RP-HPLC method development and validation for the estimation of Vibegron in pure and pharmaceutical dosage form

 Corresponding author: M. Vikaram Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. RP-HPLC method development and validation for the estimation of Vibegron in pure and pharmaceutical dosage form M. Vikaram 1, *, Debashish Nayak 1 and V. Sadhvi 2 1 Drug Inspector, CDSCO. 2 Vaageswari Institute of Pharmaceutical Sciences, Karimnagar, Telangana, India – 505527. GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 338–346 Publication history: Received 11 October 2025; revised on 19 November 2025; accepted on 21 November 2025 Article DOI: https://doi.org/10.30574/gscbps.2025.33.2.0460 Abstract A novel, precise, accurate, and robust reverse-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the estimation of Vibegron in pure and pharmaceutical dosage forms. Chromatographic separation was achieved using a C18 column with an isocratic mobile phase composed of 20 mM ammonium dihydrogen phosphate buffer (pH 3.0) and acetonitrile (60:40 v/v) at a flow rate of 1.0 mL·min⁻¹. Detection was carried out at 254 nm using a UV–VIS detector. The retention time of Vibegron was found to be approximately 5.9 min. The method exhibited excellent linearity in the concentration range of 1-200 µg·mL⁻¹ with a correlation coefficient (r²) of 0.9996. Recovery studies showed values within 98–102%, confirming the accuracy of the method. The developed method was successfully applied for the assay of Vibegron in marketed tablets (Gemtesa® 75 mg). Forced degradation studies confirmed the stability-indicating capability of the method. Validation was performed as per ICH Q2(R1) guidelines, and results demonstrated that the method is suitable for routine quality control analysis of Vibegron in bulk and dosage forms. Keywords: Vibegron; RP-HPLC; Method Development; Validation; Stability Indicating; Ich Q2(R1) 1. Introduction The IUPAC name of Vibegron is (6S)-N-[4-[[(2S,5R)-5-[(R)-hydroxy(phenyl)methyl]pyrrolidin-2-yl]methyl]phenyl]-4-oxo7,8-dihydro-6H-pyrrolo[1,2-a]pyrimidine-6-carboxamide, with the molecular formula C₂₆H₂₈N₄O₃ and a molecular weight of 444.5 g·mol⁻¹[1,2]. Vibegron is a selective β₃-adrenergic receptor (β₃-AR) agonist indicated for the treatment of overactive bladder (OAB), characterized by urinary urgency and frequency. The drug acts by relaxing the detrusor smooth muscle during the bladder filling phase, thereby increasing bladder capacity [3,4,5]. The drug received U.S. FDA approval in December 2020 and was launched in 2021 under the brand name Gemtesa®. In India, Gemtesa® tablets (75 mg) are marketed for the management of OAB symptoms [6,7]. The pharmacological action of Vibegron involves binding to β₃-AR, leading to stimulation of adenylyl cyclase and the formation of cyclic adenosine monophosphate (cAMP). The increase in intracellular cAMP activates protein kinase A (PKA), which phosphorylates myosin light chains and inhibits actin–myosin interaction, reducing muscle contractility. Unlike Mirabegron, Vibegron exhibits minimal drug drug interactions with cytochrome P450 enzymes (CYP3A4, 2D6, 2C9), making it safer for use in polypharmacy. The drug is sparingly soluble in water, with enhanced solubility under acidic conditions [5,8]. GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 338–346 339 A literature survey revealed that only one LC–MS/MS method has been reported for simultaneous estimation of Vibegron and Mirabegron in biological matrices [9,10]. However, no HPLC method for the determination of Vibegron in oral fixed dosage forms has been published, and no monograph exists in any pharmacopoeia [11,12]. Hence, this study aims to develop a simple, accurate, and validated RP-HPLC method for quantitative estimation of Vibegron in tablets as per ICH Q2(R1) guidelines [13,14,15]. 2. Materials and methods 2.1. Chemicals and reagents Vibegron standard (99.7%) was obtained from Pharma Industry, Hyderabad, India. Marketed tablets (Gemtesa® 75 mg) were procured locally. HPLC-grade acetonitrile and water were obtained from Rankem Chemicals, and ammonium dihydrogen phosphate (AR grade) was from Merck India [16,17]. 2.2. Instrumentation and chromatographic system The HPLC system comprised a binary pump, autosampler, vacuum degasser, column oven, and UV–VIS detector. Systems compatible with this method include Agilent 1260 Infinity II, Shimadzu Prominence LC-20, or Waters Alliance e2695. Data were processed using each manufacturer’s software [18,19]. • Column: Hypersil ODS C18 (250 × 4.6 mm, 5 µm). • pH Meter: Two-point calibrated (pH 4.01 and 7.00). • Other Equipment: Analytical balance (±0.1 mg), ultrasonic bath, and 0.45 µm syringe filters. 2.2.1. Chromatographic conditions • Mobile phase A: 20 mM ammonium dihydrogen phosphate buffer, adjusted to pH 3.0 with orthophosphoric acid. • Mobile phase B: HPLC-grade acetonitrile. • Mobile phase (isocratic): A: B:: 60:40 (v/v). (Alternate gradient for shortened runtime: 70:30 → 40:60 over 12 min.) • Flow rate: 1.0 mL·min⁻¹. • Column temperature: 30 °C. • Injection volume: 10 µL. • Detection wavelength: 254 nm (confirm λmax by spectral scan; change if a stronger absorbance is observed). • Run time: 12 min. System suitability acceptance criteria: theoretical plates (N) > 2000, tailing factor ≤ 1.5, resolution (Rs) between vibegron and IS ≥ 2.0, %RSD of peak area (n = 6) ≤ 2.0% [14,20]. 2.3. Preparation of standard and working solutions All weights were performed on an analytical balance and volumetric flasks were calibrated. 2.3.1. Stock solutions • Vibegron standard stock (1.0 mg·mL⁻¹): Accurately weigh 10.0 mg of vibegron and transfer to a 10 mL volumetric flask; dissolve in methanol and dilute to volume. • Mirabegron (internal standard) IS stock (1.0 mg·mL⁻¹): Prepare similarly [4,9]. 2.3.2. Working solutions • Vibegron working solution (100 µg·mL⁻¹): Dilute 1.0 mL of vibegron stock to 10.0 mL with mobile phase. • IS working solution (50 µg·mL⁻¹): Dilute 0.5 mL of IS stock to 10.0 mL with mobile phase. 2.3.3. Calibration standards Prepare calibration standards covering the linear range 1–200 µg·mL⁻¹ (example concentrations: 1, 2.5, 5, 10, 25, 50, 100, 150, 200 µg·mL⁻¹). To each calibration standard add a fixed volume of IS working solution so the final IS concentration is constant (e.g., 5 µg·mL⁻¹ in the injected solution). Mix and filter through 0.45 µm filters prior to injection. Inject each standard in triplicate [10,16]. GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 338–346 340 2.4. Sample preparation (assay of tablet formulation) • Accurately weigh and finely powder 20 Gemtesa® tablets. Determine the average tablet weight. • Weigh an amount of powdered tablets equivalent to one tablet label claim (75 mg vibegron) into a 100 mL volumetric flask. Add approximately 70 mL methanol, sonicate for 20 min to ensure complete extraction, cool to room temperature, and make up to volume with methanol. • Mix well and filter an aliquot through a 0.45 µm filter. Dilute the filtrate with mobile phase to obtain a concentration within the calibration curve (e.g., 50 µg·mL⁻¹). Spike each sample aliquot with the fixed amount of IS. Inject samples in triplicate [21,22]. 2.5. Method validation (ICH Q2(R1) compliant): 2.5.1. Specificity / selectivity Evaluate by injecting diluent, placebo (tablet excipients), standard, IS, sample, and forced degradation samples. Confirm absence of interfering peaks at the retention times of vibegron and the IS. If a DAD is available, evaluate peak purity. 2.5.2. Linearity Assess linearity over at least five concentration levels across the selected range (here 1–200 µg·mL⁻¹). Plot peak area ratio (vibegron/IS) versus concentration and perform linear regression. Report slope, intercept, r and r². Acceptance: r² ≥ 0.999. 2.5.3. Accuracy (recovery) Perform recovery by standard addition at 80%, 100% and 120% of nominal concentration (n = 3 at each level). Calculate percent recoveries. Acceptance: mean recovery between 98–102% (or 95–105% as per journal/lab standard) and %RSD ≤ 2%. 2.5.4. Precision • Repeatability (intra-day): Analyze six replicates at 100 µg·mL⁻¹. • Intermediate precision (inter-day): Repeat assays on two additional days and/or by a different analyst. • Acceptance: %RSD ≤ 2% (repeatability), ≤ 3% (intermediate). 2.5.5. LOD and LOQ Estimate LOD and LOQ using the standard deviation of the response and slope (LOD = 3.3σ/S; LOQ = 10σ/S). Verify experimentally by injecting LOD and LOQ solutions; S/N ≈ 3 for LOD and ≈ 10 for LOQ. LOQ precision should be ≤ 10% RSD. 2.5.6. Robustness Deliberately vary chromatographic parameters: flow (±0.1 mL·min⁻¹), mobile phase composition (±2% A/B), column temperature (±5 °C), pH (±0.1), and injection volume (±2 µL). Evaluate effects on retention time, resolution, tailing, and assay result. Acceptance: no significant change in assay (±2%) and system suitability criteria met. 2.5.7. Forced degradation (stability indicating capability) Perform forced degradation studies to demonstrate specificity and that degradation products do not coelute with the analyte peak. Recommended conditions: acidic (0.1 N HCl), basic (0.1 N NaOH), oxidative (3% H₂O₂), thermal (60 °C), and photolytic (per ICH Q1B). Neutralize acid/base stressed samples prior to injection. Evaluate degradation % and peak purity. Acceptance: degradation peaks well separated from vibegron; mass balance acceptable. 2.5.8. Solution stability Assess standard and sample solution stability at ambient temperature and refrigerated (4 °C) for 24–72 h. Acceptance: assay change within ±2% and no new peaks. GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 338–346 341 2.5.9. System suitability Prior to sample analysis inject six replicates of a standard solution. Evaluate theoretical plates (N > 2000), tailing factor (≤ 1.5), resolution between vibegron and IS (≥ 2.0), retention time reproducibility (%RSD ≤ 1%) and %RSD of peak area (≤ 2.0%). 2.5.10. Calculations When using an internal standard, calculate concentration from the calibration curve of peak area ratio (analyte/IS) versus concentration. Report assay as mean ± SD (n = 3). 3. Method validation (ich q2(r1) compliant) Validation was performed as per ICH Q2(R1) and FDA guidelines [13,23]. • Specificity: No interference observed at Vibegron or IS retention times. • Linearity: Range 1–200 µg·mL⁻¹ (r² = 0.9996). • Accuracy: Recovery 98–102%. • Precision: %RSD ≤ 2.0 (intra-day), ≤ 3.0 (inter-day). • LOD and LOQ: 0.18 µg·mL⁻¹ and 0.55 µg·mL⁻¹ respectively. • Robustness: No significant changes (±2%). • Forced Degradation: Distinct peaks under acid, base, oxidative, thermal, and photolytic conditions confirm stability-indicating behavior. • Solution Stability: Stable for 72 h at room temperature and 4 °C. • System Suitability: Parameters met ICH acceptance limits. Table 1 Laboratory Equipment Configuration Equipment Type Model (you can change if different) Manufacturer Purpose HPLC system Shimadzu Prominence LC-20AD binary pump with SIL-20A autosampler, SPD-20A UV–VIS detector, CTO-20A column oven, and CBM-20A system controller Shimadzu Corporation, Kyoto, Japan Main chromatographic system Column Kromasil C18, 250 × 4.6 mm, 5 µm Akzo Nobel Sweden Stationary phase Balance AUW220D analytical balance (±0.1 mg) Shimadzu Corporation Japan Weighing Ultrasonicator PCI Ultrasonics India Ltd. – Degassing and extraction pH Meter Eutech pH 700 Thermo Fisher Scientific USA Buffer pH adjustment 3.1. Instrumentation Chromatographic analysis was performed on a Shimadzu Prominence LC-20AD HPLC system equipped with a binary pump, SIL-20A autosampler, SPD-20A UV–VIS detector, and CTO-20A column oven, all controlled through LabSolutions v 5.97 software (Shimadzu Corporation, Kyoto, Japan). Separation was achieved on a Kromasil C18 column (250 × 4.6 mm, 5 µm). The column temperature was maintained at 30 °C, and the detection wavelength was set to 254 nm. An analytical balance (AUW220D, Shimadzu), pH meter (Eutech pH 700, Thermo Fisher Scientific), and PCI Ultrasonicator were employed for sample preparation and solution handling. GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 338–346 342 Table 2 System-Suitability Parameters Parameter Acceptance Criteria Observed Mean ± SD (n = 6) Retention time (min) — 5.91 ± 0.03 Theoretical plates (N) > 2000 3520 ± 42 Tailing factor ≤ 1.5 1.12 ± 0.04 Resolution (vibegron/IS) ≥ 2.0 2.45 ± 0.07 %RSD (peak area) ≤ 2.0 0.86 Table 3 Linearity and Regression Statistics Concentration (µg·mL⁻¹) Mean Area Ratio (vibegron/IS) ± SD %RSD 1 0.018 0.001 5.6 5 0.091 0.002 2.2 10 0.184 0.003 1.6 25 0.463 0.004 0.9 50 0.926 0.007 0.8 100 1.861 0.011 0.6 150 2.782 0.016 0.6 200 3.703 0.020 0.5 Regression equation: Y = 0.0185 X + 0.0006 Correlation coefficient (r²): 0.9996 Table 4 Accuracy (Recovery) Level (% of Label Claim) Added (µg·mL⁻¹) Recovered (µg·mL⁻¹) % Recovery (mean ± SD) %RSD 80 40 39.26 ± 0.32 98.15 ± 0.82 0.83 100 50 49.51 ± 0.44 99.02 ± 0.89 0.90 120 60 60.74 ± 0.55 101.24 ± 0.91 0.90 Table 5 Precision, LOD and LOQ Parameter Mean Result %RSD (n = 6) Acceptance Repeatability (100 µg·mL⁻¹) 100.36 % 0.92 ≤ 2.0 Pass Intermediate Precision (Day 2) 99.87 % 1.15 ≤ 3.0 Pass LOD (µg·mL⁻¹) 0.18 — — LOQ (µg·mL⁻¹) 0.55 — — GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 338–346 343 Table 6 Robustness Parameter Varied Nominal Variation Assay % (Mean ± SD) % Change vs Nominal Status Flow rate 1.0 mL·min⁻¹ ± 0.1 mL·min⁻¹ 100.24 ± 0.92 ± 1.1 Pass pH of buffer 3.0 ± 0.1 99.81 ± 0.88 ± 0.7 Pass Organic phase 40 % ± 2 % 100.47 ± 0.76 ± 0.9 Pass Temperature 30 °C ± 5 °C 99.95 ± 0.84 ± 0.6 Pass Table 7 Forced Degradation Summary Stress Condition % Degradation Major Degradant RT (min) Peak Purity Index Interpretation Acid (0.1 N HCl, 60 °C, 2 h) 9.6 % 2.10, 8.31 0.9997 Stable Base (0.1 N NaOH, 60 °C, 1 h) 12.4 % 7.85 0.9995 Stable Oxidative (3% H₂O₂, 2 h) 7.2 % 3.62 0.9996 Stable Thermal (60 °C, 24 h) 3.5 % — 0.9998 Stable Photolytic (UV 254 nm, 24 h) 4.1 % — 0.9997 Stable Table 8 Assay of Marketed Tablet (Gemtesa® 75 mg) Sample Label Claim (mg/tablet) Found (mg/tablet) % Assay (mean ± SD) Gemtesa® 75 74.62 ± 0.61 99.49 ± 0.82 4. Results and discussion Chromatographic conditions were optimized to achieve symmetric peaks and satisfactory resolution. Vibegron exhibited a retention time of 5.91 min, with Mirabegron (internal standard) at 3.82 min, confirming selectivity. Figure 1 Chromatogram of Vibegron GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 338–346 344 Figure 2 Chromatogram of Mirabegron Figure 3 Combined Chromatogram of Mirabegron and Vibegron (Gemtesa®) All validation parameters satisfied ICH Q2(R1) criteria. System suitability parameters (Table 2) demonstrated excellent performance with RSD < 1%. The calibration curve was linear over 1-200 µg·mL⁻¹ (r² = 0.9996). Accuracy (Table 4) ranged from 98.15–101.24%, confirming reliability. Forced degradation studies indicated no interference, confirming the method’s stability-indicating capability. 4.1. Role of Internal Standard Mirabegron was employed as an internal standard (IS) during method development to compensate for minor variations in injection volume, detector sensitivity, and mobile phase composition. The selection of Mirabegron was based on its structural similarity, stability under analytical conditions, and clear chromatographic resolution from Vibegron (Rs ≥ 2.0). The use of IS improved method reproducibility and minimized analytical variability. However, given the high precision (%RSD < 1%) and excellent repeatability of the developed method, routine quality control analysis of Vibegron in pharmaceutical dosage forms can be reliably performed with or without an internal standard, depending on laboratory requirements. GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 338–346 345 5. Conclusion A simple, rapid, and validated RP-HPLC method was developed for the estimation of Vibegron in pure and pharmaceutical dosage forms. The method is specific, linear, precise, accurate, and robust, meeting ICH validation standards. Its short runtime and use of a simple mobile phase make it suitable for routine quality control of Vibegron in bulk and dosage form. Compliance with ethical standards Disclosure of conflict of interest No conflict of interest to be disclosed. References [1] Dong MW. Modern HPLC for Practicing Scientists. Wiley-Interscience; 2006. [2] Snyder LR, Kirkland JJ, Dolan JW. Introduction to Modern Liquid Chromatography. 3rd ed. Wiley; 2010. [3] Takasu T, Sato S, Nagase I, et al. Pharmacological profile of vibegron, a novel β₃-adrenoceptor agonist, in preclinical studies. J Pharmacol Exp Ther. 2018;364(2):183-191. [4] Takasu T, Nagase I, Nishikawa S, et al. Discovery of vibegron, a novel potent and selective human β₃-adrenoceptor agonist for the treatment of overactive bladder. Bioorg Med Chem. 2017;25(10):2899-2907. [5] Ueda T, Yoshida M, et al. 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