Validation of a Separation of Diastereomers in the Pharmaceutical Industry

Abstract

Method validation is an important step in any method development and has important implications in the pharmaceutical industry. Particular efforts should be directed towards the reproducibility, sensitivity and ruggedness of each method developed. In this paper, we report the validation of an HPLC method for the separation of the L-699, 392 and its (S, R) diastereomer. This compound contains two chiral centers and a carboxyl functionality able to participate in a hydrogen bonding process. In order to obtain maximum sensitivity, the elution order of the two diastereomers was adjusted such that the minor diastereomer eluted before the major one. To achieve this elution order a nonpolar mobile phase consisting of methylene chloride and n-propanol containing quinine as a hydrogen bond acceptor was used. In order to optimize the separation, the influence of quinine concentration on the capacity and separation factor of the two diastereomers, influence of the polar modifier in the mobile phase and influence of the flow rate on the separation factor and system efficiency, were studied.

The method has been shown to be rugged giving base line separation of the two diastereomers with a limit of detection of 0.06% by weight for the minor diastereomer (S, R). The detector response of the (S, S) diastereomer was linear in the range 0.0005 to 1.1 mg/ml with an r² of 0.9996. The injection precision, linearity of the detector response and solution stability were also evaluated.