Method Development, Optimization, and Validation of the Separation of Ketamine Enantiomers by Capillary Electrophoresis Using Design of Experiments

Abstract

AbstractCapillary electrophoresis was chosen as cost-effective and robust method to separate ketamine enantiomers. For the method development, first different native and modified cyclodextrins were tested. The most promising chiral selector was α-cyclodextrin. A design of experiments (DoE) was carried out, which started with the screening of relevant factors. Based on these results, the method was optimized according to the significant factors (buffer, cyclodextrin concentration, pH value, voltage, temperature) of the screening based on the response resolution and migration time of the later migrating enantiomer. The optimized conditions consisted of a background electrolyte with 275 mM TRIS, adjusted with 85% phosphoric acid to a pH of 2.50, and 50 mM α-cyclodextrin, at a temperature of 15 °C, an applied voltage of 30 kV and an injection pressure of 1.0 psi for 10 s. A fused-silica capillary with a total length of 70 cm and an effective length to the detector of 60 cm was used. The method was validated according to ICH guideline Q2 R(1). The limit of quantification was 3.51 µg mL−1 for S-ketamine and 3.98 µg mL−1 for R-ketamine. The method showed good linearity for racemic ketamine with R2 of 0.9995 for S-ketamine and 0.9994 for R-ketamine. The lowest quantifiable content of S-ketamine found in R-ketamine was 0.45%.