Enantioselective Green HPLC Method for Simultaneous Determination of Enantiomer, and Potential Impurities in Apremilast Drug Substance

Abstract

Abstract A single reversed-phase HPLC method was developed for the quantification of seven impurities of Apremilast and its enantiomer in the drug substance. An immobilized chiral stationary phase with a chiral selector “tris (3,5-dimethyl phenyl carbamate) derivative of amylose-Chiralpak IA-3 (250 mm × 4.6 mm, 3 μm) was employed to achieve the desired separation. A mobile phase consists of buffer (0.01M NH4HCO3, PH 8.0) and acetonitrile in the ratio 50:50 (v/v) and is pumped at a flow rate of 0.4 mL/min with an isocratic elution mode. The column oven temperature is set at 25°C, and the chromatographic output is monitored at 225 nm with a total run time of 45 min. A test concentration of 500 µg/mL of Apremilast in the mobile phase is used with an injection volume of 10 µL. Induced degradation studies were carried out to study the intrinsic chemical behavior of the drug. The degradation sample solutions were utilized to demonstrate the stability-indicating nature of the developed analytical method. The obtained mass number [M+H]+ for the primary degradation product formed in acid hydrolysis was identified as 418.36 and in base hydrolysis 478.12. The two impurities were identified as impurity-5(Deacetylated impurity), and impurity-2(open ring acid impurity), respectively, and the degradation pathways were established. Following ICH Q2 and USP<1225>guidelines, complete method validation was carried out. The RSD for the drug and impurities in interday and intraday studies are less than 4.0%, and the recoveries for the impurities are between 96.1-102.1% and linearity r ≥ 0.9997. LOQ results for the drug and impurities are between 0.052 µg/mL and 0.107 µg/mL, and LOD results are between 0.016 µg/mL and 0.032 µg/mL. The greenness of the method was evaluated by using an analytical eco scale, GAPI, and AGREE, and it was found that the method is green. GRAPHICAL ABSTRACT