Stereo‐Selective Separation and Determination of Larotrectinib and Its Isomeric Impurities on a Novel Immobilized Cellulose Tris‐(3‐Chloro‐5‐Methyl Phenyl Carbamate) Chiral Stationary Phase by Using Normal Phase Liquid Chromatography

ABSTRACT A specific, sensitive, and accurate chiral analytical method was developed to separate enantiomers and isomers of Larotrectinib using chiral high‐performance liquid chromatography (HPLC). The enantiomers and diastereomers of Larotrectinib were well separated using an immobilized chiral stationary phase in normal‐phase mode operation. Larotrectinib is an orally administered medication that inhibits the activity of tropomyosin receptor kinase (TRK), a specific protein. It helps to suppress tumor cell growth and supports patient survival. Chiral separation of Larotrectinib was achieved using a novel immobilized chiral selector: cellulose‐tris‐(3‐chloro‐5‐methylphenylcarbamate) (CHIRALPAK IK‐3, 250 mm × 4.6 mm, 3 µm) with a mobile phase consisting of n‐hexane, ethanol, methanol, and triethylamine in the ratio of 50:25:25:0.1 (v/v/v/v). The column oven temperature, detection wavelength, and flow rate were maintained at 40°C, 265 nm, and 1.0 mL/min, respectively. The LOD/LOQ values for the Larotrectinib API, S, S‐isomer, R, S‐enantiomer, and R, R‐isomers were 0.012/0.038, 0.01/0.03, 0.011/0.033, and 0.014/0.044 µg/mL, respectively. The relative standard deviation (RSD) values for method and system precision were < 5% and < 2%, respectively. Mean recovery values ranged from 96.8% to 114.9%. The developed method was validated and successfully applied for the determination and identification of Larotrectinib and its enantiomers and diastereomers in the active pharmaceutical ingredient (API) using the CHIRALPAK IK‐3 chiral stationary phase.