INTRODUCTION:

Setracanozole, a synthetic antifungal agent, has gained significant attention for its remarkable efficacy in treating many fungal infections¹. It belongs to the azole class, inhibiting lanosterol 14α-demethylase, an enzyme crucial for ergosterol biosynthesis, thus disrupting the fungal cell membrane's integrity². This disruption impairs the growth and survival of various fungal pathogens, including dermatophytes, Candida species, and Aspergillus species³. Setracanozole's broad-spectrum activity makes it versatile for treating invasive candidiasis, dermatophyte infections like onychomycosis and tinea corporis, and Malassezia-related skin conditions^1-4.

Setracanozole is a azole compound featuring a triazole ring with dichlorophenyl and tetramethylcyclopropylphenyl groups (Figure-1). It exhibits a pKa around 6.7, indicating its acidic nature crucial for inhibiting fungal enzyme activity⁵. It is sparingly soluble in water but dissolves well in organic solvents like methanol, ethanol, acetonitrile⁵.

Figure 1. Chemical structure of Setracanozole nitrate

The analytical quality by design (AQbD) technique minimizes experimental time and cost while adhering to FDA and ICH criteria⁶. Pharmaceutical companies can use QbD principles to identify and reduce sources of variability, improve method robustness, and achieve performance criteria across the product lifecycle^6,7. AQbD's primary goal is to identify failure modes and create a robust design space within system suitability requirements, allowing method flexibility without revalidation or regulatory assessment when introduced during development^6-8.

Currently, many types of analytical procedures are disclosed in reputed journals to analyze Setracanozole. Several simultaneous analytical procedures were established for Setracanozole and other drugs in combined formulations^9-10. Correspondingly, a few methods were employed for Setracanozole analysis alone in marketed formulation^11-15. The reported HPLC methods recognized Many drawbacks or downsides, such as longer elution time and less sensitivity ^11-16. Besides that, adequate stability representing the HPLC procedure was not found during the in-depth exploration of literature^13-15. To full fill that gap, a stability-indicating HPLC method with efficient separation of intended analyte and degradants with shorter elution time, good sensitivity, remarkable resolution, and economical mobile phase was developed and validated as per ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use)¹⁷.

MATERIALS AND METHODS:

Setracanozole in pure form was purchased from Spectrum Pharma in Hyderabad. All analytical and HPLC grade solvents were purchased from a nearby Merck India Limited vendor. Shimadzu HPLC (LC-20A, UV detector, and LC solution software) was used for the procedure. Water (Milli-Q) and a 1 mg sensitive balance (SCALETEC-SAB224CL) were also utilized.

Method development:

The central composite design (CCD) design of randomized response surface study was used to optimize the method. Preliminary experiments are necessary to optimize the ultimate method. To confirm the method's quality and robustness, a total of two factors, or critical quality attributes (CQA), such as the volume of organic mobile phase (Methanol) and flow rate, were thought to be optimized. These factors can directly affect the dependent responses, or quality target product profile (QTPP), of the method, including retention time (RT) and Tailing factor (TF). For two CQA, the CCD yielded 13 runs with 5 centre points (Table-1). To forecast acceptable QTPP parameters with an analytical target profile, optimal circumstances were determined by considering statistical data such as ANOVA and desirability function.

Table-1. CCD with two independent variables (CQA) with two dependent responses (QTPP’s)

		Factor 1	Factor 2	Response 1	Response 2
Std	Run	A: Methanol	B: Flow rate	RT	TF
		ml	ml/min	MIN
1	12	80	0.8	3.48	2.98
2	3	90	0.8	5.6	1.54
3	2	80	1	2.98	2.78
4	5	90	1	4.2	1.35
5	8	77.9	0.9	2.58	3.15
6	6	92.1	0.9	4.87	1.15
7	1	85	0.8	5.18	2.25
8	9	85	1	4.01	2.12
9	11	85	0.9	4.71	2.06
10	10	85	0.9	4.71	2.16
11	13	85	0.9	4.71	2.07
12	7	85	0.9	4.61	2.15
13	4	85	0.9	4.69	2.16

Optimized chromatographic conditions:

Sertaconazole was successfully separated using Shimadzu C18(250 x 4.6mm, 5µ)), a mobile phase of 0.1%W/V formic acid and Methanol in a 10:90 ratio, a flow rate of 1 ml/min, an injection volume of 20µl, a detection wavelength of 245nm, and a column temperature of 30⁰C. The mobile phase was used as a diluent to create the working standard, sample, and forced degradation solution.

Preparation of standard solution:

Weigh 10mg of Sertaconazole accurately and transfer it into 100ml of volumetric flasks, add 60ml of methanol, sonicate to dissolve, and make up the volume with methanol. Pipette out 1ml of this solution into 10ml of volumetric flask and dilute to the volume with methanol to gain10µg/mL of Sertaconazole.

Preparation of test solution:

Weigh and transfer powder equivalent to 10mg of Sertaconazole into 100ml volumetric flask, add 60ml of methanol and sonicate for 5 minutes, and dilute to the volume with methanol, filter the solution through 0.45 µm Nylon filter. Pipette out 1ml of this solution into 10 ml volumetric flask and dilute it to the volume with methanol to get a concentration of about 10µg/mL.

METHOD VALIDATION:

System Suitability Test:

The system suitability test of the current method was carried out by injecting standard concentration in 6 replicates, and parameters like percentage relative standard deviation (%RSD), USP tailing factors (T), and USP plate count(N) were evaluated for the obtained chromatograms.

Linearity:

The linearity of the current method was assessed by injecting a series of working standard concentrations ranging from 2.5µg/mL to 30µg/mL of Sertaconazole into the HPLC system under optimized chromatographic conditions. A linearity graph was then plotted with concentration on the x-axis and peak area on the y-axis, and the regression coefficient (r²) value was determined to evaluate the method's linearity.

Accuracy:

The accuracy of the method was evaluated using recovery studies. Known amounts of the sample solution were spiked with the drug at three different concentration levels: 50%, 100%, and 150% of the standard concentration. Each spiked level was injected in triplicate. The percentage recovery at each concentration level was then determined by calculating the mean of the measured concentrations compared to the amount of drug spiked.

Precision:

The precision of the method was assessed for both intra-day and inter-day variability. For intra-day precision, the standard concentration was injected six times on the same day. The same concentration level was injected thrice daily for three consecutive days for inter-day precision. The percentage relative standard deviation (%RSD) was calculated for the peak areas obtained in each instance.

Specificity:

A 10 µL volume of the prepared blank solution, 100% level pure working standard solution, and standard solution with placebo were individually injected sequentially. The retention times (RT) of the two intended analytes in the individual injections of the standard solution, sample solution, forced degradation solution, and standard solution spiked with placebo were observed to assess any interference with the peak of Favipiravir in the obtained chromatograms.

Sensitivity:

Sensitivity in terms of limit of detection (LOD) and limit of quantification (LOQ) were assessed by standard deviation approach.

Formula:

LOD=3σ/S, LOQ = 10 σ/S

Where, σ is the standard deviation of the intercept of responses

S is the slope of the calibration curve

Robustness:

The method's robustness was assessed by altering the method parameters, such as flow rate (±0.1mL/min), mobile phase composition (±1ml), and maximum absorption wavelength (±2nm). To calculate the % RSD value for essential system suitability parameters, 10 µg/mL of Sertaconazole was injected three times.

Assay:

To evaluate the % purity of the marketed formulation, both standard and sample solutions containing 20µg/mL of Favipiravir were injected into the system sequentially.

Forced degradation studies:

Forced degradation studies, in which a drug substance or drug product is intentionally exposed to increasingly intense stress conditions, can be used to examine an analytical method's stability-indicating character. Forced degradation studies help to ensure the chemical stability of the drug ingredient or drug product. The degradation studies procedures were adopted asper ICH Q1A, QIB and Q2B^17-26.

Acid hydrolysis:

5ml of standard stock solution (0.1mg/mL of Sertaconazole) mixed with 5ml of 0.1N HCl and sonicate for 1hr, further neutralized with the same strength of NaOH. The above solution was diluted again to get a 10µg/ml concentration of Sertaconazole.

Base hydrolysis:

5ml of standard stock solution (0.1mg/mL of Sertaconazole) mixed with 5ml of 0.1N NaOH and sonicate for 1hr, further neutralized with the same strength of HCl. The above solution was diluted again to get a 10µg/ml concentration of Sertaconazole.

Thermal degradation:

5ml of standard stock solution (0.1mg/mL of Sertaconazole) was exposed to 105°C/75% RH condition in the heating chamber for 48 \hr. Further cooled to room temperature, the solution was diluted to get a 10µg/ml concentration of Sertaconazole.

Photodegradation:

5ml of standard stock solution (0.1mg/mL of Sertaconazole) was exposed to UV light at a wavelength of 254nm in dark control for 48hr. The resulting solution was further diluted to obtain a solution of 10µg/ml of Sertaconazole.

RESULTS AND DISCUSSION:

Optimization of chromatographic conditions by AQBD:

Optimization of the method was confirmed from the ANOVA test, where the predicted R² values of both responses (RT and TF) were in considerable harmony with the adjusted R² values; i.e., the difference is less than 0.2. Adequate precision measures the signal-to-noise ratio. A ratio of more than 4 indicates an acceptable signal, which reveals that the model can be desirable in finding the way the design space (Table 2). The surface response graphs for desirability, TF, and RT of the recommended method are shown in Figure- 2. The confirmed location of methanol (90%) and flow rate (1.0mL/min) were suggested, with a desirability of 0.981 (Figue-2). Successful separation of analytes was obtained using Shimadzu C18 (250 x 4.6mm, 5µ), column, and a mobile phase of methanol: formic acid (90:10) with 1ml/min flow rate. The RT and TF of Sertaconazole were found to be 2.21min and 1.39, respectively (Figure 3). The observed values of RT (4.20) Tailing factor (1.41) were very close to predicted values (RT: 4.17 and TF: 1.36). The deviation between predicted and observed values was less than 5%, significantly revealing that the obtained response values obey the design space. The polynomial equations suggested for this model were as follows.

RT= 4.69 + 0.82A-0.51B-0.22AB-0.48A²-0.12B²

TF=2.14-0.71A-0.086B

Table 2. Fit statistic parameters of retention time and tailing factor

Parameter	TF	Retention time
SD	0.0442	0.0637
Mean	2.15	4.33
C.V. %	2.06	1.47
R²	0.9953	0.9968
Adjusted R²	0.9943	0.9945
Predicted R²	0.9934	0.9833
Adeq Precision	95.18	71.44

Figure 3. Optimized chromatogram of Sertaconazole

Method validation:

System suitability parameters include % RSD, TF and plate count values were within the Q2 specifications ICH acceptance limits (Table-3).

Table 3: System suitability of standard solution of Sertaconazole

Peak area	RT	TF	Plate count
163021	4.201	1.41	4401
165498	4.261	1.409	4488
166932	4.212	1.398	4413
165150	4.215	1.396	4394
159871	4.209	1.42	4401
160279	4.204	1.397	4469
163458.5	4.217	1.405	4427.667
2907.119	0.022154	0.009592	40.29723
1.778506	0.52535	0.682681	0.910123

Sertaconazole has a computed R² value of 0.999 over a concentration range of 2.5 to 30µg/ml (Figure-4), indicating significant linearity within the range. The mean percentage recovery of Sertaconazole in spiked standard solutions was 100%±2, showing method accuracy (Table-4). The percentage RSD values for retention time (RT) and peak area responses from six consecutive injections of standard solutions ranged from 0.74 to 1.47 (Table-5), indicating the new method's precision. Furthermore, purposeful and modest modifications in method parameters had no significant effect on performance, with %RSD values falling within ICH acceptable limits (Table-6), demonstrating the procedure's robustness. Interference at RT Sertaconazole was not seen with the RT of blank, degradants, or placebo, demonstrating the method's specificity to Sertaconazole. Sertaconazole 's limit of detection (LOD) and limit of quantification (LOQ) are 0.071μg/ml and 0.022μg/ml, respectively. The percentage purity of Sertaconazole in tablet dosage form found to be 99.23% W/V.

Figure 4. Linear curve of Sertaconazole concentrations ranges from (2.5 to 30µg/ml)

Table 4. Percentage recovery of Sertaconazole in different levels of spiked solutions

% Level	Average amount of standard added (µg/ml)	Average amount of standard recovered (µg/ml)	% Mean recovery
50	5.00	5.082	101.64
100	10.00	9.91	99.1
150	15.00	15.263	101.75

Table 5. Precision data of standard solution of Sertaconazole

Parameter	Intraday precision		Inter-day precision
Parameter	RT	Peak area	RT	Peak area
Mean	4.206	160411	4.204	159641
SD	0.062	1193.02	0.057	2318
%RSD	1.47	0.74	1.35	1.45

Table 6. Robustness of standard solution of Sertaconazole

Parameter		Mean peak area	SD	%RSD
Column temperature (⁰C)	28	159772	1265.39	0.79
Column temperature (⁰C)	32	159233	1823.21	1.14
Detection wavelength (nm)	243	160939	1100.82	0.68
Detection wavelength (nm)	247	163547	299.29	0.18
Flow rate (ml/min)	0.8	154008	1202.80	0.78
Flow rate (ml/min)	1.2	162867	1827.36	1.12

In most stability-indicating procedures, a 20% degradation in the drug component is deemed adequate. The percentage degradation of Sertaconazole was determined by comparing the peak areas of freshly prepared and forced standard solutions. Figure 5 displays the chromatograms derived from acid, alkali degradation, blank and fresh test samples. The results of forced degradation (FD) experiments clearly establish the method's capacity to indicate stability (Table-7). Sertaconazole did not degraded considerable amount under Acid, photolytic and thermal degradation conditions, confirming its stability in these environments. However, Sertaconazole is very vulnerable to the basic climate due to increased degradation under specified conditions. As a result, it is claimed that the proposed technique for determining the stability of API and dosage forms offers stability-indicating data.

Table-7. % Degradation of standard solution of Sertaconazole at various stress conditions

Type of degradation	Degradation conditions	% Drug degraded
Acid hydrolysis	0.1N HCl for 24hr	1.08
Base hydrolysis	0.1N NaOH for 24hr	13.26
Photolytic degradation	UV light (254nm) for 48hr	1.34
Thermal degradation	105°C/75% RH for 48hr	2.1

In contrast to published strategies where the RT of Sertaconazole was more than 5min, the method's affordability has been shown by a mobile phase of methanol and 0.1% formic acid in 90:10v/v ratios with 4.2min of RT. The present methodology ought to be employed to examine more samples quickly. The approach for Sertaconazole analysis has been shown to have improved accuracy, sensitivity, and significant specificity based on the actual data for the validation parameters. The LOD and LOQ of Sertaconazole (0.07 and 0.22µg/ml) results were remarkably effective when compared to previously published methods^9,10. Stability studies are far more important criteria for assessing the quality of pharmaceutical products and pharmaceutical substances. The evaluation of Sertaconazole degradation under various stress environments illustrates the procedure's ability to determine the stability of the desired analyte. No single method has implemented the Analytical Quality by Design (QbD) approach to develop a robust HPLC method for Sertaconazole. The improvements in the proposed technique over earlier methods warrant its adoption.

CONCLUSION:

An economical, sensitive, accurate, and easy RP-HPLC approach with high sensitivity has been proposed to analyze Sertaconazole in bulk and tablet forms. The CCD optimized the method, which shows its relevance under ICH Q8 guidelines. The evaluation of analytes under various forced or stressed situations confirms the stability-indicating property of the developed method. The optimized setting of the suggested HPLC technique resulted in a shorter RT of 4.2 minutes and excellent sensitivity for Sertaconazole. As a result, the established HPLC technique is suitable for pharmaceutical applications.

ACKNOWLEDGMENT:

The authors are thankful to the Department of Pharmaceutical Analysis, CHAITANYA Deemed to be University, and GITAM Deemed to be University, Hyderabad, for continuous support.

AUTHORS’ CONTRIBUTIONS:

All the authors contributed equally to the design and frame of the work, acquisition, and interpretation of data, and manuscript preparation; all authors have read the prepared manuscript and approved it for publication.

CONFLICT OF INTEREST:

No conflict of interest from all the authors.

LIST OF ABBREVIATIONS:

CCD: Central Composite Design

CQA: Critical Quality Attributes

QTPP: Quality Target Product Profile

FD- Forced Degradation

ICH- International Committee for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use

RP- HPLC- Reverse Phase High-Performance Liquid Chromatography

LOD: Limit of Detection

LOQ: Limit of Quantification

RSD: Relative Standard Deviation

SD: Standard Deviation

RT: Retention Time

TF: Tailing Factor

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Received on 15.07.2024 Revised on 05.11.2024

Accepted on 10.01.2025 Published on 05.09.2025

Available online from September 08, 2025

Research J. Pharmacy and Technology. 2025;18(9):4225-4231.

DOI: 10.52711/0974-360X.2025.00607

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