DEVELOPMENT AND VALIDATION OF A RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF VANZACAFTOR, TEZACAFTOR, AND DEUTIVACAFTOR IN PHARMACEUTICAL FORMULATION

DEVELOPMENT AND VALIDATION OF A RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF VANZACAFTOR, TEZACAFTOR, AND DEUTIVACAFTOR IN PHARMACEUTICAL FORMULATION

Shaik Mahammad Noorulla * and Syeda Azra Tabassum

Deccan School of Pharmacy, Hyderabad, Telangana, India.

ABSTRACT: The present study reports the development and validation of a simple, precise, and robust RP- HPLC method for the simultaneous estimation of Vanzacaftor (VNC), Deutivacaftor (DEC), and Tezacaftor (TZC) in the fixed-dose combination formulation Alyftrek. Chromatographic separation was achieved using an XTERRAC18 column (250×4.6mm, 5µm) with a mobile phase consisting of potassium dihydrogen phosphate buffer and methanol (80:20, v/v). The analytes were detected at 247nm, and well-resolved peaks were obtained at retention times of 3.206 min (VNC), 5.148 min (DEC), and 6.823 min (TZC). The method was validated according to ICHQ2 (R1) guidelines. Linearity was established over the ranges of 2–6 µg/mL for VNC, 25–75µg/mL for DEC, and 10–30µg/mL for TZC with correlation coefficients (R²) close to 1. The limits of detection were 0.045 µg/mL (VNC), 0.431 µg/mL (DEC), and 0.073 µg/mL (TZC), while the limits of quantification were 0.150 µg/mL, 1.436 µg/mL, and 0.245 µg/mL, respectively. Precision studies showed %RSD values below 1%, indicating excellent repeatability. Accuracy was confirmed with recovery values between 99–100% for all three drugs. Overall, the method demonstrated suitability for routine quality control analysis, offering accurate quantification and reliable performance for simultaneous estimation of VNC, DEC, and TZC in pharmaceutical formulations.

Keywords: Vanzacaftor, Tezacaftor, Deutivacaftor, RP- HPLC, Method Development, Validation

INTRODUCTION: Cystic fibrosis (CF) is a genetic disorder characterized by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, leading to impaired chloride transport and progressive multisystem complications ^{1, 2, 3}. CFTR modulators including correctors and potentiators have significantly improved therapeutic outcomes by enhancing the quantity and functional activity of the CFTR protein at the cell surface ¹.

Vanzacaftor (VNC) and Tezacaftor (TZC) are CFTR correctors that facilitate proper protein folding and trafficking, while Deutivacaftor (DEC) is a CFTR potentiator that enhances channel gating ^{5, 6}. The combination of VNC, TZC, and DEC has been recently introduced in the pharmaceutical formulation Alyftrek for patients with responsive CFTR mutations ^{5, 6}.

Despite the growing clinical use of this triple-combination therapy, no simple and validated RP-HPLC method has been reported for the simultaneous quantification of VNC, DEC, and TZC in a single run ^{7, 8, 9}. Available analytical methods primarily focus on two-drug combinations or individual components, making them inadequate for routine quality control of this multi-component formulation ^{7, 8}. Therefore, the present study aims to develop and validate a rapid, sensitive, and reliable RP- HPLC method for the simultaneous estimation of VNC, DEC, and TZC in bulk and combined dosage forms ^{7, 8, 9, 10}.

The method is optimized for adequate peak resolution, accuracy, precision, and robustness, ensuring suitability for routine quality control and stability testing applications ^{16, 17, 18, 19, 20}.

FIG. 1: STRUCTURE OF VANZACAFTOR

FIG. 2: STRUCTURE OF DEUTIVACAFTOR   

FIG. 3: STRUCTURE OF TEZACAFTOR

Chemicals and Reagents: Vanzacaftor (VNC), Deutivacaftor (DEC), and Tezacaftor (TZC) were used as reference standards for the analysis. Methanol, sodium hydroxide (NaOH), potassium dihydrogen phosphate (KH₂PO₄), acetonitrile, and hydrochloric acid (HCl) of HPLC grade were employed throughout the study ¹⁶^, ¹⁷. All solutions were prepared using HPLC-grade water to ensure purity and minimize interference ¹⁶^, ¹⁷.

Instrumentation: Chromatographic analysis was performed on a Waters Alliance HPLC system equipped with a Photodiode Array (PDA) detector and operated using Empower 2 software (Waters Corporation, USA) ¹⁶. The system provided stable baseline performance and consistent retention for all analytes ¹⁶.

Chromatographic Conditions: Separation was achieved on an XTERRAC18 column (250×4.6mm, 5µm) using a mobile phase of potassium dihydrogenphosphate buffer and methanol (80:20, v/v). The buffer was adjusted to pH 4.8 and filtered through a 0.45µm membrane prior to use. The mobile phase was degassed by sonication. Detection was carried out at 247 nm ¹⁶.

Stockvnc, Dec & Tzcsolution: A primary stock solution was prepared by accurately weighing 4 mg of VNC, 50 mg of DEC, and 20 mg of TZC into a 100 mL volumetric flask and dissolving the mixture with a diluent consisting of 0.1MK₂HPO₄ buffer (pH3.2) and methanol in a 60:40(v/v) ratio. The flask was then made up to volume to obtain stock concentrations of40 µg/mL for VNC, 500µg/mL for DEC, and 200 µg/mL for TZC ¹⁶.

From this solution, a working standard was prepared by transferring 10 mL of the primary stock into a separate 100 mL volumetric flask and diluting to volume with the same diluent, yielding final concentrations of 4 µg/mL (VNC), 50 µg/mL (DEC), and 20 µg/mL (TZC) ¹⁶. These working concentrations were used throughout the method development and validation studies, including linearity, accuracy, precision, and sensitivity assessments ¹⁹^, ²⁰.

Preparation of Sample Stock Solution: Crush 20 tablets into a fine powder. Accuratelyweigh175 mg (containing 4 mg Vanzacaftor, 20mg Tezacaftor, and 50mg Deutivacaftor) and transfer into a100Ml volumetric flask. Add 10 mL methanol, sonicate for 20 minutes or shake for 10 minutes, then make up the volume with water.

Transfer 1 mL of this solution into a 10 mL volumetric flask and dilute with methanol. Filter through a 0.45 µm filter before HPLC injection.

Method Validation: The developed RP-HPLC method was validated in accordance with ICHQ2 (R1) guidelines ¹⁹^, ²⁰ to confirm its suitability for the quantitative determination of VNC, DEC, and TZC. The parameters evaluated included system suitability, linearity, accuracy, precision, sensitivity, and robustness ¹⁶^, ¹⁷.

Linearity: Linearity was assessed at five concentration levels (2–6 µg/mL for VNC, 25–75 µg/mL for DEC, 10–30 µg/mL for TZC). Calibration curves were constructed, and regression analysis demonstrated excellent linearity across the tested ranges ⁷^, ⁸^, ⁹.

Accuracy: Evaluated using standard-addition at 80%, 100%, and 120% levels. Percent recoveries were within 98–102% with %RSD < 2%, indicating good trueness of the method ⁷^, ⁸.

Precision: Repeatability (intra-day) and intermediate precision (inter-day) showed %RSD values <2%, confirming adequate precision ⁷^, ⁸.

Sensitivity (LOD and LOQ): Determined based on signal-to-noise ratios of 3:1 and 10:1, respectively, demonstrating high sensitivity ⁷^, ⁸.

System Suitability: Theoretical plates, tailing factor, and resolution met USP criteria, confirming chromatographic performance ¹⁶.

RESULTS AND DISCUSSION:

System Suitability Parameters:

FIG. 4: SYSTEM SUITABILITY CHROMATOGRAM

TABLE1: SYSTEM SUITABILITY PARAMETERS

Validation Parameters:

Linearity: Calibration curves for VNC, DEC, and TZC were generated by plotting their peak areas against corresponding concentrations at five different levels. The data Table 2 and Fig. 5 were analyzed using least-squares regression, confirming strong linear relationship for VNC (2–6μg/mL), DEC (25–75μg/mL), and TZC (10–30 μg/mL).

TABLE 2: LINEARITY DATA

FIG. 5: LINEARITY OF VNC, DEC, &TZC

Limit of Detection: The LOD for VNC, DEC, and TZC was determined based on a signal to noise ratio (S/N) of approximately 3:1, as per ICH Q2(R1). The LOD values obtained were:

VNC: 0.045µg/mL

DEC: 0.431µg/mL

TZC: 0.073µg/mL

TABLE 3: LOD DATA

FIG. 6: CHROMATOGRAM OF LOD

Limit of Quantification: The LOQ was calculated using the formula LOQ = 10 × (SD / slope) and confirmed experimentally using signal to noise ratio of approximately 10:1. The LOQ values obtained were:

VNC: 0.150µg/mL

DEC: 1.436µg/mL

TZC: 0.245µg/mL

TABLE 4: LOQ DATA

FIG. 7: CHROMATOGRAM OF LOQ

Precision: Solutions containing VNC (4 µg/mL), DEC (50 µg/mL), and TZC (20 µg/mL) were injected six consecutive times. The standard deviation (SD), relative standard deviation (RSD), and chromatograms for the peak responses of VNC, DEC, and TZC were calculated. The results demonstrated high accuracy in the combined analysis of VNC, DEC, and TZC.

TABLE 5: VNC, DEC & TZC PRECISION

Accuracy: Solutions of VNC (4µg/mL), DEC (50µg/mL), and TZC (20 µg/mL) were injected six times consecutively. Peak responses, assay values, and chromatograms for VNC, DEC, and TZC were calculated, showing high accuracy in their combined analysis.

TABLE 6: ACCURACY TABLE OF VNC

TABLE 7: ACCURACY TABLE OF TZC

TABLE 8: ACCURACY TABLE OF DEC

Robustness: In order to figure out how robust it was, the most important chromatographic settings were changed, and the chromatographic equipment suitability profile was watched and written down at the same time.

The following chromatographic settings were thought to be important:

• The amount of acetonitrile,
• The pH value,
• The detector nanometers,
• The rate of stream flow and
• The temperature of the column.

We figured out the peak response, the chromatographic equipment suitability profile, and the chromatograms for VNC, DEC, and TZC. For VNC, DEC, and TZC combinational analysis, the test results were pretty solid.

TABLE 9: ROBUSTNESS DATA AT DIFFERENT FLOW RATES

TABLE 10: ROBUSTNESS DATA AT DIFFERENT PH

TABLE 11: ROBUSTNESS DATA AT DIFFERENT TEMPERATURES

TABLE 12: ROBUSTNESS DATA AT DIFFERENT MOBILE PHASE COMPOSITIONS (ACN %)

TABLE 13: ROBUSTNESS DATA AT DIFFERENT WAVELENGTHS (NM)

CONCLUSION: In this study, a simple, accurate, and precise RP-HPLC method was successfully developed and validated for the simultaneous estimation of VNC, DEC, and TZC in Alyftrek.

The method exhibited excellent linearity over the selected concentration ranges, with correlation coefficients indicating strong proportionality between concentration and response. Accuracy and precision studies confirmed the reliability of the method, while robustness testing demonstrated that small deliberate variations in chromatographic conditions did not significantly affect performance. The method also showed high sensitivity, enabling the detection and quantification of all three components effectively.

Overall, the validated RP- HPLC method is rapid, reproducible, and suitable for routine quality control analysis of Alyftrek, ensuring consistent assessment of VNC, DEC, and TZC in pharmaceutical formulations.

ACKNOWLEDGEMENT: Nil

CONFLICTS OF INTEREST: Nil

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    How to cite this article:

    Noorulla SM and Tabassum SA: Development and validation of a RP-HPLC method for simultaneous estimation of vanzacaftor, tezacaftor, and deutivacaftor in pharmaceutical formulation. Int J Pharm Sci & Res 2026; 17(5): 1534-41. doi: 10.13040/IJPSR.0975-8232.17(5).1534-41.

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