Development and validation of indirect RP-HPLC method for enantiomeric purity determination of D-cycloserine drug substance.

摘要

A new chiral purity method was developed for D-cycloserine (D-cys) by reverse phase HPLC and validated. Chiral derivatizing reagents, viz., o-phthalaldehyde and N-acetyl-L-cysteine were utilized in this method. The resultant diastereomers were resolved using Zorbax SB Phenyl HPLC column under isocratic elution. A mobile phase of 95:05 (v/v), 20mM Na(2)HPO(4) (pH 7), and acetonitrile, respectively, was used with the flow rate of 1.0 mL/min and UV detection at 335 nm. The method development with different chiral stationary phases and chiral derivatization reagents were also investigated. The stability of diastereomer derivative and influence of organic modifier and pH of the mobile phase were studied and optimized. The stability-indicating capability of the method was established by performing stress studies under acidic, basic, oxidation, light, humidity and thermal conditions. The detection and quantitation limit of L-cycloserine (L-cys) were 0.015 and 0.05% (w/w), respectively. A linear range from 0.05 to 0.30% (w/w) was obtained with the coefficient of determination (r(2)) 0.998. The recovery obtained for L-cys was between 92.9 and 100.2%. This method was applied successfully in pharmaceutical analysis to determine the content of L-cys in D-cys bulk drug.