D. Gopal et al., VALIDATION OF A SEPARATION OF DIASTEREOMERS IN THE PHARMACEUTICAL-INDUSTRY, Journal of liquid chromatography, 16(8), 1993, pp. 1749-1768
Method validation is an important step in any method development and h
as important implications in the pharmaceutical industry. Particular e
fforts should be directed towards the reproducibility, sensitivity and
ruggedness of each method developed. In this paper, we report the val
idation 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 ca
rboxyl 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 be
fore the major one. To achieve this elution order a nonpolar mobile ph
ase consisting of methylene chloride and n-propanol containing quinine
as a hydrogen bond acceptor was used. In order to optimize the separa
tion, the influence of quinine concentration on the capacity and separ
ation 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 precisi
on, linearity of the detector response and solution stability were als
o evaluated.