Identification of Degradation Products and a Stability-Indicating RP-HPLC Method for the Determination of Belzutifan in Pharmaceutical Dosage Forms

Abstract

Background:

Hypoxia-inducible factor-2alpha (HIF-2α) is a protein, the selective inhibitor of which (belzutifan) is used in the treatment of cancers related to von Hippel-Lindau (VHL) disease. No analytical procedure has been so far reported in the identification of forced degradation products of belzutifan. Thus, the current project was conducted to design and approve the liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) technique to perform a full analysis of it.

Objective:

This research was to develop and confirm a strong high-performance liquid chromatography (HPLC) procedure in the quantification of belzutifan in the tablet dosage form and to utilize LC-MS/MS in the characterization of the forced degradation products of belzutifan. Approach: The separation was done by the chromatographic method utilizing a Zorbax C₁₈ column with the mobile phase comprised of acetonitrile and 0.1% formic acid (50:50, v/v), and the flow rate was 1.0 mL/min with 247nm as a detector wavelength. Belzutifan had a retention time of 2.338 min and a total reaction time of 4 min.

Results:

The derived technique showed great linearity (R2 > 0.999) in the range of concentrations (10-60 μg/ml) and good accuracy, precision, and strength. The forced degradation analysis identified specific degradation products, which were further identified using the LC-MS/MS, thus gathering significant information about the stability profile of belzutifan in stressful environments.

Conclusion:

This study developed and validated a simple, rapid, cost-effective, and highly sensitive RP-HPLC method for quantitative estimation of belzutifan in tablets. The method complied with ICH guidelines, demonstrating excellent linearity, accuracy, specificity, robustness, and stability. Linearity was achieved over a wide range, with no interference from excipients, confirming its reliability. Relative standard deviations (RSD) were below 2%, indicating precision and reproducibility. Owing to its simplicity and efficiency, the method is suitable for routine quality control in pharmaceutical laboratories.

INTRODUCTION

The pharmaceutical industry relies heavily on accurate and efficient analytical methods to ensure the quality, safety, and efficacy of drug formulations. High-performance liquid chromatography (HPLC) has emerged as one of the most widely used techniques for quantifying and quality assessment of pharmaceutical compounds due to its precision, sensitivity, and versatility. This study focuses on developing and validating a novel HPLC method for quantitatively determining Belzutifan, a drug used to treat certain cancers, in tablet dosage form. Belzutifan is a selective inhibitor of hypoxia-inducible factor 2α (HIF-2α). This transcription factor plays a crucial role in the growth of certain cancers,[1] including those associated with von Hippel–Lindau (VHL) disease. Identified in the 1990s by researchers at UT Southwestern Medical Centre, HIF-2α is critical for the progression of various cancers. Belzutifan works by preventing the complex formation between HIF-2α and HIF-1β, a necessary step for HIF-2α activation, thereby inhibiting tumor growth in VHL-associated cancers. Belzutifan received FDA approval on August 13, 2021,[2] for treating VHL-associated cancers. Upon oral administration, it blocks the function of HIF-2α, preventing its heterodimerization and binding to DNA, which results in the suppression of downstream genes involved in hypoxic signaling. This inhibition leads to reduced cell growth and survival in HIF-2α-expressing tumor cells. Overexpression of HIF-2α is linked to tumorigenesis, and by targeting this transcription factor, Belzutifan can effectively treat solid tumors associated with VHL disease. In healthy individuals, HIF-2α is degraded by VHL proteins under normal oxygen conditions. Still, during hypoxia, it translocates into the nucleus, forming a complex with HIF-1β, triggering gene expression that promotes cellular proliferation and angiogenesis. Belzutifan is also known to be effective in treating Pacak–Zhuang syndrome,[3] a rare disorder associated with VHL disease. Clinical trials over three years showed no serious safety concerns.[4,5] The chemical structure of Belzutifan is 3-{[(1S,2S,3R)-2,3-difluoro-1-hydroxy-7-methanesulfonyl-2,3-dihydro-1H-inden-4-yl]oxy}-5-fluoro benzonitrile [Figure 1]. Given its increasing clinical importance, ensuring the quality and consistency of Belzutifan formulations is essential. However, limited analytical methods, including HPLC–MS[6,7,8] and UPLC,[9] have been reported in the literature to quantify and analyze Belzutifan in various formulations, underscoring the need for a validated, rapid, and cost-effective HPLC method. This study aims to develop a simple yet robust analytical procedure that can provide reliable results, ensuring compliance with regulatory standards. The method was optimized for high sensitivity and reproducibility while minimizing analysis time and operational costs. This makes the proposed method suitable for routine quality control and large-scale analysis of Belzutifan tablets in pharmaceutical manufacturing settings. In addition to method development, this study emphasizes thorough validation of key performance parameters, including linearity, accuracy, specificity, robustness, and stability. By adhering to ICH guidelines, the validated method demonstrates its applicability and reliability for routine use in quality control laboratories.

Figure 1.

Chemical structures of Belzutifan

MATERIALS AND METHODS

Reagents and chemicals

Acetonitrile (HPLC mark), formic acid (HPLC mark), and water (HPLC mark) were obtained from Merck India Ltd., Mumbai, India. Belzutifan was obtained from Cipla Pharmaceutical Company, Mumbai. HPLC software of Empower version 3.0 was used. Waters HPLC with a quaternary pump and PDA detector with Empower 3.0 software were employed.

Equipments

HPLC (High-performance liquid chromatography)

The Waters e-2695 quaternary pump, PDA detector 2998, and Empower 3.0 chromatographic software were utilized.

LC–MS/MS

An HPLC system (Waters Alliance e2695 model) was coupled to a QTRAP 5500 triple quadrupole mass spectrometer (Sciex).

Chromatographic conditions

Chromatographic separation of Belzutifan was performed in isocratic mode at ambient temperature utilizing a Zorbax C18 column, 150 mm × 4.6 mm, 3.5 µm, with a mobile phase consisting of a 50:50 combination of acetonitrile and 0.1% formic acid. The injection volume was 20 μL, and at 247 nm, the eluents were regulated for a duration of 4 minutes.

Preparation of 0.1% formic acid

Transfer 1 ml of formic acid to 1000 ml of HPLC-grade water and mix well.

Preparation of mobile phase

Mix acetonitrile and 0.1% formic acid in a ratio of 50:50 and filter through a 0.45-µm membrane filter paper.

Note: Mobile phase is used as diluent.

Preparation of belzutifan standard solution

Weigh 40 mg of Belzutifan working standard into a 100-ml volumetric flask and dilute volume with diluent. Further diluted 5 ml into 50 ml with diluents.

Preparation of sample solution

Transfer 10 tablets of Belzutifan (each tablet containing 40 mg of Belzutifan) into a 100-ml volumetric flask and add 70 ml of diluent; sonicate to dissolve and get the desired volume. Subsequently, 5 ml of the aforementioned solution was put into a 50-ml volumetric flask and diluted to the mark with diluent.

Validation procedure[10,11,12,13,14,15,16,17,18,19,20,21,22,23]

In accordance with ICH Q2 (R1) criteria, the analytical method was validated concerning characteristics, including system appropriateness, method precision, specificity, intermediate precision, linearity, robustness, limit of detection (LOD), limit of quantification (LOQ), and the stability of forced degradation.

Suitability

System suitability parameters were computed to evaluate the system’s performance. The parameters encompass the quantity of USP plates, USP tailing, and % RSD, which can be computed and determined to be within the acceptable limits [Figure 2].

Figure 2.

System suitability chromatogram of Belzutifan

Specificity

Specificity refers to the capacity to evaluate unambiguously in the presence of an analyte, together with additional contaminants and components such as excipients, which may be presumed to exist in the sample and standard solution. The verification was conducted by analyzing chromatograms of both the blank standard and the standard formulated with Belzutifan.

Accuracy

Accuracy refers to the closeness of the test result to the genuine value. The research evaluated the treatment at three distinct concentration levels. A minimum of three injections have been administered at each stage, and the % recovery, standard deviation, and relative standard deviation (RSD) have been calculated.

Linearity

The linearity of an analytical method is its ability to obtain the results directly proportional to the concentration of the analyte. Seven series of standard solutions were chosen to assess the linearity range. A calibration curve was constructed by plotting peak area against the concentration of the standard solution, and the regression equations were determined using the least squares method.

LOD and LOQ

LOD is the minimum analyte concentration in a sample that can be found, whereas LOQ denotes the minimum analyte concentration that can be quantified with sufficient accuracy and precision. The limits of detection (LOD) and quantification (LOQ) were individually established based on the calibration curves. The limit of detection (LOD) and limit of quantification (LOQ) were determined in accordance with ICH recommendations as 3.3σ/s and 10σ/s, respectively, where σ/s denotes the signal-to-noise ratio.

Robustness

Analytical procedure robustness measures its ability to remain unaffected by small but deliberate variations in the method’s parameters and indicates its reliability during normal use. The robustness study was conducted by injecting the standard solution into the HPLC system and modifying the flow rate (±0.2 ml/min) and organic phase (±10 percent) of chromatographic conditions.

Degradation

Stress degradation must not compromise the peaks observed in the forced degradation chromatogram. The stress degradation studies were performed in accordance with ICH recommendations. The degradation peaks must be distinctly separated, with a minimum resolution of 1.0 between them, and the peak purity of the primary peaks must be satisfactory.

RESULTS AND DISCUSSION

Method development and optimization

The RP-HPLC system was initially optimized using a Zorbax C₁₈ column, 150 mm × 4.6 mm, 3.5 µm column. A mixture of acetonitrile and 0.1% formic acid, with a ratio of 50:50 v/v, was more appropriate to satisfy the system suitability parameters. Table 1 summarizes the optimized chromatographic conditions.

Table 1.

Optimized chromatographic conditions

Conditions	Parameters
Stationary phase	Zorbax C18column, 150 mm × 4.6 mm, 3.5 μm
Mobile phase	Acetonitrile and Belzutifan in the ratio of 50:50 v/v
Wavelength	247 nm
Injection volume	20 μL
Column temperature	Ambient
Flow rate	1.0 ml/min
Run time	4 min

Tests for method validation

System suitability

The HPLC system was stabilized for 60 min to get a stable baseline. Six replicate injections mixture containing 40 µg/ml of Belzutifan was performed and assessed to check the system’s suitability. From six replication injections, the system suitability parameters were evaluated. The research concludes that the system suitability and outcomes of the used HPLC system are summarized in Table 2.

Table 2.

System suitability results

System suitability parameter	Acceptance criteria	Belzutifan
% Relative Standard Deviation	NMT 2.0	0.95
USP Tailing	NMT 2.0	1.42
USP Plate count	NMT 2000	2256

USP: United states pharmacopeia

Specificity

There was no interference from the blank during Belzutifan’s retention time. The method is specific [Figure 3].

Figure 3.

Blank chromatogram of Belzutifan

Linearity

A series of linear solutions containing Belzutifan at six different concentrations (10, 20, 30, 40, 50, and 60 µg/mL) were prepared to demonstrate the linearity of the devised test method. Throughout the Belzutifan concentration series, the calibration curves were linear. Table 3 and Figure 4 list the linearity values. According to the calibration curve, Belzutifan’s coefficient of correlation is 0.9992.

Table 3.

Linearity data of Belzutifan

Conc. (μg/ml)	Area
10	831393
20	1499261
30	2194419
40	2906102
50	3816581
60	4506553
Correlation coefficient	0.9992
Slope	74631.70
Intercept	11664.57

Figure 4.

Linearity plot of Belzutifan

Accuracy

The precision of Belzutifan is reliant upon recovery experiments conducted at three distinct dilution levels (50%, 100%, and 150%). The test process involved injecting the test solutions in three preparations for each spike level, thus facilitating the assay. The observed shared recovery values fell within the range of 98.5% to 101.9%; the data are presented in Table 4.

Table 4.

HPLC results of the accuracy of Belzutifan

Concentration (μg/ml)	Mean±SD	% RSD	% Recovery
50	99.9±1.33	1.330	99.9
100	99.8±055	0.550	99.8
150	101.2±071	0.700	101.2

SD=Standard deviation, USP=United states pharmacopeia

LOD and LOQ

The limits of detection (LOD) and quantification (LOQ) were individually established by the calibration curve approach; LOD and LOQ of the substances were computed by sequentially injecting progressively lower concentrations of standard solutions utilizing the devised HPLC method. The limit of detection (LOD) values for Belzutifan were recorded at 0.225 µg/mL and 0.25 µg/mL, with corresponding signal-to-noise (s/n) values of 0.12. LOQ values were 0.25 µg/mL, and the corresponding s/n values were 0.4.

Robustness

As per ICH norms, small but intentional differences were made in the method’s parameters, for instance, the change in the flow rate (±0.2 ml/min) and organic phase (±10%) to check the method’s capacity to remain unaffected. The results are shown in Table 5.

Table 5.

Results of the robustness of Belzutifan

Change in parameter	% RSD of Belzutifan
Flow Plus (1.2 ml/min)	1.15
Flow Minus (0.8 ml/min)	0.96
Org Phase Plus (52:48)	0.75
Org Phase Minus (60:40)	0.96

RSD=Relative standard deviation

Forced degradation studies of belzutifan

The proposed analytical approach is applicable for release and stability investigations, facilitating effective evaluations, and qualifies as a stability-indicating method. The forced degradation study was performed in accordance with ICH principles, encompassing acid, base, peroxide, reduction, heat, and hydrolysis degradation. The chromatograms indicate that the selected medicines remained stable under the imposed stress conditions, although degradation peaks were observed. The degradation samples were analyzed by LC–MS [Table 6].

Table 6.

Results of forced degradation

Stress condition	% Degradation	% Assay
Acid	12.6	87.4
Alkali	11.9	88.1
Peroxide	14.7	85.3
Reduction	4.7	95.3
Thermal degradation	0.8	99.2
Hydrolysis	10.4	89.6

Acid degradation

During acid degradation, the chosen samples were hydrolyzed with 1 N HCl for three hours at 60°C, resulting in a 12.6% degradation of Belzutifan as determined by HPLC, and yielding four degradation products: DP1, DP2, DP3, and DP4.

Alkali degradation

The alkali degradation of specific samples commenced with 1 N NaOH, resulting in an 11.9% degradation of Belzutifan as determined by HPLC, and yielding three degradation products: DP3, DP4, and DP5.

Peroxide degradation

The sample’s peroxide breakdown was examined in 30% hydrogen peroxide, and an HPLC analysis revealed that 14.7% of Belzutifan was present. Four degradation products—DP2, DP3, DP4, and DP6—were produced.

Reduction degradation

A 30% sodium bisulfate solution was used to study the reduction degradation of specific pharmaceuticals. Using HPLC, 4.7% of Belzutifan degradation was found, and three degradation products—DP3, DP4, and DP7—were produced.

Thermal degradation

After six hours of exposure to 105°C, the sample underwent thermal deterioration, and HPLC revealed a 0.8% degradation of Belzutifan. However, there was no degradation residue.

Hydrolysis degradation

In HPLC water, hydrolysis-induced degradation of Belzutifan was found, resulting in a 10.4% degradation rate. There were no degradation products produced.

MS/MS degradation product

Figure 5 illustrates the fragmentation mechanism of the m/z-402 degradation product 1 under acidic degradation conditions. The spectrum has significant output at m/z 264, m/z 186, and m/z 102. The proposed structures were confirmed using precise mass measurements and MS/MS analyses. The fragmentation mechanism of degradation product 2 at m/z 346, identified under alkaline degradation conditions, is illustrated in Figure 6. The spectrum has significant output at m/z 186 and m/z 102. The proposed structures were validated using precise mass measurements and MS/MS analyses. Figure 7 illustrates the fragmentation mechanism of degradation product 3 with a mass-to-charge ratio of 386 under peroxide degradation conditions. The spectrum displays significant emission at m/z 264, m/z 186, and m/z 102. The proposed structures were confirmed using precise mass measurements and MS/MS analyses.

Figure 5.

Proposed fragmentation mechanism of DP1 (m/z-402.0385)

Figure 6.

Proposed fragmentation mechanism of DP2 (m/z-346.0429)

Figure 7.

Proposed fragmentation mechanism of DP3 (m/z-386.0436)

Figure 8 illustrates the fragmentation mechanism of the m/z-324 degradation product 3 under reductive degradation conditions. The spectrum has significant output at m/z 186 and m/z 102. The proposed structures were validated using precise mass measurements and MS/MS analyses.

Figure 8.

Proposed fragmentation mechanism of DP4 (m/z-324.0609)

CONCLUSION

This study effectively established and validated an innovative, straightforward, speedy, economical, and highly sensitive HPLC technique for the quantitative analysis of Belzutifan in tablet form. The method’s design prioritizes practical applicability, offering a combination of minimal operational costs and reduced analysis time, making it a valuable tool for pharmaceutical laboratories engaged in high-throughput sample analysis.

Validating key analytical parameters, including linearity, accuracy, specificity, robustness, and stability, all of which conformed to ICH (International Council for Harmonization) guidelines, underscored the method’s robustness and precision. The linearity was demonstrated over a wide concentration range, indicating the method’s capability to detect and quantify varying analyte levels with high accuracy. Additionally, the method exhibited strong specificity, ensuring no interference from excipients or other formulation components, enhancing its reliability in complex pharmaceutical matrices.

All evaluated parameters’ relative standard deviations (RSD) were consistently below 2%, reflecting minimal variability and high reproducibility across multiple analyses. The low RSD suggests that the procedure is dependable and yields consistent findings across many operators, instruments, and laboratories. Such reproducibility is critical for routine quality control, where consistency and accuracy directly impact product safety and efficacy.

The established HPLC method is an effective, scalable, and reliable instrument for the routine quality control and analysis of Belzutifan in pharmaceutical formulations. Its simplicity and efficiency make it well suited for deployment in research and industrial settings, ultimately contributing to enhanced quality assurance in pharmaceutical manufacturing.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.