Development and Validation of a Stability‐Indicating RP‐HPLC Method for Simultaneous Determination of Diazoxide and Its Related Impurities in Capsule Dosage Form: Application to Forced Degradation Studies

ABSTRACT

Diazoxide is a benzothiadiazine‐derivative potassium channel opener that carries FDA orphan‐drug designation for the management of persistent hyperinsulinemic hypoglycemia of infancy (PHHI), a life‐threatening neonatal condition with few pharmacological alternatives. Despite renewed clinical interest in this molecule, the analytical literature still lacks a validated, stability‐indicating reversed‐phase HPLC method capable of resolving diazoxide alongside all four of its related impurities, namely deschloro diazoxide, sulfonamide urea, benzene sulfonamide, and urea impurity, in a complex capsule matrix. A gradient RP‐HPLC method was developed on a Fortis Endurus C18‐Tri Alpha column (250 mm × 4.6 mm, 3 µm) using a binary mobile phase system: 0.1% aqueous formic acid as Phase A, and a ternary blend of acetonitrile, methanol, and water (40:40:20, v/v/v) as Phase B, with UV detection at 254 nm. Full validation followed ICH Q2(R2) guidelines, covering specificity, linearity, precision, accuracy, LOD/LOQ, and robustness. Relative response factors (RRF) were calculated individually for each impurity. All five analytes resolved cleanly, with PDA purity angles well below purity thresholds, confirming spectral homogeneity. Linearity was established over 0.050 to 0.786 µg/mL ( r ² ≥ 0.9971). Precision yielded %RSD ≤ 0.50% across six replicate injections. Accuracy ranged from 91.9% to 105.1% recovery at LOQ, 100%, and 150% spike levels. Both LOD and LOQ were uniformly achieved at 0.017 µg/mL and 0.050 to 0.052 µg/mL, respectively. This is the first RP‐HPLC method to report experimentally determined relative response factors for all four related impurities together with a full ICH Q1A(R2) forced‐degradation evaluation, validated specifically for the diazoxide capsule dosage form.