Method Development and Validation for Determination of p-Toluenesulfonoxypropyl-(+)-Dihydrotetrabenazine Enantiomeric Purity by HPLC on a Chiral Stationary Phase

Abstract

A precise and sensitive HPLC method was developed for the determination of p-toluenesulfonoxypropyl-(+)-dihydrotetrabenazine [TsOP-(+)-DTBZ] enantiomeric purity. The effects of mobile phase composition and column temperature on the enantioresolution and analysis time of the enantiomers were investigated. The optimized chiral separation method was performed on a Phenomenex Chirex 3014 column (250 mm × 4.6 mm, particle size 5 μm) using a mobile phase consisting of n-hexane/1,2-dichloroethane/ethanol/trifluoroacetic acid/triethylamine (64/32/4/0.01/0.005 v/v/v/v/v) at a flow rate of 0.8 mL min⁻¹ and a column temperature of 25 °C. The enantiomers were monitored with UV detection at 280 nm, and baseline separation with a resolution higher than 2.0 was achieved within 20 min. The method was validated in terms of linearity, precision, accuracy, robustness, limit of detection (LOD) and limit of quantification (LOQ). The linearity range for the determination of each enantiomer was 0.002–2.0 mg mL⁻¹ (n = 6). Additionally, the LOD and LOQ of TsOP-(+)-DTBZ were 0.15 and 0.50 μg mL⁻¹; those for its enantiomer were 0.18 and 0.60 μg mL⁻¹, respectively. Furthermore, this validated method was successfully applied to analyze three different TsOP-(+)-DTBZ bulk samples. The results demonstrated that the proposed method was simple, rapid, accurate and robust for routine enantiopurity analysis of TsOP-(+)-DTBZ in pharmaceutical research.