Development and Validation of related Substances method for Deflazacort Suspension by High Performance Liquid Chromatography using AQbD Approach

Vikram Gharge, Pranav Bang, Sachin Khopade, Krishna Kinage, Balasaheb Jadhav

Research and Development, Zuventus Healthcare Limited, Hinjawadi, Pune – 411057, Maharashtra, India.

*Corresponding Author E-mail: Vikram.gharge@zuventus.com

ABSTRACT:

The stability-indicating method for Deflazacort Suspension was developed using the Analytical Quality by Design (AQbD) approach, focusing on process control and understanding. A multilevel factorial design was used to optimize the gradient mode's time and mobile phase ratio. Sophisticated software like Design Expert and Minitab aided in chromatographic condition optimization. The method utilizes a Zorbax Eclipse XDB C18 column (150mm x 4.6mm, 5µm particle size), with a flow rate of 1.0 mL/min, and monitoring the analyte with a UV/PDA detector at of 245nm wavelength known for its reliability. It employs two mobile phases: A, consisting of Water, Tetrahydrofuran, and Acetonitrile (91:3:6, v/v/v), and B, comprising Water, Tetrahydrofuran, Acetonitrile, and Methanol (4:2:74:20, v/v/v/v). Statistical analysis confirms the importance of these components in achieving effective separation and detecting related substances. The validation of method was done as per ICH guidelines. Linearity, specificity, Limit of Detection (LOD), Limit of Quantification (LOQ), precision, accuracy, solution stability, and robustness parameters were validated. The method was validated to ensure reliability and accuracy, demonstrating linearity from 0.02 to 1.2ppm with a correlation coefficient (r2 > 0.99) at 245nm. This confirms the method's ability to precisely quantify related substances in Deflazacort Suspension. The stability of Deflazacort Oral Suspension was rigorously assessed, including a forced degradation study. Critical system suitability parameters, tailing factor, and theoretical plate, met acceptable limits, confirming the method's efficiency and resolution. Successful separation of degradation peaks from each other and the main peak was achieved during the study. The method's robustness was confirmed with variations under 2%, ensuring consistent and reproducible results despite minor changes in conditions. Peak purity analysis showed no co-eluting peaks, confirming the method's specificity. These findings validate the stability-indicating nature of the chromatographic method for Deflazacort Oral Suspension analysis, ensuring its reliability for quality control.

KEYWORDS: Deflazacort Oral Suspension, Design expert, HPLC, Minitab, Quality by Design, Related substances.

INTRODUCTION:

Deflazacort is chemically described as [2-[(1S,2S,4R,8S,9S,11S,12S,13R)-11-hydroxy-6,9,13-trimethyl-16-oxo-5-oxa-7-azapentacycloicosa-6,14,17-trien-8-yl]-2-oxoethyl] acetate. It is characterized by its non-polar nature, with a pKa of 14.74 and a log P of 2.56. This compound exhibits insolubility in water while demonstrating solubility in Methanol, Acetonitrile, and Acetone. Deflazacort features various functional groups, including the ketone group, ester group, hydroxyl group, among others¹. (Refer Figure 1).

Figure. 1 Deflazacort

Deflazacort, a prednisolone derivative, effectively treats conditions like rheumatoid arthritis and asthma due to its anti-inflammatory and immunosuppressive properties, with a favourable safety profile compared to other glucocorticoids². It is also valuable in organ transplants, helping prevent rejection by modulating immune responses^3-7. Available in tablet and oral suspension forms, Deflazacort can be accurately analysed using various techniques^8-11, including High-Performance Liquid Chromatography (HPLC)^12-13, spectrophotometry^14-15, LC/MS¹⁶, and High-Performance Thin-Layer Chromatography (HPTLC), with reversed-phase HPLC with UV detection noted for its reliability and precision^17-18.

Various methods have been developed for quantifying Deflazacort in pharmaceutical formulations, highlighting the need to tailor analytical techniques for specific applications^19-20. This study aimed to create a stability-indicating HPLC method for Deflazacort Suspension, guided by Analytical Quality by Design (AQbD) principles in line with ICH guidelines. AQbD emphasizes a systematic approach to managing critical method variables, enhancing performance, robustness, and adaptability for ongoing refinement while ensuring compliance with system suitability requirements^21-24.

A reverse-phase C18 column measuring 150mm x 4.6 mm (with 5µm particle size) was employed alongside a mobile phase A composed of Water, THF and Acetonitrile in a ratio of 91:3:6 (v/v), and a mobile phase B consisting of Water, Tetrahydrofuran, Acetonitrile, and Methanol in a ratio of 4:2:74:20 (v/v/v/v). The flow rate was maintained at 1.0mL/min, with detection at 245 nm, demonstrating excellent selectivity, precision, and linearity from the Limit of Quantification (LOQ) to 150%. Preliminary experiments confirmed the suitability of this reversed-phase HPLC method for analyzing potential impurities in Deflazacort. This study aims to develop, optimize, and validate an HPLC technique for routine analysis of oral suspension formulations containing Deflazacort, using an Analytical Quality-by-Design (AQbD) approach to ensure consistent and reliable results while addressing variations in formulation and environmental factors^25-28.

MATERIAL AND METHOD:

Materials:

Acetonitrile, Tetrahydrofuran, and Methanol sourced from Zuventus Healthcare Ltd. were utilized in the study. All solutions were prepared using purified water to ensure optimal quality. The drug substance Deflazacort and Cortimax® 5mg/5mL, a 30mL bottle of the commercial formulation by Zuventus Healthcare Ltd., were employed in the experiment.

Instrumentation:

A reversed-phase HPLC system was used, featuring a Waters setup with Empower software and a Shimadzu system with Lab Solution software. The Waters system included a Zorbax Eclipse XDB C18 column (150mm x 4.6mm, 5µm) and a quaternary pump system with a UV detector and photo-diode array connected to Empower 3 for signal monitoring. For Analytical Quality by Design (AQbD) trials, Design Expert version 11.0 and Minitab version 18.0 were employed to systematically explore critical method variables, optimizing performance and robustness.

Chromatographic conditions

A Zorbax Eclipse XDB C18 HPLC column (150mm x 4.6mm, 5µm particle size) was employed for chromatographic separation. The mobile phase consisted of two components: mobile phase A, a mixture of Water, Tetrahydrofuran and Acetonitrile in a ratio of 91:3:6 (v/v/v), and mobile phase B, a blend of Water, Tetrahydrofuran, Acetonitrile, and Methanol in a ratio of 4:2:74:20 (v/v/v/v). The flow rate was maintained at 1.0 mL/min. The gradient program was programmed as follows: initially, the system started with 85% of solvent A, then at 2.0 minutes, it remained at 85% A, followed by a linear gradient to 75% A at 15 minutes, 60% A at 25 minutes, 20% A at 30 minutes, holding at 20% A until 35 minutes, returning to 85% A at 36 minutes, and maintaining at 85% A until 40 minutes. For detection, a UV detector was set at a wavelength of 245nm. The column temperature was held constant at 40°C, while the sample temperature was maintained at 5°C to ensure stability. The injection volume for each sample was set at 50µL.

Method:

Standard preparation:

A standard solution was prepared containing 0.8ppm of Deflazacort, 1.6ppm of Deflazacort 21-Alcohol, and 0.8 ppm of Deflazacort Impurity A.

Sample preparation:

Ten grams of the sample were measured and placed in a 50mL volumetric flask. Approximately 10mL of methanol was added, and the mixture was sonicated for 10minutes. After that, 20mL of Acetonitrile was introduced, followed by another 10minutes of sonication with shaking. The volume was then brought up to the mark with acetonitrile and mixed thoroughly. The solution was filtered through a 0.45µm nylon filter, discarding the first 5mL of the filtrate. Finally, 4.0mL of the filtered solution was transferred to a 10mL volumetric flask, and the volume was adjusted to the mark with water before mixing.

AQbD approach for method development:

We identified the Analytical Target Profile (ATP) and Critical Quality Attributes (CQA) based on a thorough understanding of method parameters. To optimize the method systematically, we used a factorial design approach, summarized in Table 1.

Table-1: - Analytical QbD Parameters

QTPP

CQA

Factorial Design

1.Retention time

2.Resolution

1.Composition of the mobile phase

2.gradient programme

1.Multilevel Factorial Design exploratory approach

Analytical method validation:

Method validation is crucial for confirming the appropriateness of an analytical process. Following the ICH Q2 (R1) guidelines, the HPLC method used to analyse Deflazacort's impurity profile was validated by assessing several parameters, including Accuracy, Precision, Specificity, Limit of Detection (LOD), Limit of Quantification (LOQ), Linearity, Range, and Robustness. Linearity was evaluated from the LOQ up to 150%, and correlation coefficients were calculated. Precision was assessed through repeatability tests using six samples, while accuracy was evaluated through a recovery study conducted at three different levels. The method's robustness was tested by making slight changes to the chromatographic conditions to gauge its resilience.

RESULTS:

Method development and optimization:

A Zorbax Eclipse XDB C18 HPLC column (250mm x 4.6mm, 5µm) was initially used. Mobile phase A was a 92:8 (v/v) mixture of Water and Acetonitrile, while mobile phase B consisted of Water, Acetonitrile, and Methanol in a 4:75:21 (v/v) ratio. The flow rate was set to 1.0 mL/min, with samples and standards prepared in Acetonitrile. However, an unknown peak was observed overlapping with placebo peaks. (Refer to Figure 2).

To enhance the separation of the placebo peaks, Tetrahydrofuran (THF) was incorporated into the mobile phase. In the revised method, mobile phase A was adjusted to a 92:6:2 (v/v) ratio of Water, Acetonitrile, and THF, while mobile phase B was configured to a 4:74:2:20 (v/v) ratio of Water, Acetonitrile, Methanol, and THF. The other chromatographic conditions remained the same, resulting in successful separation of the placebo peaks from the unknown peak. (Refer to Figure 3).

A Zorbax Eclipse XDB C18 HPLC column (150mm x 4.6 mm, 5 µm) was used to tackle several challenges. The main objectives were to reduce Deflazacort's retention time, enhance resolution between the placebo and unknown peak, and improve the shape of the unknown peak. (Refer to Figure 4).

To improve peak shape, optimization trials for the diluent revealed that a mixture of Methanol, Acetonitrile, and Water was effective. The wavelength was also optimized to 245nm to reduce interference from placebos. Final chromatographic conditions were refined using an Analytical Quality by Design (AQbD) approach, with further parameter optimization conducted through a multilayer factorial design method for comprehensive enhancement of method parameters.

Method Development by AQbD:

Analytical target profile:

The Analytical Target Profiles (ATPs) chosen for optimizing the HPLC chromatographic conditions were resolution and retention time.

Critical quality attributes:

The time vs. concentration of mobile phase A in the gradient program is identified as a critical quality parameter.

Factorial design:

The multilevel factorial design was chosen as the preferred approach to develop the proposed HPLC method. Table 2 illustrates the optimization of various parameters.

Design space:

By employing a multilevel factorial design with two factors in Minitab, we identified the design space, resulting in eight runs. Interaction and main effect plots, along with a quadratic design model, were utilized. Time and concentration were measured against two responses: retention time and resolution between placebo and Deflazacort Unknown Imp. The data were summarized using the proposed multilevel factorial design.

Table 2. Parameter optimization for analytical method by making use of multilevel factorial design

Standard Order	Factor 1 Time	Factor 2 Concentration of Mobile phase A	Response 1 Retention Time of Deflazacort	Response 2 Resolution between placebo and Deflazacort unknown impurity
1	15	61	19.21	1.69
2	15	65	21.64	2.31
3	15	70	27.11	3.18
4	15	79	39.60	5.64
5	20	61	22.34	3.09
6	20	65	24.63	3.81
7	20	70	29.68	4.59
8	20	79	39.82	6.07

Optimized condition:

The process of developing the optimal conditions involved fine-tuning the HPLC settings and anticipating responses. Additionally, Minitab software (version) was utilized to analyse each response obtained from various experimental runs. The observed response value was determined by conducting HPLC experiments at specific time points and concentrations. Subsequently, the D Optimal was calculated by comparing the observed value to the expected values. The final chromatographic conditions are as follows: employing a Zorbax Eclipse XDB C18 HPLC column with dimensions of 150mm x 4.6mm and a particle size of 5µm, with the column temperature set at 40°C. The flow rate was maintained at 1.0mL/min, and the gradient program was set as follows: time / % of solvent A: 0/85, 2.0/85, 20/79, 25/60, 30/20, 35/20, 36/85, 40/85. The UV detector wavelength was set at 245nm, and a 50µL injection volume was used. The sample temperature was kept at 5°C (Refer to Figure no. 6).

Method validation results:

System suitability:

Various parameters were calculated and compared against the method's specifications, as detailed in Table 3. Deflazacort and related impurities served as reference standards for evaluating these characteristics. The system's acceptability was then assessed and compared to the criteria outlined in the ICH Q2 (Rl) guidelines²⁹.

Table 3: - System suitability test (SST) data. (Average of 6 Observations)

Analyte	Rt	%RSD	RRT	Rs	Tf	Rf
Deflazacort	25.48	0.06	1.0	5.6	1.04	1.0
Deflazacort 21- Alcohol	14.58	0.03	0.57	3.8	1.06	1.02
Deflazacort Impurity A	22.07	0.04	0.87	6.9	1.02	0.99

Rt; Retention Time in Min, %RSD-Reproducibility, RRT- Relative Retention Time, Rs-Resolution, Tf- Tailing Factor, Rf- Relative Retention Factor

Precision:

When evaluating the system's precision through six replicate injections of the standard solution, Deflazacort, Deflazacort 21-alcohol, and Deflazacort Impurity A exhibited %RSDs of 0.22%, 0.11%, and 0.15%, respectively. To further assess the method's precision, six separate sample solutions were injected, and the % RSD of each replicate injection's peak response was determined (Table 4), with a maximum allowed % RSD of 5%. The precision assessment revealed sample peaks at 25.48, 14.58, and 22.07 for Deflazacort, Deflazacort 21-Alcohol, and Deflazacort Impurity A retention times respectively. Table 4 demonstrates that both intra- and inter-day precisions fell within the range of 0.20–0.69% and 0.34–0.87% (%RSD), respectively.

Table 4: - System Precision data.

Name of Analyte	Deflazacort	Deflazacort 21-Alcohol	Deflazacort Impurity A
Peak Area (Mean) n=6	100104	191577	99577
SD (Standard Deviation)	181.0	152.4	223.1
%RSD	0.22	0.09	0.15
Intra-day precision (RSD%) n=6	0.69	0.20	0.54

Linearity:

For quantification, the peak areas of each compound were recorded. A linear relationship was identified within the concentration ranges of 0.020–1.2µg/mL for Deflazacort, 0.020–2.4µg/mL for Deflazacort 21-Alcohol, and 0.020–1.2µg/mL for Deflazacort Impurity A, as shown by plotting the peak areas against concentration. Evaluation metrics, including slope, intercept, and correlation coefficient, were calculated, leading to the construction of standard curves. (Figure 7) The calibration equations and correlation coefficients for Deflazacort and its related impurities are detailed in Table 6.

Table No. 7: - Results obtained with force degradation of sample solution

Sr. No.	Stress condition	Peak purity Index	Single point threshold	Peak purity criteria of Deflazacort peak	% Deflazacort 21-alcohol	% of Deflazacort Impurity A	% Any individual impurity	% Total impurities
1	As such sample	1.000	0.999	Pass	BDL	BDL	0.056	0.056
2	Acid degradation: 5 mL 0.1M HCl, keep Test solution in water bath at 70°C for 60 minutes.	1.000	0.999	Pass	1.103	BDL	1.950	3.379
3	Alkali degradation: 5mL 0.01M NaOH, keep Test solution at room temperature for 10 minutes.	1.000	0.999	Pass	BDL	BDL	BDL	BDL
4	Oxidation degradation: 5ml 3.0% H₂O₂, keep Test solution in water bath at 70°C for 60 minutes.	1.000	0.999	Pass	8.483	BDL	1.666	11.197
5	Hydrolysis degradation (solid state): 5mL water, keep Test solution in water bath at 70°C for 60 minutes.	1.000	0.999	Pass	BDL	BDL	0.049	0.049
6	Heat degradation (solid state): Expose the sample at 60°C for 24 hours in vacuum oven.	1.000	0.999	Pass	BDL	BDL	BDL	BDL
7	Humidity degradation: Expose the sample at 75% RH for 24 hours.	1.000	0.999	Pass	BDL	BDL	BDL	BDL
8	Photolytic degradation: keep in a photo stability chamber for exposure of about 1.2 million-lux hours and near UV at 200-watt hrs/m².	1.000	0.999	Pass	BDL	BDL	0.102	0.203

Robustness studies:

In robustness and ruggedness studies, a sample solution of Deflazacort at a concentration of 80ppm was employed. Robustness was examined by deliberately introducing slight variations in intrinsic method parameters such as pump flow rate, mobile phase composition, and column temperature. The %RSD for peak area was found to be less than 2% when these parameters were altered. Please refer to Table No. 6 for details.

Force degradation study:

For a deeper comprehension of the stability of drug substances and drug products, as well as to identify degradation pathways and products, forced degradation studies are employed. This research assists in developing analytical methodologies and aids in formulating the most stable product. Please refer to Table No. 7 for the results obtained from the forced degradation of the sample solution under various conditions.

DISCUSSION:

Analytical quality-by-design principles were implemented in developing an HPLC method for quantifying Deflazacort impurities in oral suspension. Retention time and resolution were identified as the analytical target product profiles, with time and mobile phase concentration recognized as critical quality factors influencing them. Minitab Software Version 17.0 was utilized to apply Multilevel Factorial Design for these factors, with time at two levels and mobile phase concentration at four separate levels. Through risk assessment analysis, essential factors affecting the analytical target profile were identified. Variables such as flow rate and column temperature underwent robustness testing in chromatographic separation, while variability in column selection, instrument configuration, injection volume, and gradient program remained constant.

The quality-by-design methodology successfully developed the HPLC method. In the optimized method, an Agilent Zorbax Eclipse XDB C18 column (150 mm x 4.6 mm, 5µm) was used, employing a gradient program with Water, Tetrahydrofuran, and Acetonitrile in a proportion of 91:3:6 as mobile phase A, and Water, Tetrahydrofuran, Acetonitrile, and Methanol in a proportion of 4:2:74:20 as mobile phase B. The flow rate was set at 1mL/min, with a retention time of approximately 25.5 minutes for Deflazacort. The method exhibited linearity in the range of 0.020 – 1.2ppm, with a correlation coefficient exceeding 0.99. The optimized approach demonstrated accuracy, with a repeatability and precision of less than 2% RSD. The LOD and LOQ were determined to be 0.02ppm and 0.081ppm, respectively. According to ICH recommendations, the percentage recovery of samples met the acceptance requirements, ranging from 90 to 110%.

CONCLUSION:

The development of HPLC methods using a quality-by-design approach for Deflazacort has been well-documented. This process began with identifying method goals based on the analytical target product profile and involved exploring critical components such as time and mobile phase concentration through a multilevel factorial design. This approach allowed for optimization of interrelationships among parameters affecting chromatographic separation. Validation confirmed that all parameters met acceptance criteria, demonstrating that the method for Deflazacort impurity determination is linear, specific, accurate, and robust. The quality-by-design approach minimized the risk of failure during validation and transfer. Using Minitab software facilitated a more efficient method development compared to conventional methods. Statistical analysis indicated that the procedure is repeatable, selective, accurate, and robust, making it suitable for routine quality control in the pharmaceutical industry.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors are grateful to Zuventus Healthcare Ltd. for providing laboratory facilities for this research work.

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Received on 21.06.2024 Revised on 13.09.2024

Accepted on 18.11.2024 Published on 28.02.2025

Available online from March 04, 2025

Asian Journal of Pharmaceutical Analysis. 2025; 15(1):31-39.

DOI: 10.52711/2231-5675.2025.00006

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Analyte	Calibration equation Y=area, x= µg/mL	Correlation coefficient (r²)	LOD (µg/mL)	LOQ (µg/mL)	Accuracy (%)
Deflazacort	125,627.8255x - 80.6323	0.9999	0.020	0.081	98.9-99.6
Deflazacort 21- Alcohol	120,554.2406x + 2,576.6282	0.9995	0.019	0.080	99.2-99.8
Deflazacort Impurity A	130,336.8757x + 352.4006	0.9982	0.019	0.079	99.0-99.3

Flow rate		Mobile phase composition		Column oven temperature
0.9mL/min	1.1mL/min	+	-	38 °C	42 °C
0.42*	0.48*	0.49*	0.56*	0.47*	0.38*