Development and Validation of a Stability-Indicating RP-HPLC Method with Dual-Detection for Simultaneous Estimation of Sodium Alendronate and Cholecalciferol in Fixed-Dose Combination Tablets Using Quality by Design Principles
Year: 2026
Type: article
Abstract: Background: The fixed-dose combination (FDC) of Sodium Alendronate (SAL) and Cholecalciferol (CHO) is widely employed in the pharmacological management of osteoporosis. The analytical quantification of these two drugs in a single dosage form presents significant challenges due to their extreme physicochemical dissimilarity — SAL lacks a chromophore and exhibits end-absorption below 220 nm, while CHO possesses a strong characteristic absorption at 265 nm and is prone to photolytic and oxidative degradation. No regulatory-compliant, stabilityindicating RP-HPLC method incorporating Analytical Quality by Design (AQbD) principles has been reported for this binary system. Methods: An AQbD-driven method development strategy was adopted. Critical Method Parameters (CMPs) — mobile phase pH, buffer concentration, and gradient slope — were systematically optimized using a Box-Behnken Design (BBD). The final HPLC method employed an Agilent Zorbax Eclipse Plus C18 column (100 mm × 4.6 mm, 3.5 μm) with a mobile phase of 10 mM phosphate buffer (pH 3.0) containing 10 mM sodium hexane sulfonate (ionpairing agent) and acetonitrile under gradient conditions at 1.0 mL/min. A novel dual-detection system was utilized: SAL was detected via fluorescence after pre-column derivatization with FMOC-Cl (λex = 330 nm, λem = 445 nm), while CHO was simultaneously monitored via Diode Array Detection (DAD) at 265 nm. Method validation was performed as per ICH Q2(R1) guidelines. Results: The optimized method achieved excellent chromatographic resolution between SAL (Rt ≈ 4.2 min) and CHO (Rt ≈ 10.5 min) with resolution >12.8. Linearity was demonstrated over 20–120 μg/mL for SAL and 2–12 μg/mL for CHO, with correlation coefficients ≥0.9998. Precision studies showed %RSD <1.05% for all parameters. Accuracy (recovery) ranged from 99.1–100.9% for SAL and 98.9–101.1% for CHO. Forced degradation studies under five stress conditions (acid, alkali, oxidative, thermal, and photolytic) conclusively demonstrated the stabilityindicating nature of the method. CHO was found to be labile under acidic, oxidative, and photolytic conditions, while SAL exhibited relative stability to most stress conditions. Conclusion: A robust, selective, and sensitive RP-HPLC method with dual-detection has been successfully developed and validated for the simultaneous determination of SAL and CHO. The AQbD framework established a well-defined Method Operable Design Region (MODR) ensuring consistent method performance. The method is suitable for routine quality control analysis and stability testing of SAL+CHO FDC tablets.
Institution: Oriental Universiteti
Language: English
Subfield: Analytical Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: bronze
OpenAlex ID: W7164035751