases [32]. However, to the best of our knowledge, no one has tried to co-encapsulate the two drugs in polymeric micelles.

Several methods were used to analyze either curcumin or dexamethasone alone in their various formulations; however, to the best of our knowledge, no simple, validated method has been reported for the simultaneous quantification of both compounds in co-loaded polymeric micelles. This is particularly important because both drugs are highly hydrophobic, exhibit overlapping solubility challenges, and are prone to matrix interference in excipient-rich systems.

In general, reverse-phase HPLC (RP-HPLC) is the most frequently used method for the analysis of curcumin or dexamethasone alone, employing C18 columns, and mobile phase mixtures of methanol, acetonitrile, or water [33,34]. Curcumin is usually detected at wavelengths between 419 and 425 nm [35,36], whereas dexamethasone is commonly detected between 239 and 254 nm [37,38]. Moreover, A reverse-phase HPLC method was developed for the simultaneous determination of curcumin and the disodium phosphate salt of dexamethasone in nanodispersions [32]. The method employed an Eclipse XDB C8 column, with a mobile phase consisting of a 42:58% (v/v) mixture of acetonitrile and water. Detection was carried out using a PDA detector at 281 nm for both drugs [32]. However, it is worth mentioning that dexamethasone differs from its diphosphate form in terms of chemical structure, as it lacks the O-phosphate group at position 21, which renders it neutral and more hydrophobic at physiological pH, compared to the negatively charged, more water-soluble phosphate salt [39]. This essentially means that the retention behavior of dexamethasone in HPLC will differ, and the two forms cannot be analyzed using the same HPLC method.

This project aims to develop and validate a reverse-phase HPLC method, for the simultaneous determination of curcumin and dexamethasone in polymeric micelle nanoparticles. The method we present offers a short runtime (<7 min), isocratic elution with common solvents, and compliance with ICH validation standards, allowing the precise and reliable quantification of these compounds when combined in similar drug delivery systems, and is especially useful in resource-limited laboratories, where advanced UHPLC or MS systems might not be available.

2. Materials and methods

2.1. Chemicals and reagents

Curcumin was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dexamethasone, used in this study, was kindly provided by Prof. Hatem Al Khatib, from the Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, University of Jordan. Soluplus^® was obtained from BASF (Ludwigshafen, Germany), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was obtained from Biosynth (Switzerland). HPLC-grade methanol was obtained from Macron Fine Chemicals (Center Valley, PA, USA). Chloroform AR grade was obtained from Alpha Chemika (India). All reagents were used without further purification.

2.2. Method development

2.2.1. Instrumentation

The chromatographic analysis was performed using a Shimadzu Prominence-I LC-2030C 3D Plus system equipped with a photodiode array (PDA) detector (Shimadzu Corporation, Kyoto, Japan). Data acquisition and processing were carried out using LabSolutions software version 5.92 (Shimadzu Corporation).

2.2.2. Chromatographic conditions

Separation of curcumin and dexamethasone was achieved using a Universil HS C18 column (25 × 4.6 mm, 5 µm; Fortis Technologies, UK). The mobile phase consisted of methanol and acidic water (pH 3.5, adjusted with concentrated acetic acid). Isocratic elution with mobile phase ratios ranging from 85:15 to 70:30 (methanol: acidic water) was evaluated to optimize separation. The flow rate was maintained at 1.0 mL/min, and the column temperature was set at 30 °C. Detection was performed using a photodiode array detector, with an injection volume of 10 µL.

2.2.3. Sample preparation

Stock solutions (1 mg/mL) of either curcumin or dexamethasone alone were prepared by dissolving 2 mg of each analyte in 2 mL methanol HPLC grade. A standard working solution, containing a mixture of curcumin and dexamethasone was prepared by mixing equal volumes of the respective stock solutions to achieve a final concentration of 0.5 mg/mL for each analyte. The following calibration curve standard solutions (0.0078, 0.03125, 0.01563, 0.0625, 0.125, and 0.25 mg/mL) were prepared by mixing appropriate volumes of the previously prepared working standard with methanol of HPLC grade, up to 1 mL. All solutions were stored at room temperature and protected from light to ensure stability.

2.2.4. System suitability

System suitability testing was performed to ensure the reliability and efficiency of the developed HPLC method. Key parameters assessed included retention time, resolution, tailing factor, and the number of theoretical plates. To verify system performance, these parameters were evaluated using replicate injections (n = 4) of the standard mixture at a concentration of 0.25 mg/mL.

2.2.5. Method validation

The proposed HPLC method was validated according to ICH guidelines for the following parameters:

2.2.5.1. Linearity and range

The linearity of the method was assessed by analyzing standard mixture solutions of curcumin and dexamethasone at concentrations ranging from 0.0078 to 0.25 mg/mL for each analyte, in triplicate. Mean area under the curve (AUC) values were plotted against concentration to construct calibration curves for both analytes. The coefficient of determination (R²) was calculated to evaluate the goodness of fit.

2.2.5.2. Repeatability

Repeatability was assessed by computing the relative standard deviation (RSD%) for AUC values obtained from each calibration curve sample analyzed, using the following formula:

$$\text{RSD}\%=\left(\text{Standard Deviation}/\text{Mean}\right)\ast 100$$ (1)

2.2.5.3. Precision

• Intraday precision: In the same day, mixture solutions at three concentration levels for each analyte (0.25, 0.0625, and 0.01563 mg/mL) were analyzed in triplicate. RSD% for AUC values was computed.

• Inter-day precision: Mixtures at three concentration levels for each analyte (0.25, 0.0625, and 0.01563mg/mL) were analyzed in triplicate on three separate days. The RSD% for AUC values each day was calculated to assess repeatability.

2.2.5.4. Accuracy

Accuracy was determined by analyzing standard mixtures within the concentration range of 0.25–0.01563 mg/mL. Using the calibration curve equations, the actual concentrations were calculated. Percent recoveries were computed as:

$$\%\text{\hspace{0.17em}Recovery\hspace{0.17em}}=\hspace{0.17em}\left(\text{Actual\hspace{0.17em}\hspace{0.17em}Concentration}/\text{Theoretical\hspace{0.17em}\hspace{0.17em}Concentration}\right)\hspace{0.17em}\ast 100$$ (2)

Mean Recovery values for each analyte, and RSD% of the recovery values at each concentration level were also computed to evaluate the consistency of the results.

2.2.5.5. Robustness

Robustness was assessed by analyzing mixture solutions at three concentration levels for each analyte (0.25, 0.0625, and 0.01563 mg/mL) in triplicate under deliberately varied chromatographic conditions. Parameters including wavelength (±5 nm), column temperature (±5 °C), and flow rate (±0.1 mL/min) were altered to examine the method’s resilience. RSD% for AUC values was calculated to ensure that the method performance was unaffected by these minor changes.

2.2.5.6. Limit of detection (LOD) and limit of quantification (LOQ)

LOD and LOQ were calculated for each analyte from the calibration curve, based on the standard deviation of the response (σ) and slope (S), according to ICH guidelines Q2(R1) [40], using the following formulae:

$$\text{LOD}=\text{3}.3\mathrm{\sigma }/\mathrm{S}$$ (3)

$$\text{LOQ }=\text{1}0\mathrm{\sigma }/\mathrm{S}$$ (4)

2.3. Preparation and characterization of polymeric micelle nanoparticles

2.3.1. Preparation of polymeric micelle nanoparticles

The polymeric micelle nanoparticle formula was optimized based on a pilot study performed previously (data unpublished). Micelles were prepared using the solvent evaporation method [41], where 50 mg Soluplus^®, 4.14 mg DOPE, 2 mg dexamethasone, and 2 mg curcumin were dissolved in 2 mL of organic solvent mixture consisting of methanol and chloroform in 1:1 (v/v) ratio. The solvent was then evaporated by gradually reducing the pressure from 350 mbar to 100 mbar at 50 rpm and 40 °C, using a Büchi Rotavapor R-300 (Switzerland), until a uniform thin film was formed. The thin film was kept under vacuum overnight to remove any organic solvent residues, and then rehydrated using 5 mL of deionized water (pH 7) at 35 °C for 20 minutes under stirring. The sample was subsequently ultrasonicated for 1 minute using a Bandelin SONOPLUS ultrasonic homogenizer (Germany). The formed nanoparticles were separated from unencapsulated drugs by centrifugation for 50 min at 10,000rpm and 4 °C. The pellet was resuspended in 5 mL of deionized water, and the supernatant and resuspended pellet were stored at 4 °C for characterization.

2.3.2. Characterization of nanoparticles

Fresh nanoparticle dispersions were characterized for particle size, PDI, and zeta potential by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK). Samples were diluted (20:980 µL) with deionized water before measurement to ensure optimal scattering intensity before analysis.

To determine the encapsulation efficiency (EE%), the direct method was initially attempted by disrupting micelles using various organic solvent mixtures (acetonitrile, methanol, and chloroform), followed by analysis of the samples using the developed RP-HPLC method. However, this consistently resulted in overlapping interference from Soluplus^® at the retention time of curcumin, which yielded inaccurate results and encapsulation efficiency values exceeding 100%. Therefore, to eliminate matrix effects, it was decided to use the indirect method to measure EE%. Nanoparticles were separated from free (unencapsulated) drugs using centrifugation, and the supernatant was analyzed in triplicate for free drug content, using the developed reverse-phase HPLC method. EE% was calculated using the following formula:

$$\text{EE}\%=\hspace{0.17em}\left[\left(\text{Amount\hspace{0.17em}\hspace{0.17em}of\hspace{0.17em}\hspace{0.17em}drug\hspace{0.17em}initially\hspace{0.17em}\hspace{0.17em}added\hspace{0.17em}\hspace{0.17em}in\hspace{0.17em}\hspace{0.17em}the\hspace{0.17em}\hspace{0.17em}formula\hspace{0.17em}}-\text{\hspace{0.17em}Amount\hspace{0.17em}\hspace{0.17em}of\hspace{0.17em}\hspace{0.17em}free\hspace{0.17em}drug}\right)/\text{Amount\hspace{0.17em}\hspace{0.17em}of\hspace{0.17em}\hspace{0.17em}drug\hspace{0.17em}\hspace{0.17em}initially\hspace{0.17em}added}\right]\hspace{0.17em}\ast 100.$$ (5)

3. Results and discussion

3.1. Conditions optimization

The scope of this study was limited to the quantification of curcumin and dexamethasone in micellar formulations; therefore, the method was not developed as a stability-indicating assay.

Selection of chromatographic parameters was based on maximizing separation, minimizing run time, and ensuring reproducibility in excipient-rich samples. A C18 column was chosen due to its well-documented suitability for hydrophobic analytes, such as curcumin and dexamethasone, which provides optimal retention and peak symmetry. Methanol was selected as the organic modifier because it offered a good peak shape and reproducibility, while maintaining a low back pressure. Acidification of the aqueous phase to pH 3.5 enhanced curcumin stability and resulted in sharper peaks for dexamethasone, consistent with their respective ionization behaviors. A simple, isocratic elution (80:20 methanol:aqueous) was favored over gradient elution for short, routine analysis. The chosen flow rate (1.0 mL/min) and column temperature (30 °C) provided consistent retention times without excessive back pressure, ensuring robustness in day-to-day use. Detection was performed using a photodiode array detector at wavelengths of 425 nm for curcumin and 254 nm for dexamethasone. The injection volume was 10 µL. Both peaks were sharp and symmetrical, with retention time of 4.15 min for dexamethasone and 5.1 min for curcumin, and a total run time of 7 minutes (Figure 2).

Figure 2.

Chromatograms of a standard mixture of curcumin and dexamethasone (0.25 mg/mL each) at 425 and 254 nm, showing peaks of curcumin at 5.1 min and dexamethasone at 4.152 min.

A system suitability test was performed to ensure the efficiency of the developed method in simultaneous determination of the target analytes. All parameters were within acceptable limits (Table 1). Retention time values were consistent, resolution was >2, tailing factor was < 2, while theoretical plates were >2000.

Table 1.

System suitability test for analyte mixture at 0.25 mg/mL (n = 4).

Parameter	Curcumin	Dexamethasone
Retention time (min)	Mean = 5.1035	Mean = 4.15525
	%RSD = 0.238108	%RSD = 0.143558
Resolution	2.5
Tailing factor	1.095	1.197
Number of theoretical plates	2334	2709

3.2. Method validation

The proposed method was validated in accordance to ICH guidelines [40]. Standard solutions, containing a mixture of curcumin and dexamethasone, at six concentration levels between 0.0078 and 0.25 mg/mL, were analyzed in triplicate. However, since the limit of quantification (LOQ) was experimentally found to be higher than 0.0078 mg/mL for both analytes, linearity was considered across the concentration range of 0.015–0.25 mg/mL. Calibration curves were constructed by plotting average AUC values against concentration. The equations of the regression lines obtained were: y = 1 × 10^–8 × – 348522 for Curcumin and y = 2 × 10^–7 × – 54547 for dexamethasone. Where y is the area under the curve and x is the concentration (Figure 3).

Figure 3.

Calibration curves for curcumin and dexamethasone with corresponding equations and R² values.

The repeatability of the proposed method was evaluated by calculating the relative standard deviation (RSD%) of peak area values obtained from triplicate injections of calibration curve standard mixtures. The RSD% values for all measurements were found to be below 2%, demonstrating the method’s high repeatability and reliability.

Intraday and interday precision were assessed by analyzing standard mixtures at three concentration levels (0.25, 0.0625, and 0.01563 mg/mL) in triplicate on the same day or on three different days. All RSD% values computed were < 2%, indicating that the proposed method was precise.

The robustness of the method was evaluated by analyzing standard mixtures at three concentration levels (0.25, 0.0625, and 0.01563 mg/mL) in triplicate under slightly varied conditions, including temperature adjustments (±5 °C), flow rate variations (±0.1 mL/min), and wavelength shifts (±5 nm). All RSD% values for the analyzed conditions were within acceptable limits, confirming the method’s robustness under these minor variations.

Standard mixtures at three concentration levels, representing low, medium, and high (0.01563, 0.0625, and 0.25), were analyzed in triplicate to evaluate the accuracy of the method by calculating the percent recoveries (Table 2). The mean percent recovery for curcumin at the concentration levels tested was found to be 98.7%, while the mean percent recovery for dexamethasone at the concentration levels tested was 101.7%. All %RSD values for each concentration were < 2%, indicating the method’s accuracy and reliability for quantifying both curcumin and dexamethasone across the tested concentration range.

Table 2.

Accuracy evaluation of curcumin and dexamethasone quantification.

Curcumin [LOD= 0.0035 mg/mL, LOQ= 0.0106 mg/mL]
Mixture concentration (mg/mL) (n = 3)	Mean recovery (%)	%RSD
0.25	98.6	0.13
0.0625	96.5	0.29
0.01563	99.8	0.82
Dexamethasone [LOD= 0.0029 mg/mL, LOQ = 0.0088 mg/mL]
Mixture concentration (mg/mL) (n = 3)	Mean recovery (%)	%RSD
0.25	104.3	0.06
0.0625	103.9	0.17
0.01563	96.8	1.4

Limits of detection (LOD) and limits of quantitation (LOQ) were calculated based on the calibration equation of either analyte. LOD values were 0.0035 and 0.0029 mg/mL, while LOQ values were 0.0106 and 0.0088 mg/mL for curcumin and dexamethasone, respectively (Table 2). Table 3 summarizes the validation parameters tested, with all results meeting the acceptance criteria in accordance with ICH Q2(R1).

Table 3.

Summary of method validation parameters for curcumin and dexamethasone according to ICH Q2(R1) guidelines.

Parameter	Acceptance criteria (ICH Q2R1/USP)	Obtained values (this study)	Compliance
Linearity (R²)	R² ≥ 0.99 over at least 5 concentration levels	Curcumin: 0.9999 (0.015–0.25 mg/mL) Dexamethasone: 0.9999 (0.015–0.25 mg/mL)	Pass
Accuracy (Recovery %)	95–105% mean recovery at low, medium, and high levels	Curcumin: 96.5–99.8% (mean 98.7%) Dexamethasone: 96.8–104.3% (mean 101.7%)	Pass
Precision (RSD %)	RSD ≤ 2% (repeatability and intermediate precision)	Intra- and inter-day RSD < 2% for all levels	Pass
Repeatability (RSD %)	RSD ≤ 2% for replicate injections	RSD < 2% across calibration levels	Pass
Robustness	No significant change under minor variations (±0.1 mL/min flow, ±5 nm wavelength, ±5 °C temperature)	RSD < 2% under all tested variations	Pass
LOD (mg/mL)	Not specified; typically < 1/3 of LOQ	Curcumin: 0.0035 Dexamethasone: 0.0029	Meets expectation
LOQ (mg/mL)	Demonstrated precision at the lowest quantifiable level	Curcumin: 0.0106 Dexamethasone: 0.0088	Meets expectation

3.3. Preparation and characterization of polymeric micelle nanoparticles

A hybrid polymer-lipid micelle system was successfully formulated using the amphiphilic graft copolymer Soluplus^®, known for its solubilizing capacity, and DOPE phospholipid at a molar ratio of 1:10 to encapsulate two hydrophobic drugs, curcumin and dexamethasone. This drug combination was selected due to its complementary anti-inflammatory, antioxidant, and anticancer properties. The co-delivery of these agents within a single nanocarrier offers the potential for synergistic therapeutic effects, particularly in the treatment of multifactorial diseases such as cancer, where simultaneous modulation of inflammatory and proliferative pathways is crucial. Freshly prepared micelles exhibited an average particle size of 89.59 ± 1.38 nm, with a polydispersity index (PDI) of 0.259 ± 0.04, indicating a narrow size distribution and uniformity. The surface charge, as indicated by the zeta potential, was −12.93 ± 1.8 mV, reflecting moderate stability due to electrostatic repulsion.

Encapsulation efficiency EE% was determined by employing the developed RP-HPLC method, using an indirect approach, by measuring the amount of free (unencapsulated) drug and comparing it to the initial amount of loaded drug. In fresh nanoparticles, EE% values obtained for both drugs were 78.84 ± 0.05% for curcumin and 54.33 ± 0.05% for dexamethasone, demonstrating the suitability of the developed HPLC method for quantifying both drugs in the polymeric micelle nanoparticle formulation.

It is noteworthy that attempts to apply a direct solvent-disruption method produced unreliable values (>100% EE) due to interference from Soluplus^® at the curcumin retention time. By contrast, the indirect centrifugation-based approach avoided this interference and yielded consistent results, confirming its suitability for polymeric micelle formulations.

While the present method was not subjected to forced degradation experiments, our objective was to provide a practical, validated tool for the routine quantification of curcumin and dexamethasone in nanoparticle formulations. Future work may extend the method to incorporate stress-testing conditions, thereby establishing full stability-indicating capability.

4. Conclusions

This study successfully developed and validated a reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous determination of curcumin and dexamethasone in nanoparticle formulations. The method demonstrated excellent linearity, precision, accuracy, and robustness, meeting the requirements of ICH guidelines. The method was successfully applied to characterize a polymeric micelle nanoparticle formula that co-encapsulates two hydrophobic drugs, demonstrating satisfactory encapsulation efficiencies.

Unlike many previously reported methods that require gradient elution, uncommon mobile phases, or detection at a single wavelength, the presented method provides baseline separation at dual wavelengths (425 nm for curcumin and 254 nm for dexamethasone) in under 7 minutes using widely available solvents. These features make the method highly relevant for routine encapsulation efficiency, drug loading, and release studies in polymeric drug delivery research, and offer a practical alternative to more complex stability-indicating or MS-based methods.

It is important to note that this method was not intended as a comprehensive stability-indicating assay, but rather as a reliable and accessible analytical tool for quality control of nanoparticle formulations. Incorporation of forced degradation studies to confirm stability-indicating performance represents an important direction for future research.

Funding Statement

Deanship of Scientific Research, the University of Jordan.

Author contributions

D Haj-Ali: carried out the experiments, conceptualization, data curation, software and writing-original draft preparation. H. Azzam: carried out the experiments and curated the data. K Aiedeh contributed to supervision, validation, and review of the original draft. W Alshaer: contributed to conceptualization, methodology, investigation, project administration, supervision, data validation, and review – editing the original draft.

Disclosure statement

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Financial disclosure

This research was supported by the Deanship of Scientific Research at the University of Jordan as a Ph.D. fund awarded to Dana Haj-Ali, a Ph.D. candidate.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article [and/or] its supplementary materials.