A QbD-based Stability Indicating Study for the Development and Validation of Vonoprazan Fumarate by HPTLC Method

Pratibha L. Ayanar, Kumudini R. Pawar*

Department of Pharmaceutical Quality Assurance, Abhinav Education Society’s College of Pharmacy, Narhe, Pune, Maharashtra, India 411041.

*Corresponding Author E-mail: kumudiniphd30@gmail.com

ABSTRACT:

A stability-indicating high-performance thin-layer chromatography (HPTLC) approach was produced and validated for the quantitative determination of Vonoprazan Fumarate in bulk and tablet dosage form utilizing a Quality by Design approach. Method was optimized through systematic risk evaluation and experimental design to ensure robustness and reliability. Chromatographic separation was achieved using HPTLC plates coated by silica gel 60 F254 with mobile phase consisting of Ethyl Acetate: n-Hexane: Glacial Acetic acid (5:4:1v/v/v) by response surface methodology. The identification was done at λmax 215 nm. Developed method demonstrated linearity (r² > 0.999) over the concentration range of 200–1200 ng/spot. The LOD and LOQ were found to be 2.96 ng/spot and 8.98 ng/spot, respectively. Accuracy was demonstrated by recovery studies at three concentration levels, yielding mean recoveries between 99.05to 103.48%. Intra-day % RSD (1.08 to 1.90%) and inter day % RSD (1.09 to 1.32 %), precision studies showed %RSD values below 2%, indicating the method’s repeatability and reliability. Forced degradation studies under acidic, basic, oxidative, thermal, and photolytic conditions confirmed the stability-indicating studies of the method, as degradation products were well resolved by analyte peak. The method was validated as per International Council for Harmonization Q2(R1) guidelines for accuracy, precision, specificity, and robustness. The application of Box-Behnken Design principles ensured method robustness and reproducibility, making it suitable for regular analysis of quality control for Vonoprazan Fumarate in pharmaceutical products. This study highlights significance for systematic analytical method development in regulatory compliance and quality assurance.

KEYWORDS: Vonoprazan Fumarate, HPTLC, Stability-indicating method, QbD, Box-Behnken Design, Validation, Pharmaceutical Analysis.

INTRODUCTION:

GERD is characterized by the backward flow of stomach acid into the esophagus, leading to symptoms such as heartburn, regurgitation, and, in more severe cases, esophageal damage.¹

In February 2015, Japan became the first country to approve vonoprazan fumarate, a potassium-competitive acid blocker (P-CAB).²Vonoprazan, has shown enhanced effectiveness compared with Proton pump inhibitors in a treatment of certain gastric acid-related problems.³

In the context of first-line Helicobacter pylori eradication, regimens that incorporate vonoprazan shows higher eradication rates than those based on proton pump inhibitors (PPIs). ⁴ Vonoprazan fumarate has a longer duration of effect, a stronger acid suppression, and a quicker onset of action when compared to proton pump inhibitors.⁵

Fig 1. Chemical structure of Vonoprazan Fumarate

Chemical structure of vonoprazan Fumarate is shown in Fig. 1. In India, Vonoprazan Fumarate is marketed under Brand Name Vonogress by La Renon Healthcare Pvt. Ltd. A once daily regimen of 10 or 20 mg of Vonoprazan Fumarate is administered orally. ⁶ The literature study indicates that some analytical techniques, such UV⁷, HPLC⁸, and stability indicating HPTLC methods⁹, was established for the measurement of Vonoprazan Fumarate into bulk and in tablets, However, no reports have yet been made regarding the HPTLC method's stability in terms of the quality-by-design approach.

The principles of Quality by Design (QbD) are detailed in guidelines established by the ICH, specifically in Q8 (R2): Pharmaceutical Development^,10 Q9: Quality Risk Management, and Q10: Pharmaceutical Quality System^.11The ICH Q10 guidelines give a thorough framework for evaluating pharmaceuticals, which should be integrated at all phases of pharmaceutical manufacturing to guarantee quality. ¹²

A separate study was conducted to develop a stability-indicating HPTLC method by using Box-Behnken Design for Vonoprazan Fumarate, in which the drug was subjected to various forced degradation conditions; the method successfully resolved the degradation products, confirming its suitability for routine quality control analysis. From our research work the findings underline the importance of HPTLC in forced degradation study, as it provides reliable and efficient methods for monitoring medication stability and assuring pharmaceutical product quality.

MATERIALS AND METHODS:

Materials:

API of Vonoprazan Fumarate was a gift sample from Dr. Reddy's Lab Pvt Ltd., Hyderabad, India. Ethyl Acetate, N- hexane, Glacial acetic acid and Methanol were acquired from Loba Chemicals, Mumbai. Vonoprazan Fumarate tablet named Vonogress (10mg) was purchased from local market.

Instrumentation:

Using a Hamilton (100μL) sample syringe (CAMAG, Switzerland) and a 6mm Linomat 5 applicator, samples were applied to plates in 6 mm-wide bands. For the analysis, 20 x 20cm, 0.1mm thick aluminum silica gel 60 F254 HPTLC plates (Merck, Germany) were utilized. A 20 x 20cm twin trough chamber (CAMAG, Switzerland) was utilized to develop and validate the HPTLC method. Separated dots on TLC plates were observed using the UV-Visible chamber (CAMAG, Switzerland). The densitometric scanning was carried out at 215nm using the TLC scanner 3 (CAMAG, Switzerland) in a reflectance mode. The Win CATS program, version 1.4.3.6336, simplifies scanner control and data processing.

Chromatographic Conditions:

An appropriate amount of API and sample solutions was spread on the HPTLC plate in a series of 6 mm band length. Plates were prewashed before chromatography with methanol and activated at 100℃ 10 minutes. Mobile phase consists of Ethyl acetate: n-Hexane: Glacial acetic acid (5:4:1 v/v/v). Saturation time of mobile phase was maintained for 15 minutes with a linearly upward development was done over the distance of 80mm. The plates were allowed to air dry after development. A CAMAG TLC Scanner 3, set to a wavelength of 215nm, was used for scanning, with a deuterium lamp as the radiation source. The data was obtained from the scan by using win CATS software.

Preparation of standard solutions:

A standard solution is prepared by dissolving 10 mg of the drug in 10 mL of methanol, yielding a concentration of 1000 µg/mL, which is then appropriately diluted to obtain working concentrations ranging from 200 to 1200 ng/spot.

Preparation of sample solution:

Twenty tablets of Vonoprazan Fumarate (10 mg) were accurately weighed and ground into a fine powder. The mean weight of the tablets was determined, and powder equal to ten milligrams of vonoprazan fumarate was transferred to a 10 mL volumetric flask with 10mL of methanol. The obtained solution has a concentration of 1000 µg/mL. Then solution was filter through 0.45 µm Whatman filter paper.

Method Development and Optimization of the HPTLC Method utilizing the QbD approach:

Risk Assessment and Impact Analysis Using ICH Q8(R2) and Q9 Guidelines

To ensure robust method development for the stability-indicating HPTLC assay of Vonoprazan fumarate, a systematic risk assessment and impact analysis were carried out in compliance with the guidelines provided in ICH Q8(R2): Pharmaceutical Development and ICH Q9: Quality Risk Management.

1. Analytical Target Profile (ATP):

The ability of the method to precisely, accurately, and specifically quantify Vonoprazan Fumarate in dosage form and bulk with sufficient resolution from its degradation products under stress conditions was defined as the ATP. The choice of method parameters that are essential to attaining method performance was guided by the ATP.¹³

2. Determining the Critical Method Parameters (CMPs) and Critical Method Attributes (CMAs):

An initial risk assessment was done in order to identify variables that might have an impact on the performance of the method.¹⁴

The following were determined based on preliminary experimental screening and existing knowledge:

CMAs: Resolution between degradation products and analyte, peak symmetry, and retention factor (Rf).

CMPs: Solvent front migration distance, band length and saturation time.

3. Ranking and Filtering of Risk:

Each CMP was assessed for detectability, probability of occurrence, and possible impact on the CMA using Ishikawa (Fishbone) diagram (Fig2). The following criteria were ranked and scored:

Saturation time:

Because it affects solvent migration and resolution, saturation time carries a high risk.

Band length: Has a medium risk of influencing quantification accuracy and spot size.

Solvent front: High risk, essential for peak development and separation effectiveness.

This assessment led to the inclusion of all three CMPs in the ensuing experimental design.¹⁵

Fig 2. The Ishikawa (fishbone) diagram to identify potential variables for the HPTLC method development

4. Evaluation of Impact Making use of Design of Experiments (DoE):

A Box-Behnken Design (BBD) was used to measure and simulate the effect of particular CMPs on method performance. The DoE used second-order polynomial regression to examine the effects of CMPs, both individually and in combination, on the response variable (Rf value).¹⁶ Typically, optimizing study designs looked at the components at 3 levels (-1, 0, +1) or fewer (Table 1).¹⁷Interaction plots revealed either antagonistic or synergistic effects between variables, and ANOVA results validated model significance (p < 0.0001).¹⁸

Several factors were assessed in the initial trials to establish the HPTLC method. The Ethyl acetate volume was determined to be between 4 and 6mL. Additionally, the n- hexane volume was set within the range of 3 to 5 mL, while the Glacial acetic acid was adjusted to a range of 0.01 to 1 mL. A total of 17 experiments were conducted. Observe response for all experimental runs are recorded in Table 2.

Table 1. Chromatographic considerations for the study design of Box-Behnken

Sr No	Factor	Lower Limit (-1)	Intermediate (0)	Upper Limit (+1)
1	Saturation Time (min)	10	15	20
2	Band Length(mm)	4	6	8
3	Solvent Front(mm)	70	80	90

Table 2. Responses observed for 17 analytical trials using the Box-Behnken design of Vonoprazan Fumarate

Std	Experimental Trials	Factor A: Saturation Time (min)	Factor B: Band length (mm)	Factor C: Solvent front (mm)	Rf
10	1	15	8	70	0.49
1	2	10	4	80	0.46
7	3	10	6	90	0.47
14	4	15	6	80	0.52
15	5	15	6	80	0.52
16	6	15	6	80	0.52
2	7	20	4	80	0.54
13	8	15	6	80	0.52
8	9	20	6	90	0.60
4	10	20	8	80	0.77
11	11	15	4	90	0.50
6	12	20	6	70	0.66
9	13	15	4	70	0.48
12	14	15	8	90	0.51
17	15	15	6	80	0.52
5	16	10	6	70	0.53
3	17	10	8	80	0.45

5. Method Operable Design Region (MODR) establishment:

The MODR, a multidimensional combination of CMP ranges that consistently yielded acceptable method performance (resolution, Rf, peak symmetry), was defined using overlay and perturbation plots. According to ICH Q8, the MODR is a design space where modifications are exempted from regulatory notification.¹⁹

6. Strategy for Control:

A control strategy was suggested based on the risk analysis and MODR:

Mobile phase ratio (5:4:1 v/v/v)

Saturation time: 15 minutes

Band length: 6 mm

Solvent front distance: 80 mm

Validation of DoE‑ based HPTLC Technique:

The technique was verified in accordance with ICH Q2(R1) standards. ²⁰

A comprehensive HPTLC method validation for Vonoprazan Fumarate was performed covering linearity, precision, accuracy, sensitivity, robustness, and specificity. Linearity was demonstrated at a concentration from 200–1200 ng/band by transferring 0.2–1.2 µL of a stock solution into 10 mL volumetric flasks, followed by calibration using linear regression (with high R² values) from six repeated measurements.²¹ Three concentrations was analyzed to validate intraday and interday precision at multiple time points and on consecutive days, with low % RSD values.²² Assay precision was evaluated through six replicate analyses of a 600 ng/band tablet solution,²³ while accuracy was established using three different level at 80%, 100%, and 120% levels, showing acceptable recovery percentages and minimal variability.²⁴ Sensitivity parameters, LOD and LOQ, was determine by linearity equation using values of slop and standard deviation.²⁵Method's robustness was confirmed by varying mobile phase compositions and detection wavelengths,²⁶ and specificity was ensured through comparing spectra of solvent, mobile phase, pure API, and tablet sample, verifying peak purity at multiple points along the band.²⁷

Force degradation study:

In these forced degradation studies for Vonoprazan Fumarate, the drug was exposed to numbers of stressful conditions such as acidic (0.01 N HCl for 60 min), alkaline (0.01 N NaOH for1 h), oxidative (3% H2O2 for1 h), neutral (distilled water for1 h) and thermal (exposure at 60°C for 30 minute). This comprehensive approach ensures that potential degradation pathways are identified, and the stability profile of Vonoprazan fumarate under different stress conditions is thoroughly evaluated.

RESULT AND DISCUSSION:

Chromatographic development:

Several mobile phases were tested in varied ratios for method optimization. By following several experiments, a mobile phase comprising EA: N-Hexane: GAA (5:4:1 v/v/v) was chosen because it produced sharp peaks without any fronting and tailing shown in figure 3.

The effectiveness of HPTLC densitometry dependent on the specific wavelength used to detect the analytes. In this study, the developed HPTLC plate was scanned within the UV range of wavelength 200-400 nm using a TLC scanner III (CAMAG). The optimal estimation wavelength for Vonoprazan Fumarate was chosen 215 nm, as depicted in fig 4.

Figure 3: Optimized Densitogram of a standard

Figure 4: UV Spectra of Vonoprazan Fumarate solution of vonoprazan

Risk Assessment and Impact Analysis:

The ATP encompassed the summary of quality characteristics of the targeted method. ATP elements set up for developing a robust HPTLC method for Vonoprazan Fumarate. The retardation factor (Rf) should be optimum (0.4 to 0.7) desirable for attaining the optimal method.

Determination of critical method attributes (CMA):

In the initial trials aimed at developing the mobile phase, several parameters such as the mobile phase ratio, development distance, and saturation time were assessed. It was determined during these preliminary trials that the mobile phase ratio (5:4:1v/v/v) emerged as the only critical parameter in the development process.

Risk assessment:

The scanning time was modified in increments of 10, 15, and 20 minutes. The saturation time for the HPTLC plate was adjusted to 10, 15 and 20 minutes. Band lengths were varied at 4 mm, 6 mm and 8 mm. The volume of the mobile phase, a critical factor in method development, was selected from 5 mL, 10 mL, and 15 mL. The dimensions of the saturation chamber (Twin trough chamber) were varied to 10 cm × 10 cm, 20 cm × 10 cm, and 20 cm × 20 cm. Different solvent fronts, including 70 mm, 80 mm, and 90 mm, were tested for the development of the HPTLC method. The HPTLC plate was scanned at wavelengths of 213 nm, 215 nm, and 217 nm.

Evaluation of Impact Making use of Design of Experiments (DoE):

Table 3: Chromatographic characteristics

Sr No	Parameters	Analytical conditions
1.	Stationary phase	TLC aluminum plate pre-coated with silica gel 60 GF₂₅₄
2.	Mobile phase	Ethyl Acetate: n-Hexane: GAA (5:4:1 v/v/v)
3.	Detection wavelength	215nm
4.	Saturation time	15min
5.	Band width	6mm

API and tablet powder solutions containing Vonoprazan fumarate was put onto HPTLC plates and developed using linear ascending method in twin trough glass chamber. After complete drying, the produced HPTLC plates was scanned with 215 nm. The final optimized chromatographic conditions included a mobile phase ratio Ethyl acetate: n- hexane: Glacial Acetic acid (5:4:1 v/v/v), saturation time 15 minutes, band length 6 mm and solvent front 80 mm. These conditions produced Rf value of 0.52and highest resolution (Table 3).

A typical second-order regression Equation obtained from Sate Ease design expert is Y=0.6375−0.0825*A −0.0225*B+0.0110*C+0.0021*A²−0.0044*B²−0.000075*C²+0.0060*AB+ (very small terms for AC and BC). Where, Y =Rf value A (Saturation Time), B (Band Length) and C (Solvent front) had positive impact on Rf value. Saturation time (Factor A) shows negative impact on Rf value. The validation of model was confirmed through analyzing of the variations (ANOVA) shown in Table 4 the probability value was determined to be < 0.0001, indicate that model parameters are significant when P-values are < 0.0500. The significance of the model is indicated by its F-value of 7.18.

Surface plot (fig 5) shows a generally positive correlation between both saturation time and band length, with Rf value. This means that as saturation time and band length increase, the Rf value tends to increase as well. The steepest increase in Rf value appears to occur with a combination of higher saturation times and longer band lengths. The plot can be used to identify optimal conditions for achieving a desired Rf value within the tested ranges of saturation time and band length. The MORD plot (fig 6) show contour lines are not parallel to the axes, indicating an interaction between saturation time and band length. This means the effect of one variable on the Rf value depends on the level of the other variable.

The Perturbation plot, (fig 7) also known as a sensitivity analysis plot, aids in comparing the impact of each component at a specific location inside the design space. It shows how, when all other elements are maintained constant, the reaction varies as each one deviates from the original point. An overlay image depicts the effect of three factors saturation time, band length, and solvent front on the retardation factor (Rf) in a chemical process, likely chromatography. Each graph (figure 8) shows the relationship between one of these factors and Rf, whereas a central value is maintained for all other parameters.

Table 4. Response of ANOVA by Box-Behnken design for Vonoprazan

Source	Sum of squares	Df	Mean square	F-value	P-value
Model	0.0625	3	0.0208	7.18	0.0044	Significant
A-Saturation time	0.0544	1	0.0544	18.77	0.0008
B-Band length	0.0072	1	0.0072	2.48	0.1391
C-Solvent front	0.0008	1	0.0008	0.2758	0.6083
Residual	0.0377	13	0.0029
Lack of fit	0.0377	9	0.0042
Pure Error	0.0000	4	0.0000
Cor Total	0.1002	16

Figure 5: surface plots of interaction between saturation time (A) and Band Length (B)

Figure 6: MODR plot for Vonoprazan Fumarate

Figure 7: Plot of Perturbations for factors A,B and C

Figure 8: Overlay of three factors of vonoprazan fumarate

Validation of DoE -Based HPTLC Technique:

The procedure was verified in accordance with ICH Q2(R1) standards.

Vonoprazan fumarate concentration and area of peak were found to be linearly related across the 200–1200 ng/band range. The regression coefficient (R2) value was used to demonstrate that the calibration curve was linear, and the y-intercept was determined to be 0.9995 and Y=3.7854x+165.53respectively shown in fig 9.it is concluded that response is linear to the concentration. Vonoprazan fumarate's LOD and LOQ were determined as 2.96 ng/band and 8.98 ng/band, correspondingly, demonstrating the new method's sensitivity. The % RSD for intraday is found to be 1.08 to 1.90% and for inter day % RSD was found to be 1.09 to 1.32 %, which denote the precision of method. The measured %RSD for concentrations were below 2%, signifies the better precision of the developed method. The concentration of Vonoprazan fumarate in the tablets, as a percentage of the label claim, was found to be accurate accord with the label claims through testing. This implies that there had been no interference from any of the excipients that are often found in tablets. It was discovered that the drug content was 101.90±1.77. The percentage recovery was found to be 99.05to 103.48%, which signify the accuracy of technique. It was discovered that the developed approach was highly specific for vonoprazan fumarate. For robustness, it was found that the developed approach was not significantly affected by little variations in the mobile phase volume and wavelength. the percentage RSD was found less than 2, the approach was deemed reliable. Table 5 displays an overview of every validation parameter.

Fig. 9. Calibration curve for Vonoprazan Fumarate

Table 5: An overview of the validation parameters

Sr. No.	Validation parameters	Vonoprazan fumarate
1.	Linearity equation R²	Y=3.785x +165.53 R²= 0.999
2.	Range	200-1200 ng/band
3.	Precision	(% RSD)
	Intra-day	1.08 to 1.90%
	Inter-day	1.09 to 1.32 %,
4.	% Assay (Mean ± %RSD)	101.90±1.77
5.	Accuracy	Mean ± %RSD
	80 %	100.2±0.228
	100 %	99.05±0.52
	120 %	103.48±1.17
6.	Limit of detection	2.96 ng/band
7.	Limit of quantitation	8.98 ng/band
8.	Specificity	Specific
9.	Robustness	Robust

Forced degradation study:

Vonoprazan Fumarate showed varying degree of degradation under various stress conditions, with the densitograms revealing additional peaks corresponding to degradation products (Table 6). Acidic, alkaline, oxidative, neutral and thermal conditions resulted in degradation percentage 19.31 and 11.52%, 14 and 17.76%,15.89%, 20.51% and 12.32%, respectively (Table 6), each showing distinct Rf values for the degradation products alongside the main peak of Vonoprazan Fumarate, as illustrated in Fig 10.

Table 6. Summary of % degradation of standard Vonoprazan Fumarate

Conditions	Temperature and time	Degradation % of Vonoprazan
Acid (0.01N HCl)	At room temp. for 1 hr	19.31 and 11.52%
Base (0.01N NaOH)	At room temp. for 1hr	14.0 and 17.16%
Oxidative (3% H2O2)	At room temp. for 1hr	15.89%
Neutral	At room temp. for 1 hr	20.51%
Thermal	60 ͦ C for 30 min	12.32%

CONCLUSION:

A quality by design methodology is used to develop a stability indicating high performance thin layer chromatographic approach for the analysis of Vonoprazan fumarate, that employes precoated silica gel 60 F254 HPTLC aluminum sheets as the stationary phase. The mobile phase was optimized using Ethyl acetate: n-hexane: Glacial acetic acid (5:4:1 v/v/v) as mobile phase. The chromatogram showed a clear, sharp and symmetrical peak with an Rf value of 0.52. Densitometric assessments are carried out in the absorbance mode at 215 nm. The calibration curve's peak area depends upon a concentration of 200–1200 ng/band was established. It was discovered that the calibration curve's coefficient of correlation was 0.999. According to the standards of the ICH, the approach was validated. The Vonoprazan fumarate was also subjected to force degradation investigations on acid, base, oxidation, neutral and thermal degradation. With noticeably differing Rf values, degradable products that were extracted from the original drugs were well resolved. This approach is easy to use, accurate, sensitive, and precise. It is applicable to the quantification of Vonoprazan fumarate in API and its formulation.

CONFLICT OF INTEREST:

It is hereby declared that None of the authors have any conflicts of interest.

ACKNOWLEDGMENTS:

Authors are thankful to Principal, Abhinav Education Society’s College of Pharmacy (Pharm), Narhe Pune. The HPTLC facilities provided by the Principal of the Dr. D.Y. Patil Institute of Pharmaceutical Sciences and Research Pimpri, Pune, is also appreciated by the authors.

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Received on 13.07.2025 Revised on 11.12.2025

Accepted on 02.02.2026 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):3317-3324.

DOI: 10.52711/0974-360X.2026.00472

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