Development and Validation of a Stability-Indicating HPLC Method for Vonoprazan with LC-MS Characterization of Degradation Products

Bhairavi  Saraf*¹, Preeti Mehta¹, Rahul Somani²

¹Department of Chemistry, School of Basic and Applied Sciences, Sangam University,

Bhilwara (Rajasthan)-311001 India.

²Alkem Laboratories Ltd, Mumbai, India.

*Corresponding Author E-mail: saraf.bhairavi@gmail.com

ABSTRACT:

Vonoprazan, is a potassium-competitive acid blocker used in the form of fumarate for the treatment of gastroduodenal ulcers and reflux esophagitis and can be combined with antibiotics for the eradication of Helicobacter pylori. Despite its clinical significance, limited stability-indicating analytical methods have been reported for its determination. This study addresses this gap by establishing a robust isocratic HPLC method, developed and validated under ICH Q1A(R2) and ICH Q1B guidelines, for the accurate and sensitive determination of vonoprazan, with characterization of its degradation products via LC-MS. In accordance with the guidelines, forced degradation was conducted under acidic, basic, oxidative, photolytic, and thermal stress conditions to ensure the separation of vonoprazan from its degradation products. Chromatographic separation was achieved using a FINEPAK SIL C-18 column and an acetonitrile:0.1% formic acid mobile phase (70:30, v/v), with detection at 266 nm. The method demonstrated excellent linearity (2–12 µg/mL, R² = 0.9981), precision (%RSD < 2), and accuracy (99.909–100.263%), with LOD and LOQ values of 0.086 and 0.261 µg/mL, respectively. Vonoprazan degraded under acidic, alkaline, and oxidative conditions, while it was stable under photolytic and thermal stress conditions. LC-MS identified two major degradation products, and their structures and proposed pathways were elucidated. The proposed HPLC method provides simple, accurate and reproducible quantitative analysis for the determination of vonoprazan in the presence of its degradants and suggests the need to protect vonoprazan from hydrolysis and oxidation during formulation and storage.

KEYWORDS: Degradation Pathways, Forced Degradation, HPLC, ICH Q1A(R2), LC-MS, LC-QTOF-MS, Stability Indicating, Vonoprazan.

INTRODUCTION: 

Vonoprazan fumarate (Figure 1), 1-(5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H–pyrrol−3-yl)-N-methyl methanamine fumarate, is an orally bioavailable potassium-competitive acid blocker¹. Literature survey revealed that few methods have been reported for the analysis of vonoprazan for process-related impurities and related substances detection^2-5.

Also, simple analytical^6-12 and bioanalytical^13-16 methods have been reported for vonoprazan estimation either alone or in combination with other drugs. The previously conducted forced degradation studies on vonoprazan which followed ICH Q1A(R2) guidelines and confirmed its degradation under various stress conditions (acidic, alkaline, oxidative), however, none of the previous studies provide detailed LC-MS-based identification or mechanistic elucidation of the resulting degradation products. As summarized in Table 1, the present study addresses this gap by developing and validating a robust, isocratic stability-indicating HPLC method for vonoprazan, coupled with LC-MS characterization of its major degradation products. It also proposes degradation pathways under ICH-specified stress conditions, thereby offering new insights into the compound’s degradation behavior and overall chemical stability profile.

Figure 1: Structure of Vonoprazan

Table 1: Comparison with reported vonoprazan methods and proposed methodology

Parameter	Luo et al. (2018)^3	Kanaan et al. (2024) ^4	Abuothman et al. (2024) ^5	Salva et al. (2024)^10	El-Hamd MA et al. (2024) ^16	Present Study
Primary Focus	10 related substances + degradation	Multiple VPZ impurities (Six Sigma optimization)	N-nitroso VPZ determination	VPZ+clarithromycin+amoxicillin	VPZ quantification with factorial design	Stability + degradant structural ID
Technique	RP-HPLC	RP-LC	LC-ESI-MS/MS	RP-UPLC	Spectrofluorimetry	RP-HPLC + LC-QTOF-MS
Detection	UV 230 nm	UV 266 nm	MS/MS (MRM)	UV 210 nm	Fluorescence (Ex/Em 465/530 nm)	UV 266 nm + QTOF-MS
Mobile Phase	Gradient: Buffer-MeOH-ACN / Buffer-ACN	ACN:H2O (optimized ratio)	Gradient 0.1% formic acid + ACN	Buffer (pH 3.8): ACN (60:40)	N/A (derivatization-based)	Isocratic: ACN/0.1% formic acid (70:30)
Degradant Identification	Degradation extent quantification only	Impurity profiling only	Specific N-nitroso compound	Forced degradation (no structural ID)	No degradation study	Complete LC-MS structural elucidation
Matrix Coverage	Bulk drug	Bulk + tablets	Tablets + raw materials	Physical mixture	Plasma + tablets	Bulk drug + tablets
Run Time	~30 min	~15 min	3.65 min	<3 min	N/A	~6 min
Greenness Assessment	Not reported	Six Sigma quality standards	Not reported	Not reported	Not reported	Not assessed
ICH Compliance	Q1A(R2), Q1B	Q2(R1)	Q2(R1)	Q2(R1)	Q2(R1)	Q1A(R2), Q1B, Q2(R1)
Unique Contribution	First comprehensive impurity method	Optimization-focused	Specific nitrosamine analysis	Triple combination analysis	Ultra-sensitive fluorometric	Degradation pathway elucidation

Key: ACN: Acetonitrile, ESI: Electrospray Ionization, Water: H₂O, ICH: International Council for Harmonisation, ID: Identification, LC: Liquid chromatography, MeOH: Methanol, RP-HPLC: Reversed-phase High-performance liquid chromatography, QTOF: Quadrupole Time-of-Flight, VPZ: Vonoprazan.

MATERIALS AND METHODS:

Chemicals and reagents:

Vonoprazan drug was received as gift sample from a reputed Indian Pharmaceutical Company and the product in its tablet form for assay was obtained from local market. Acetonitrile, methanol, HCl, NaOH, 30% H₂O₂, were procured Merck Laboratories., Mumbai and water (HPLC grade) with conductivity below 0.05 µS/cm was obtained using the Extrapure Lablink water purifier System.

HPLC Instrument specification:

The chromatographic separation was conducted on JASCO system with PU 2080 Plus Intelligent HPLC pump, and UV-2075 UV-VIS detector operated by Borwin chromatography software (version 1.50).

The analysis was performed using FINEPAK SIL C-18 column (250 mm ´ 4.6 mm, 5 µm) and Mobile Phase: Acetonitrile: 0.1 % Formic Acid in Water (70:30 v/v) with flow rate of 1.0 ml/min; injector of 20 µl and detection at wavelength of 266 nm.

LC-MS instrument details and chromatographic conditions:

LC-MS was carried out using Agilent 1260 Infinity II with Agilent 6540 UHD Accurate-Mass Q-TOF LCMS. Mass spectra were recorded in positive mode with Dual AJS ESI ion source. [Ion Source Parameters: Gas Temp: 300 °C; Gas Flow: 8 l/min; Nebulizer: 35 psi; Sheath Gas Temp: 350 °C; Sheath Gas Flow: 11 l/min; Capillary Voltage: 3500 V; Nozzle Voltage: 1000 V; Fragmentor Voltage: 150 V].

Preparation of solutions:

Preparation of Vonoprazan Standard Drug Solution and working standard solution:

A 1000 µg/mL vonoprazan stock solution was made by dissolving 10 mg of the drug in 10 mL of acetonitrile. This stock was then diluted to create a 100 µg/mL working solution, which was further diluted as needed to reach the final desired concentrations.

Preparation of stress degradant solutions:

To develop a better understanding of stress degradation, previous research on the hydrolytic, acid, and photolytic degradation of various drugs using RP-HPLC and LC-MS/MS was reviewed^17-20. The degradation experiments in the present study were in accordance with ICH Q1A(R2) and ICH Q1B guidelines^21-22. Degradation studies were conducted under stress conditions including acidic, basic, oxidative, UV light, and heat to assess the ability to separate vonoprazan from its degradation products. Table 2 outlines the stress degradant solutions and conditions used for conducting forced degradation analysis:

Validation of the analytical method²³

The developed chromatographic method was evaluated in accordance with ICH Q2(R1) guidelines, the details are summarized in Table 3.

Table 2: Solution and conditions used for conducting forced degradation studies

Stress Condition	Stress degradant solutions and Conditions	Post-Stress Dilution Steps	Final Concentration
Acid Hydrolysis	2 N HCl at room temperature for 24 hours.	Neutralize, dilute to 100 µg/mL, then dilute again.	10 µg/mL
Alkaline Hydrolysis	2 N NaOH at room temperature for 24 hours.	Neutralize, dilute to 100 µg/mL, then dilute again.	10 µg/mL
Oxidative	30% w/v H₂O₂ at room temperature for 24 hours.	Dilute to 100 µg/mL, then dilute again.	10 µg/mL
Photolytic	Expose solid drug to UV light (≥200 Wh/m²) and white light (≥1.2 million lux hours) using Newtronic NLPS4SI Photostability chamber	Dissolve to make 1000 µg/mL, then dilute.	10 µg/mL
Thermal	Expose solid drug to 100°C in a hot air oven for 8 hours.	Dissolve to make 1000 µg/mL, then dilute.	10 µg/mL

Table 3: Method Validation Parameters and Procedures

Parameter	Description of Method
Specificity	The specificity of the method was ascertained by peak purity profiling studies as well as injecting blank.
Linearity and Calibration	A stock solution (1000 µg/mL) was used to prepare a 100 µg/mL solution. This was further diluted to create six solutions over a concentration range of 2-12 µg/mL to establish the relationship between peak area and concentration.
Precision	The precision of the method was demonstrated by intra-day and inter-day variation studies. In the Intra-day studies, 3 replicates of 3 different concentrations in linear range were analyzed in a day and percentage RSD was calculated. For the inter day variation studies, 3 different concentrations were analyzed on 3 consecutive days and percentage RSD was calculated.
Assay	Twenty tablets were weighed, and average weight was determined. The tablets were crushed into fine powder and tablet powder equivalent to 10 mg of vonoprazan (Label claim: 20 mg vonoprazan per tablet; Vocab 20; Hetero Healthcare, India) was accurately weighed and transferred into 10 ml volumetric flask and volume made up with acetonitrile. The solution was filtered, and first dilution was conducted to obtain the solution of 100 μg/mL. Further dilution of 4 µg/mL was prepared with mobile phase. To get the test results 6 replicates of same concentration were evaluated.
Accuracy	Accuracy was evaluated using the standard addition method by spiking a sample solution of the marketed formulation with known amounts of the active pharmaceutical ingredient (API) at 50%, 100%, and 150% levels. Recovery was calculated from the peak area.
Detection Limit (LOD) and Quantification Limit (LOQ)	Based on the standard deviation of the y-intercept and slope of the calibration curve, LOD and LOQ of developed HPLC method were calculated according to ICH guidelines.
Robustness	Robustness of the method was checked by carrying out the analysis under conditions during which mobile phase composition (± 2% Composition), detection wavelength (± 1 nm), flow rate (± 0.05 mL/min) were altered and the effect on the area were noted.

RESULTS AND DISCUSSION:

Method Development:

The developed HPLC method successfully achieved separation of vonoprazan from its degradation products using a FINEPAK SIL C-18 column and a mobile phase of acetonitrile:0.1% formic acid in water (70:30 v/v) with appropriate system suitability parameters. The retention time for vonoprazan was consistent at 3.731 ± 0.264 min (Figure 2).

Figure 2: Chromatograph of vonoprazan under optimized chromatographic conditions

The analytical method was validated and demonstrated excellent specificity, with no matrix interference and peak purity indices >0.999. Linearity was established across a 2–12 µg/mL range, showing a strong correlation (R² = 0.9981). The method proved highly precise, with all intra-day and inter-day %RSD values below 1%. The LOD and LOQ were determined to be 0.086 µg/mL and 0.261 µg/mL, respectively. Accuracy was confirmed with recovery values between 99.909% and 100.263% for the marketed formulation. Furthermore, the method was robust, as the %RSD remained below 2% despite slight variations in chromatographic parameters. The method was accurate and robust for the marketed formulation, with recovery ranging from 99.909–100.263% and the %RSD for robustness testing remaining below 2%.

Forced Degradation and LC-MS characterization^24-33

Vonoprazan was subjected to forced degradation studies under ICH-specified stress conditions. The stress conditions were optimized to yield drug recovery in the range of 70–90%, enabling effective observation of degradation behavior. Notable degradation occurred under acidic with drug recovery of 91.84% with one peak of degradation product (DP1) at RT 4.833 min, under alkaline with drug recovery of 76.43% with one peak of DP1 at RT 4.892 min, and oxidative with drug recovery of 82.19% conditions with second degradation product (DP2) at RT 5.573 min, indicating the drug’s susceptibility to hydrolytic and oxidative stress (chromatograms are shown in Figure 3-A to 3-C). In contrast, under thermal conditions the drug recovery was 98.94% and photolytic conditions the drug recovery was 99.18%, confirming no degradation and stability of vonoprazan. Thus, the forced degradation studies conducted under ICH-specified conditions revealed that vonoprazan is susceptible to acidic, alkaline, and oxidative stress, while showing stability under thermal and photolytic conditions. These results clearly demonstrate the chemical instability of vonoprazan in hydrolytic and oxidative environments and provide essential insights into its degradation pathways.

Figure 3-A: Chromatogram of drug (10 µg/mL) under acid hydrolysis

Figure 3-B: Chromatogram of drug (10 µg/mL) under alkaline Hydrolysis

Figure 3-C: Chromatogram of drug (10 µg/mL) under oxidation

LC-MS analysis further confirmed and validated the presence of degradation products (as shown in Figure 4-A to 4-C) and enabled their structural elucidation. Two major degradation products were identified:

· DP1: Resulting from both acid and alkaline hydrolysis, observed at retention times approximately 4.833 and 4.892 minutes.

· DP2: Formed under oxidative stress, eluting at retention time approximately 5.573 minutes.

Figure 4-A: LC-MS Chromatogram of vonoprazan (10µg/mL) under acid hydrolysis

Figure 4-B: LC-MS Chromatogram of vonoprazan (10µg/mL) under alkaline hydrolysis

Figure 4-C: LC-MS Chromatogram of vonoprazan (10µg/mL) under oxidation

Mass spectral data supported the molecular fragmentation (as shown in Figure 5-A to 5-C) of these degradation products was observed as follows-

· DP1: Resulting from both acid and alkaline hydrolysis, observed at retention times approximately 4.833 and 4.892 minutes, with a protonated molecular ion [M+H]+ at m/z 204.2753 and 204.2757, respectively (Figure 5-A and Figure 5-B).

· DP2: Formed under oxidative stress, eluting at retention time approximately 5.573 minutes, showing a major ion at m/z 330.2840 (Figure 5-C).

Figure 5-A: Mass spectrum of acid degradant DP1 (RT – 4.909 min)

Figure 5-B: Mass spectrum of alkali degradant DP1 (RT – 4.779 min)

Figure 5-C: Mass spectrum of oxidation degradant DP2 eluted at (RT - 5.573 min)

The degradation pathways were proposed for each degradation product (as shown in Figure 6 to Figure 7) and is expected as below-

· DP1: Nominal molecular formula C₁₂H₁₃FN₂⁺ (Figure 6).

· DP2: Nominal molecular formula C₁₆H₁₁FN₂O₃S⁺ (Figure 7).

The mechanism of degradation through acid and alkaline hydrolysis and oxidation was mapped based on mass-to-charge ratios and known fragmentation behavior has been proposed for understanding of how vonoprazan degrades through hydrolysis and oxidation.

Figure 6: Acid/Alkali treated vonoprazan Degradation [DP1] Pathway

Figure 7: Peroxide treated vonoprazan Degradation [DP2] Pathway

This study addresses a critical gap in vonoprazan's stability profile by using LC-MS to identify and structurally characterize two key forced degradation products (DP1 and DP2), which were tentatively identified as known impurities. In parallel, a stability-indicating HPLC quantification method was validated. Together, this work provides a robust analytical framework for the quality control of vonoprazan-containing pharmaceuticals. Based on the proposed nominal molecular formula and the commercially listed impurities of vonoprazan, the degradation products DP1 and DP2 may corresponds to Vonoprazan Impurity 5 (SZ CAT No: SZ-V030010, CAS No: 1610043-62-3) and Vonoprazan Sulfonyl Aldehyde Impurity (SZ CAT No: SZ-V030007, CAS No: 881677-11-8) respectively^34-35.

Toxicological Consideration:^36-40

As per the review of existing literature and structural analysis DP1 is formed via N-dealkylation, its structure lacks any alerts typically associated with DNA reactivity. It is classified as a Class 5 impurity under ICH M7, indicating no structural concern for genotoxicity. Qualification is only needed if its concentration exceeds the threshold of Toxicological Concern (TTC). Similarly, DP2 is an oxidation product (sulfoxide or sulfone), its structure is also considered non-genotoxic. The risk of forming harmful nitrosamines is minimal due to the absence of necessary precursors in the formulation. Both degradation products appear structurally consistent with non-genotoxic compounds. To further confirm this assessment, a toxicological risk assessment in silico analysis in accordance with ICH M7(R2) guidelines is suggested. The presence of sulfoxide/sulfone groups can potentially form mutagenic agent/s under specific oxidative conditions; a careful evaluation of excipient compatibility and storage conditions is crucial to mitigate any long-term degradation risks.

CONCLUSION:

To the best of our knowledge, this study not only develops and validates a stability-indicating HPLC method for vonoprazan but also addresses the critical gap in the current analytical and stability literature for vonoprazan by structurally characterizing its probable degradation products using LC-MS and proposes degradation pathways under ICH-specified stress conditions. The developed HPLC method is simple, accurate, and robust, enabling reliable quantification of vonoprazan even in the presence of its degradation products. Comprehensive forced degradation studies revealed that vonoprazan is susceptible to hydrolytic (acidic and alkaline) and oxidative stress, while remaining stable under thermal and photolytic conditions. LC-MS analysis led to the identification of two major degradation products, and plausible degradation pathways were proposed. These findings highlight the importance of protecting vonoprazan from hydrolysis and oxidation during formulation development and storage. The validated method holds practical utility for routine quality control and long-term stability testing in pharmaceutical analysis.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

Authors are thankful to Venture Centre, Pune for their assistance with the LC-MS analysis.

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Received on 18.07.2025      Revised on 25.11.2025

Accepted on 07.02.2026 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):3239-3245.

DOI: 10.52711/0974-360X.2026.00461

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