Description:Technical Field of the Invention
The present invention relates to a Shikonin-loaded mucoadhesive microemulsion for intranasal administration and to a reverse-phase ultra-performance liquid chromatographic method for quantitative determination of Shikonin in the mucoadhesive microemulsion and biological matrices.
More particularly, the invention relates to a Shikonin-loaded oil-in-water mucoadhesive microemulsion comprising castor oil, a surfactant–cosurfactant mixture comprising Tween 80 and propylene glycol, water and chitosan, wherein the formulation provides nanoscale droplets and high Shikonin entrapment efficiency.
The invention further relates to an Analytical Quality by Design-driven reverse-phase UPLC method employing an acetonitrile and 0.1% formic acid aqueous phase as mobile phase for quantification of Shikonin in pharmaceutical formulations and biological matrices.
Background of the Invention
Schizophrenia is a chronic and debilitating neuropsychiatric disorder characterized by disturbances in perception, cognition, emotion, and behavior. The research identifies several limitations associated with existing therapeutic approaches, including poor patient compliance, inadequate brain targeting, systemic side effects, and variable pharmacokinetic behavior.
Shikonin is a bioactive naphthoquinone compound obtained from the roots of Lithospermum erythrorhizon and related botanical sources. The research describes various biological activities of Shikonin, including anti-inflammatory, antioxidant, neuroprotective, and anticancer activities, indicating its potential application in neurological disorders. However, the therapeutic application of Shikonin is limited by its poor water solubility, low systemic availability, and inadequate ability to cross the blood–brain barrier.
Intranasal administration provides a non-invasive approach for delivering therapeutic agents from the nasal cavity towards the brain. In this context, mucoadhesive microemulsions offer a formulation strategy for enhancing the solubilization of lipophilic drugs, prolonging nasal residence, and facilitating drug delivery.
The research also establishes the need for a robust, sensitive, and reproducible analytical method for accurate quantification of Shikonin in complex pharmaceutical formulations and biological matrices. Accordingly, the present invention provides a Shikonin-loaded mucoadhesive microemulsion together with an Analytical Quality by Design (AQbD)-driven RP-UPLC method for the quantitative determination of Shikonin in the formulation and biological matrices.
Objects of the Invention
The main object of the invention is to provide a Shikonin-loaded mucoadhesive microemulsion for intranasal administration.
Another object of the invention is to provide a method for preparation of the Shikonin-loaded microemulsion.
Another object of the invention is to provide an AQbD-driven RP-UPLC method for quantification of Shikonin in the mucoadhesive microemulsion and biological matrices.
Yet another object of the invention is to provide a Pharmacokinetic evaluation of brain and plasma exposure of Shikonin following intranasal administration Shikonin-loaded mucoadhesive microemulsion.
Summary of the Invention
The present invention provides a Shikonin-loaded mucoadhesive microemulsion comprising Shikonin incorporated in an oil-in-water microemulsion containing castor oil in an amount of 5–10% and a surfactant–cosurfactant mixture in an amount of 20–40%, wherein the surfactant–cosurfactant mixture comprises Tween 80 and propylene glycol in equal proportions, and wherein chitosan is incorporated as a mucoadhesive polymer. In an embodiment, the present invention provides a method for the preparation of Shikonin-loaded mucoadhesive microemulsion, wherein the microemulsion is prepared by an aqueous titration technique. Shikonin is incorporated into the optimized castor oil and surfactant–cosurfactant mixture, followed by gradual dropwise addition of distilled water under mild agitation at room temperature. The resulting microemulsion is subjected to probe ultrasonication at 20 kHz for approximately 5–15 minutes. Chitosan is dissolved in 1% v/v aqueous acetic acid and gradually incorporated into the optimized microemulsion under continuous stirring to obtain the mucoadhesive microemulsion. In an embodiment, the present invention provides a Shikonin-loaded mucoadhesive microemulsion, wherein the developed mucoadhesive microemulsion exhibited a mean droplet size of 73.8 ± 0.63 nm, polydispersity index of 0.267 ± 0.085, zeta potential of 32.68 ± 0.157 mV and entrapment efficiency of 87.9 ± 0.25%. In an embodiment, the present invention provides a AQbD-driven RP-UPLC method for quantitative determination of Shikonin in the mucoadhesive microemulsion and biological matrices, wherein the invention further provides an AQbD-driven RP-UPLC method employing a C18 column, an acetonitrile and 0.1% formic acid aqueous phase in an 80:20 v/v ratio, a flow rate of 1.0 mL/min, column temperature of 30°C and detection at 254 nm. In an embodiment, the analytical method was developed using risk assessment, Taguchi screening and Box–Behnken response surface methodology. In an embodiment, the optimized method exhibited linearity over 2–12 µg/mL, correlation coefficient of 0.9997, LOD of 0.396 µg/mL and LOQ of 1.191 µg/mL. In an embodiment, the validation results showed precision and robustness with %RSD values below 2%, and recovery between 98.16% and 100.56%. In an embodiment, the Pharmacokinetic evaluation in male Wistar rats demonstrated higher brain and plasma exposure following intranasal administration of the Shikonin-loaded mucoadhesive microemulsion. In an embodiment, the formulation produced a brain Cmax of 245.20 ± 0.25 ng/g at 2 h and plasma Cmax of 195.40 ± 0.28 ng/mL at 2 h. In an embodiment, the corresponding brain and plasma AUC0–t values were 2400.0 ± 210.0 h·ng/g and 2200.0 ± 140.0 h·ng/mL, respectively.
Brief Description of drawings
In the drawings accompanying the specification, Figure 1 shows Representative UPLC chromatogram of SHK (10 µg mL⁻¹) obtained under optimized chromatographic conditions.
In the drawings accompanying the specification, Figure 2 shows Representative UPLC chromatogram of Shikonin (10 µg mL⁻¹) in the prepared mucoadhesive microemulsion under optimized chromatographic conditions.
In the drawings accompanying the specification, Figure 3 shows Representative UPLC chromatograms of SHK in (a) brain homogenate and (b) rat plasma following intranasal administration of the mucoadhesive microemulsion.
In the drawings accompanying the specification, Figure 4 shows Plasma and brain concentration–time profiles of SHK following intranasal administration of SHK-loaded MME and oral administration of the reference drug. Data are presented as mean ± SD (n = 6).
In the drawings accompanying the specification, Figure 5 shows Assessment of method “greenness” and “whiteness” using (a) ComplexGAPI, (b) AGREE, (c) AGREEprep, and (d) BAGI.
Detailed description of the Invention
The present invention provides a Shikonin-loaded mucoadhesive microemulsion for intranasal administration and an AQbD-driven RP-UPLC method for quantitative determination of Shikonin in the developed microemulsion and biological matrices. The Analytical Quality by Design (AQbD)-driven reverse-phase ultra-performance liquid chromatographic (RP-UPLC) method for quantitative determination of Shikonin in a mucoadhesive microemulsion and biological matrices, comprising: (a) performing risk assessment for identification of critical analytical variables; (b) screening analytical variables using a Taguchi L8 orthogonal array; (c) optimizing the identified critical analytical variables using a Box–Behnken response surface methodology; and (d) quantitatively determining Shikonin using optimized chromatographic conditions comprising a C18 column, an acetonitrile and 0.1% v/v formic acid in water mobile phase in an 80:20 v/v ratio, a flow rate of 1.0 mL/min, a column temperature of 30 °C and detection at 254 nm.
The Shikonin-loaded mucoadhesive microemulsion for intranasal administration comprising Shikonin, castor oil in an amount of 5–10%, a surfactant–cosurfactant mixture in an amount of 20–40%, distilled water and chitosan, wherein the surfactant–cosurfactant mixture comprises Tween 80 and propylene glycol in equal proportions, and wherein the microemulsion comprises nanoscale droplets. The Shikonin-loaded mucoadhesive microemulsion, wherein 10 mg of Shikonin is incorporated into the castor oil and surfactant–cosurfactant mixture. The Shikonin-loaded mucoadhesive microemulsion, wherein the microemulsion has a mean droplet size of 73.8 ± 0.63 nm, polydispersity index of 0.267 ± 0.085, zeta potential of 32.68 ± 0.157 mV and entrapment efficiency of 87.9 ± 0.25%. The present invention provides a process for preparation of the Shikonin-loaded mucoadhesive microemulsion. The process for preparation of the Shikonin-loaded mucoadhesive microemulsion, comprising incorporating Shikonin into a mixture comprising castor oil and the surfactant–cosurfactant mixture, gradually adding distilled water dropwise under mild agitation at room temperature to form an oil-in-water microemulsion, subjecting the microemulsion to probe ultrasonication at 20 kHz for approximately 5–15 minutes, dissolving chitosan in 1% v/v aqueous acetic acid and gradually adding the chitosan solution to the microemulsion under constant stirring to obtain the mucoadhesive microemulsion. The process wherein the surfactant–cosurfactant mixture comprises Tween 80 and propylene glycol in equal proportions and the resulting microemulsion is transparent and isotropic. The present invention provides a reverse-phase ultra-performance liquid chromatographic method for quantitative determination of Shikonin in the mucoadhesive microemulsion and biological matrices. The reverse-phase ultra-performance liquid chromatographic method for quantitative determination of Shikonin in the mucoadhesive microemulsion and biological matrices, comprising chromatographic separation using a C18 column, an acetonitrile and 0.1% v/v formic acid in water mobile phase in an 80:20 v/v ratio, a flow rate of 1.0 mL/min, a column temperature of 30°C and detection at 254 nm. The method, wherein the method is developed using an Analytical Quality by Design approach comprising risk assessment using an Ishikawa diagram and failure mode and effects analysis, screening using a Taguchi L8 orthogonal array and optimization using Box–Behnken response surface methodology. The method, wherein the method exhibits a linearity range of 2–12 µg/mL with a correlation coefficient of 0.9997, a limit of detection of 0.396 µg/mL and a limit of quantification of 1.191 µg/mL. The method, wherein the method exhibits accuracy with recovery of 98.16 ± 1.25% at 50%, 100.56 ± 0.96% at 100% and 99.67 ± 0.271% at 150% concentration levels, with precision and robustness having %RSD values below 2%. The Shikonin-loaded mucoadhesive microemulsion, wherein intranasal administration in male Wistar rats at 30 mg/kg provides a brain Cmax of 245.20 ± 0.25 ng/g at 2 hours and a brain AUC0–t of 2400.0 ± 210.0 h·ng/g, and a plasma Cmax of 195.40 ± 0.28 ng/mL at 2 hours and a plasma AUC0–t of 2200.0 ± 140.0 h·ng/mL.
MATERIALS AND METHODS
Shikonin was obtained from Sigma-Aldrich (Mumbai, India). Castor oil was purchased from Loba Chemie Pvt. Ltd. (Mumbai, India). the method is suitable for analysis in formulations and biological matrices. Co-surfactants utilized in the study were procured from HiMedia Laboratories Pvt. Ltd. (Mumbai, India). All additional chemicals and reagents employed during the investigation were of analytical or pharmaceutical grade and were utilized as received without any further purification.
Instrumentational Attributes and Analytical Setup
Chromatographic evaluation was conducted using a Shimadzu Prominence LC-30AD UPLC system integrated with a quaternary solvent delivery unit, an autosampler, a temperature-controlled column compartment, and a photodiode array (PDA) detector. The separation of SHK was performed using a Phenomenex Luna C18 column (150 × 4.6 mm, 5 μm particle size). The mobile phase consisted of acetonitrile and 0.1% (v/v) formic acid in water mixed in an 80:20 (v/v) ratio and pumped under isocratic conditions at a flow rate of 1.0 mL min⁻¹. Before use, the mobile phase was passed through a 0.45 μm PVDF membrane filter and degassed using ultrasonication to remove dissolved gases. An injection volume of 10 µL was applied for both standard and sample preparations. Detection of the analyte was carried out at a wavelength of 254 nm using the PDA detector, which also enabled evaluation of peak purity and spectral consistency throughout the chromatographic analysis.

Preparation of Standard Solutions
A dilute acidic aqueous phase containing 0.1% formic acid was prepared by adding a measured quantity of concentrated formic acid to ultrapure water, followed by mixing thoroughly. The resulting solution was subsequently filtered through a 0.22 μm membrane to remove any particulate matter. For the standard preparation, an accurately weighed quantity of 10 mg of SHK was transferred into a 10 mL volumetric flask. It was then dissolved in the mobile phase with the assistance of sonication to ensure complete dissolution, yielding a stock solution with a concentration of 1000 µg mL⁻¹. From this stock solution, a secondary solution of
lower concentration (100 µg mL⁻¹) was obtained through appropriate dilution using the same mobile phase as diluent. Subsequent dilution of this intermediate solution provided a working solution containing 10 µg mL⁻¹ of SHK.
Calibration standards were then prepared by further diluting the intermediate solution to obtain concentrations ranging from 2 to 12 µg mL⁻¹. Each standard solution was freshly prepared prior to analysis and injected three times to confirm the repeatability and reliability of the analytical results. Chromatographic analysis was performed under optimized conditions, with the injection volume maintained
at 10 µL. Detection was conducted at a wavelength of 254 nm, and the column temperature was consistently controlled at 30 °C throughout the run.

Preparation of Shikonin-loaded mucoadhesive microemulsion
The present invention provides a Shikonin-loaded mucoadhesive microemulsion intended for intranasal delivery. For preparation of the microemulsion, 10 mg of Shikonin is incorporated into a blend comprising castor oil in an amount of 5–10% and a surfactant–cosurfactant mixture (Smix) in an amount of 20–40%. The Smix comprises Tween 80 and propylene glycol mixed in equal proportions. The components are continuously stirred using a magnetic stirrer to facilitate dissolution of Shikonin and formation of a uniform mixture. Distilled water is subsequently introduced gradually in a dropwise manner while maintaining mild agitation at room temperature. Gradual addition of the aqueous phase results in spontaneous formation of a transparent and isotropic oil-in-water microemulsion. The resulting microemulsion is subjected to probe ultrasonication at 20 kHz for approximately 5–15 minutes to enhance homogeneity and reduce droplet-size variation. Chitosan is incorporated into the microemulsion as a mucoadhesive polymer. Chitosan is initially dissolved in 1% v/v aqueous acetic acid under continuous stirring to obtain a clear solution. The chitosan solution is then gradually added to the optimized microemulsion under constant stirring to provide uniform dispersion. The resulting system is mixed until a homogeneous mucoadhesive microemulsion is obtained. The formulated samples are examined for visual appearance, including clarity and signs of phase separation, and are preserved in well-sealed glass vials. Physicochemical characterization
The developed mucoadhesive microemulsion is characterized for particle size, polydispersity index, zeta potential and entrapment efficiency.
Particle size, polydispersity index and zeta potential are determined using dynamic light scattering. Entrapment efficiency is evaluated by ultracentrifugation followed by quantification of unentrapped Shikonin in the supernatant using the developed UPLC method. The optimized mucoadhesive microemulsion exhibits:
Parameter Result
Mean droplet size 73.8 ± 0.63 nm
Polydispersity index 0.267 ± 0.085
Zeta potential 32.68 ± 0.157 mV
Entrapment efficiency 87.9 ± 0.25%

These results demonstrate nanoscale droplets, relatively narrow size distribution, positive surface charge and high Shikonin incorporation.

Quality by Design development
Risk assessment is performed using an Ishikawa diagram and failure mode and effects analysis. Factors having a risk priority number above 60 are categorized as high-risk factors and selected for further investigation.
A Taguchi L8 orthogonal array is employed for screening seven analytical factors at two levels. The chromatographic responses evaluated include retention time, tailing factor and theoretical plate count. The screening identifies organic phase composition, mobile phase flow rate and column temperature as the most influential variables. These three critical method parameters are subsequently optimized using response surface methodology with a Box–Behnken design.
The investigated ranges are:
• organic phase composition: 75–85%;
• flow rate: 0.9–1.1 mL/min; and
• column temperature: 25–35 °C.
The responses are retention time, tailing factor and theoretical plate count. Retention time is minimized, tailing factor is targeted towards unity and theoretical plate count is maximized. The optimized chromatographic conditions obtained by the desirability function comprise acetonitrile and 0.1% v/v formic acid in water in an 80:20 v/v ratio, a flow rate of 1.0 mL/min and column temperature of 30 °C. Verification using six replicate injections showed experimentally measured values differing by less than 5% from predicted values.
AQbD-Assisted RP-UPLC Method Development
Optimization of Critical Method Parameters
Following the results obtained from the risk evaluation and initial factor screening, three analytical parameters were recognized as having a major influence on chromatographic behavior. These included the proportion of organic solvent in the mobile phase (X₁), the mobile phase flow velocity (X₂), and the operating temperature of the column (X₃). Prior to this stage, a Taguchi orthogonal array-based screening design was employed to systematically evaluate a broader set of potential method variables, enabling efficient identification of the most influential CMPs while minimizing the number of experimental runs. The selection of these three variables was based on their statistically significant effects on chromatographic responses observed during the screening phase. To systematically determine the optimal conditions for these variables, response surface methodology (RSM) was applied using a Box–Behnken experimental design (BBD). The choice of BBD was justified by its efficiency in fitting quadratic response surfaces, requirement of fewer experimental runs compared to central composite designs, and its suitability for exploring interaction effects among variables without including extreme factor combinations that could compromise chromatographic performance. In this optimization approach, the three selected factors were examined at three coded levels (− 1, 0, and + 1). The design matrix generated fifteen experimental trials, which incorporated several center-point experiments to evaluate the adequacy of the mathematical model and to estimate experimental error. During this optimization process, all other chromatographic parameters were kept unchanged, while the analyte concentration remained constant in each run to maintain uniform experimental conditions (Table 2). The selected responses, defined as CAAs, included RT, TF, and TP, representing chromatographic efficiency, peak symmetry, and column performance, respectively. These response parameters were specifically chosen as CAAs to guide the optimization process, where RT was minimized to ensure rapid analysis, TF was targeted toward unity to
achieve symmetrical peak shape, and TP were maximized to enhance column efficiency and resolution. The investigated ranges for the CMPs were as follows: organic phase composition (75–85%), flow rate (0.9–1.1 mL min⁻¹), and column temperature (25–35 °C). Data generated from the experimental design trials were subjected to mathematical modeling using a second-order (quadratic) regression approach. The adequacy and significance of the model were examined using analysis of variance (ANOVA), which enabled assessment of the main effects, curvature (quadratic terms), and interaction effects among the studied variables. This statistical model enabled prediction of chromatographic behavior and assisted in defining the most appropriate operational conditions. A desirability function approach was further employed to simultaneously optimize all selected responses,
ensuring a balanced trade-off between rapid analysis and optimal chromatographic performance. The optimization strategy focused on achieving rapid analysis while maitaining high-quality chromatographic performance, including reduced retention time, improved peak shape, and enhanced column efficiency. By integrating these responses during optimization, the final analytical conditions were established to provide consistent and dependable chromatographic outcomes. Consequently, a stable and reliable UPLC procedure was obtained for accurate determination of SHK in pharmaceutical formulations as well as biological matrices.

Preliminary Method Optimization
The UPLC method for the quantification of SHK in MME and biological matrices was developed using a systematic QbD approach to ensure robust and reliable analytical performance. Initial method development experiments focused on assessing the impact of mobile phase composition and chromatographic conditions on peak shape, resolution, and overall system efficiency. Acetonitrile was selected as the organic modifier due to its superior elution strength and ability to produce sharper peaks with improved detector response compared to methanol. Preliminary trials using water as the aqueous phase resulted in broad and poorly resolved peaks, indicating inadequate control over analyte ionization and chromatographic behavior. Subsequent modification of the aqueous phase with 0.1% orthophosphoric acid led to partial improvement in peak characteristics; however, significant peak tailing and reduced theoretical plate counts were still observed, suggesting suboptimal chromatographic performance. To overcome these limitations, orthophosphoric acid was replaced with 0.1% (v/v) formic acid. This modification resulted in a marked improvement in peak symmetry, reduced tailing, and enhanced column efficiency. The improved performance can be attributed to better pH control and the compatibility of formic acid with reversed-phase chromatographic systems, particularly under UPLC conditions. These findings established the suitability of the selected mobile phase system for further method optimization within the QbD framework.
Validation of analytical method
The developed RP-UPLC method is validated for linearity, sensitivity, precision, accuracy, system suitability and robustness. The method exhibits linearity over 2–12 µg/mL with a correlation coefficient of 0.9997. The LOD is 0.396 µg/mL and the LOQ is 1.191 µg/mL. System suitability demonstrates %RSD values of 0.981 for peak area, 0.725 for retention time, 0.963 for tailing factor and 1.054 for theoretical plate count. Intra-day and inter-day precision exhibit %RSD values of 0.987 and 0.692, respectively. Robustness testing provides %RSD values of 1.056 for mobile phase ratio, 1.245 for flow rate and 1.314 for detection wavelength. Accuracy studies provide recoveries of 98.16 ± 1.25%, 100.56 ± 0.96% and 99.67 ± 0.271% at 50%, 100% and 150% levels, respectively.
Risk Assessment and Identification of Critical Factors
A systematic risk evaluation was performed to determine and rank the critical factors that could potentially affect the efficiency and reliability of the developed UPLC analytical method for SHK. Based on preliminary experimental findings and established chromatographic principles, more than thirty potential factors were initially identified. These variables were systematically categorized into six domains—instrumentation, method parameters, materials, measurement, personnel, and environmental conditions—and represented using an Ishikawa (fishbone) diagram.

Establishment of Optimized Chromatographic Conditions
The optimal chromatographic conditions for the UPLC analysis of SHK were determined by applying RSM integrated with a desirability function approach. This statistical optimization strategy was executed using Design-Expert® software (Version 13.0, Stat-Ease Inc., USA). The optimization criteria were defined to minimize RT and TF while maximizing TP, thereby achieving an optimal balance between analysis speed, peak symmetry, and column efficiency. The composite desirability function yielded a value close to unity, indicating that the selected conditions provided the most favorable compromise among the targeted responses.
The corresponding overlay plot delineated a robust design space in which all critical method parameters met the predefined acceptance criteria, confirming the method’s operational flexibility. According to the outcomes of the desirability assessment and the predictions generated by the statistical model, the optimal chromatographic parameters were identified. The recommended conditions included a mobile phase composed of acetonitrile and 0.1% (v/v) formic acid in water in an 80:20 (v/v) ratio, a mobile phase flow rate of 1.0 mL min⁻¹, and maintaining the column temperature at 30°C during analysis. These conditions were selected not only on the basis of statistical optimization but also considering their practicality and reproducibility for routine analytical applications. Method verification under the optimized conditions was performed using six replicate injections. The experimentally measured values of RT, TF, and TP differed by less than 5% from the values predicted by the statistical model. This close correspondence demonstrates the precision, predictive capability, and robustness of the developed models. Representative chromatograms (Fig. 1) demonstrated sharp, well-resolved, and symmetrical peaks without interference, consistent with system suitability requirements. Additional chromatographic assessments further confirmed baseline stability and reproducibility across repeated analyses. Overall, the optimized method exhibited excellent robustness, precision, and reliability, the method is suitable for analysis in formulations and biological matrices.

Method Validation and Performance Characteristics
The proposed UPLC analytical procedure was rigorously validated following the recommendations outlined in ICH guidelines Q2(R1) and Q2(R2) to confirm its appropriateness for the accurate measurement of SHK in MME formulations as well as in biological samples. Various validation characteristics were systematically examined, including system suitability, calibration linearity, detection sensitivity, precision, accuracy, and robustness, and the results are presented in Table 3. Assessment of system suitability verified stable and reproducible chromatographic behavior. Six consecutive injections produced highly consistent results, with %RSD values below 2% for parameters such as RT, peak area, TF, and TP, demonstrating reliable system performance and repeatability. Calibration studies confirmed a linear analytical response within the concentration range of 2–12 µg mL⁻¹, yielding a correlation coefficient (R²) of 0.999, which reflects a strong proportional relationship between SHK concentration and detector signal. In addition, the method showed satisfactory analytical sensitivity, with the LOD calculated as 0.396 µg mL⁻¹ and the LOQ determined to be 1.191 µg mL⁻¹, indicating the method’s capability to detect and quantify low levels of the analyte.
Method precision was investigated at three different concentration levels by examining both repeatability and intermediate precision. The variability observed in all measurements remained minimal, with %RSD values consistently below 2%, indicating that the analytical procedure provides highly reproducible results. The stability of the method against small operational changes was evaluated through robustness testing. Minor intentional modifications were introduced in key chromatographic parameters, such as organic solvent proportion (± 2%), mobile phase flow rate (± 0.1 mL min⁻¹), and detection wavelength (± 2 nm). These controlled alterations did not cause noticeable variations in RT, peak shape, or column performance, demonstrating that the method maintains stable performance even under slightly altered conditions.
To verify the accuracy of the procedure, recovery experiments were carried out at 50%, 100%, and 150% of the target concentration. The percentage recovery values were found within the range of 98.16% to 100.56%, while the associated %RSD values remained below 2%, confirming the correctness and reliability of the measurements. Taken together, the overall validation outcomes establish that the
developed UPLC method possesses excellent precision, accuracy, sensitivity, and robustness, thereby confirming its suitability for routine quantitative determination of SHK in both pharmaceutical formulations and biological matrices.
Application to Formulation Analysis and Physicochemical Characterization
Preparation and Characterization of SHK-Loaded Mucoadhesive Microemulsion
The validated UPLC method was successfully applied for the quantification of SHK in a MME formulated using the aqueous titration approach. The developed formulation resulted in nanosized droplets with a mean particle size of 73.8 ± 0.63 nm, indicating efficient formation of a fine and stable dispersion. The PDI was found to be 0.267 ± 0.085, suggesting a relatively narrow size distribution and good
uniformity of the dispersed phase. The zeta potential of the formulation was measured to be 32.68 ± 0.157 mV, reflecting adequate electrostatic stabilization and contributing to the physical stability of the system. The Entrapment efficiency (EE) was determined to be 87.9 ± 0.25%, demonstrating effective incorporation of SHK within the microemulsion system. The optimized surfactant–cosurfactant (Smix) ratio likely facilitated efficient solubilization and minimized drug leakage during formulation, thereby enhancing drug loading capacity. These physicochemical characteristics confirm the suitability of the developed MME as a stable and efficient carrier system for SHK. Furthermore, the successful application of the validated UPLC method highlights its reliability for routine quantitative analysis in formulation development and advanced drug delivery studies.
Recovery Assessment in Mucoadhesive Microemulsion
The validated UPLC method was successfully applied for the quantitative determination of SHK in the optimized MME formulation. Triplicate analysis yielded recovery values in the range of 97.15 ± 0.67% to 99.96 ± 0.35%, with %RSD values consistently below 2%, demonstrating high accuracy and precision of the method. The representative chromatogram (Fig. 2) exhibited a well-resolved and symmetrical peak corresponding to SHK, with no observable interference from formulation excipients, confirming the selectivity of the method in complex nanoparticulate systems. These results collectively demonstrate the reliability and consistency of the developed UPLC method, supporting its suitability for routine quantitative analysis of SHK in MME formulations.
In Vivo Pharmacokinetic Evaluation and Brain Distribution
Pharmacokinetic investigations were performed to assess the systemic exposure and brain uptake of SHK following intranasal administration of the SHK-loaded MME, in comparison with an orally administered formulation (reference treatment) to enable quantitative evaluation of route-dependent differences in drug disposition. Male Wistar rats were randomly assigned to two groups: the reference group received chlorpromazine via the oral route, whereas the test group was treated with SHK-loaded MME administered intranasally at a dose of 30 mg kg⁻¹ once daily for
seven consecutive days. Blood and brain samples were collected at predefined intervals ranging from 0.5 to 24 h post-administration. SHK concentrations in plasma and brain homogenates were quantified using the validated UPLC method, with representative chromatograms presented in Fig. 3a and 3b. All pharmacokinetic parameters were calculated using non-compartmental analysis, and results are expressed as mean ± SD (n = 6). Statistical comparisons between groups were performed using an unpaired Student’s t-test, with p < 0.05 considered statistically significant. The pharmacokinetic analysis demonstrated enhanced systemic absorption and brain delivery of SHK following intranasal administration of the MME formulation. In brain tissue, the MME group exhibited a maximum concentration (C_max) of 245.20 ± 0.25 ng g⁻¹ at a time to reach maximum concentration (T_max) of 2 h, whereas the reference group showed a lower C_max of 210.75 ± 0.30 ng g⁻¹ with a delayed T_max of 4 h. This represents an approximately 1.16-fold increase in brain C_max and a two-fold reduction in T_max, indicating faster and enhanced brain uptake via the intranasal route (p < 0.05). A similar trend was observed in plasma, where the MME formulation achieved a C_max of 195.40 ± 0.28 ng mL⁻¹ at 2 h, compared to 175.60 ± 0.22 ng mL⁻¹ at 6 h in the reference group, corresponding to a ~ 1.11-fold increase in systemic exposure and significantly earlier absorption (p < 0.05), indicating more rapid and efficient systemic absorption via the intranasal route. Figure 3 Representative UPLC chromatograms of SHK in (a) brain homogenate and (b) rat
plasma following intranasal administration of the mucoadhesive microemulsion

The pharmacokinetic results are summarized below:
The area under the concentration–time curve (AUC₀–t) further confirmed the improved bioavailability of SHK. The brain AUC₀–t for the MME formulation (2400.0 ± 210.0 h•ng g⁻¹) was notably higher than that of the reference group (2050.0 ± 230.0 h•ng g⁻¹), reflecting an approximate 1.17-fold enhancement in brain exposure (p < 0.05). Similarly, plasma AUC₀–t values were increased in the MME-treated group (2200.0 ± 140.0 h•ng mL⁻¹) compared to the reference treatment (1850.0 ± 160.0 h•ng mL⁻¹), indicating a ~ 1.19-fold increase in systemic bioavailability (p < 0.05). A summary of pharmacokinetic parameters is provided in Table 4, and the corresponding concentration–time profiles for plasma and brain are illustrated in Fig. 4. Additionally, brain-to-plasma concentration ratios were found to be higher in the intranasal MME group compared to the oral reference, further supporting enhanced nose-to-brain targeting efficiency. The observed enhancement in pharmacokinetic performance can be attributed to the nanoscale droplet size and mucoadhesive nature of the microemulsion system. These features likely contribute to prolonged nasal residence time, improved permeation across the nasal mucosa, and facilitated direct transport to the brain via the olfactory pathway. The statistically significant improvements in C_max, T_max, and AUC collectively validate the superiority of the intranasal MME system over conventional oral administration. Collectively, these findings highlight the potential of the developed MME as an effective platform for nose-to-brain delivery of SHK for neurological applications.
Integrated Assessment of Method Greenness and Whiteness
Environmental assessment of the analytical method procedure was carefully examined using several widely accepted green and white analytical evaluation frameworks. Employing multiple assessment tools provided a comprehensive understanding of the method’s environmental profile. This strategy ensured that, in addition to achieving reliable analytical efficiency, the procedure also complies
with modern sustainability concepts and current regulatory recommendations that encourage the development of environmentally conscious analytical techniques. The ComplexGAPI was initially applied to provide a comprehensive, visual assessment of the method’s environmental impact by considering factors such as sample preparation, solvent consumption, instrumentation, and pre-analytical steps. The resulting pictogram indicated a predominantly green profile, comprising eight green, six yellow, and one red segment, along with a low E-factor value of 2 (Fig. 5a). These results reflect reduced reagent usage, minimal waste generation, and an overall low environmental burden. To quantitatively determine the sustainability profile of the analytical procedure, the AGREE metric-which is derived from the twelve principles of green analytical chemistry—was applied. The evaluation produced an overall AGREE score of 0.71, and the associated radial visualization displayed a predominantly green pattern, signifying that the developed method largely conforms to established green chemistry guidelines (Fig. 5b). In addition, the AGREEprep assessment tool was employed to specifically examine the environmental footprint associated with the sample preparation stage, enabling a more detailed evaluation of the sustainability aspects related to this part of the analytical workflow. The protein precipitation procedure adopted for biological samples demonstrated efficient solvent usage and low waste generation, yielding a score of 0.63 (Fig. 5c), thereby supporting its suitability as a sustainable sample preparation strategy. Furthermore, the ESA was employed to further examine the environmental profile of the method. This semi-quantitative evaluation system estimates the greenness of an analytical procedure by assigning penalty points related to the use of hazardous chemicals, energy requirements, and the amount of waste generated during analysis. Based on this assessment, the proposed method obtained an Eco-Scale value of 75, which places it analytical methods. A comprehensive evaluation of the method was also performed using the BAGI, which considers the combined influence of analytical effectiveness, environmental friendliness, and practical usability. Through this assessment, the developed method achieved a BAGI value of 72.5, reflecting a well-balanced integration of performance efficiency, ecological responsibility, and operational practicality (Fig. 5d). Overall, the results obtained from ComplexGAPI, AGREE, AGREEprep, ESA, and BAGI collectively confirm that the proposed UPLC analytical approach maintains strong analytical reliability while minimizing its environmental impact. These findings highlight the method as a sustainable, efficient, and dependable analytical platform, making it highly suitable for routine quantitative determinations in both pharmaceutical analysis and bioanalytical studies.
Conclusion
In this study, a reliable and environmentally conscious RP-UPLC method was developed and validated for the quantitative determination of SHK in MME formulations and biological matrices using an AQbD-driven approach. The method demonstrated acceptable accuracy, precision, sensitivity, and robustness in accordance with ICH guidelines, supporting its suitability for routine analysis. Pharmacokinetic investigations suggested that intranasal administration of SHK-loaded MME may enhance systemic and brain exposure compared to oral administration, with improvements likely attributable to nanoscale droplet size and
mucoadhesive properties enabling improved nasal residence and potential nose-to-brain transport. The method also demonstrated good environmental performance based on green analytical chemistry assessment tools. However, the study is limited by evaluation in a single animal model with a relatively small sample size, absence of long-term toxicity and biodistribution data, and lack of detailed mechanistic
insights into nose-to-brain transport. Further preclinical and clinical studies are therefore required to confirm the therapeutic relevance and translational potential of the proposed system. Overall, the proposed method provides a robust and sustainable analytical platform with potential application in pharmaceutical and CNS-targeted drug delivery research.
 
, Claims:We claim:
1. An Analytical Quality by Design (AQbD)-driven reverse-phase ultra-performance liquid chromatographic (RP-UPLC) method for quantitative determination of Shikonin in a mucoadhesive microemulsion and biological matrices, comprising:
(a) performing risk assessment for identification of critical analytical variables;
(b) screening analytical variables using a Taguchi L8 orthogonal array;
(c) optimizing the identified critical analytical variables using a Box–Behnken response surface methodology; and
(d) quantitatively determining Shikonin using optimized chromatographic conditions comprising a C18 column, an acetonitrile and 0.1% v/v formic acid in water mobile phase in an 80:20 v/v ratio, a flow rate of 1.0 mL/min, a column temperature of 30 °C and detection at 254 nm.
2. A Shikonin-loaded mucoadhesive microemulsion for intranasal administration, comprising Shikonin, castor oil in an amount of 5–10%, a surfactant–cosurfactant mixture in an amount of 20–40%, distilled water and chitosan, wherein 10 mg of Shikonin is incorporated into the castor oil and surfactant–cosurfactant mixture; wherein the surfactant–cosurfactant mixture comprises Tween 80 and propylene glycol in equal proportions, and wherein the microemulsion comprises nanoscale droplets.
3. The Shikonin-loaded mucoadhesive microemulsion as claimed in claim 2, wherein the microemulsion has a mean droplet size of 73.8 ± 0.63 nm, polydispersity index of 0.267 ± 0.085, zeta potential of 32.68 ± 0.157 mV and entrapment efficiency of 87.9 ± 0.25%.
4. A process for preparation of the Shikonin-loaded mucoadhesive microemulsion as claimed in claim 2, comprising incorporating Shikonin into a mixture comprising castor oil and the surfactant–cosurfactant mixture, gradually adding distilled water dropwise under mild agitation at room temperature to form an oil-in-water microemulsion, subjecting the microemulsion to probe ultrasonication at 20 kHz for approximately 5–15 minutes, dissolving chitosan in 1% v/v aqueous acetic acid and gradually adding the chitosan solution to the microemulsion under constant stirring to obtain the mucoadhesive microemulsion.
5. The process as claimed in claim 4, wherein the surfactant–cosurfactant mixture comprises Tween 80 and propylene glycol in equal proportions and the resulting microemulsion is transparent and isotropic.
6. A reverse-phase ultra-performance liquid chromatographic method for quantitative determination of Shikonin in the mucoadhesive microemulsion and biological matrices, comprising chromatographic separation using a C18 column, an acetonitrile and 0.1% v/v formic acid in water mobile phase in an 80:20 v/v ratio, a flow rate of 1.0 mL/min, a column temperature of 30 °C and detection at 254 nm.
7. The reverse-phase ultra-performance liquid chromatographic method for quantitative determination of Shikonin as claimed in claim 6, wherein the method is developed using an Analytical Quality by Design approach comprising risk assessment using an Ishikawa diagram and failure mode and effects analysis, screening using a Taguchi L8 orthogonal array and optimization using Box–Behnken response surface methodology.
8. The reverse-phase ultra-performance liquid chromatographic method for quantitative determination of Shikonin as claimed in claim 6, wherein the method exhibits a linearity range of 2–12 µg/mL with a correlation coefficient of 0.9997, a limit of detection of 0.396 µg/mL and a limit of quantification of 1.191 µg/mL.
9. The reverse-phase ultra-performance liquid chromatographic method for quantitative determination of Shikonin as claimed in claim 6, wherein the method exhibits accuracy with recovery of 98.16 ± 1.25% at 50%, 100.56 ± 0.96% at 100% and 99.67 ± 0.271% at 150% concentration levels, with precision and robustness having %RSD values below 2%.
10. The Shikonin-loaded mucoadhesive microemulsion as claimed in claim 2, wherein intranasal administration in male Wistar rats at 30 mg/kg provides a brain Cmax of 245.20 ± 0.25 ng/g at 2 hours and a brain AUC0–t of 2400.0 ± 210.0 h·ng/g, and a plasma Cmax of 195.40 ± 0.28 ng/mL at 2 hours and a plasma AUC0–t of 2200.0 ± 140.0 h·ng/mL.