DUAL ANALYTICAL EVALUATION OF EVEROLIMUS 2% BHT: HPLC METHOD VALIDATION AND LC-MS IDENTIFICATION OF UNCHARACTERIZED IMPURITIES

Abstract

Creating a robust and precise reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the quantitative analysis of Everolimus 2% BHT API. The chromatographic separation was performed using a C18 column which is 250mm length × 3.0 mm internal diameter size and 5 µm of particle size, with an inflow rate of 1.1 mL/min. The discovery was carried out at 210 nm, using a 10 µL injection volume, and the total runtime was 55 minutes.

The mobile phase consisted of two components: Mobile Phase A, a 60:40 (v/v) mixture of 0.27 g/L potassium dihydrogen phosphate buffer and acetonitrile, and Mobile Phase B, which was 100% acetonitrile and also used as the diluent. Both standard and sample concentrations were   10 mg/mL. The retention time (Rt) for Everolimus and BHT were found to be 15.52 minutes and 18.51 minutes, respectively.

System suitability parameters such as %RSD, resolution, theoretical plates, and tailing factor complied with ICH Q2(R1) guidelines. During accelerated stability testing, an unknown impurity was detected at Rt 8.01 minutes (RRT 0.53), which was identified via LC-MS as Seco-Everolimus acid, a product of acid degradation. All validation parameters, including specificity, linearity, precision, accuracy, sensitivity, robustness, and forced degradation studies, were within acceptable limits, confirming the method's reliability for routine analysis.