Nonequivalence behavior studies for the direct determination of enantiomeric purity and absolute configuration of timolol by NMR.

Direct determination of both the enantiomeric purity and absolute configuration of timolol was accomplished utilizing 1H NMR (400 MHz) spectroscopy with fast diamagnetic chiral solvating agent to dissimilarly perturb the spectra of enantiomeric solutes. Nonequivalence behavior was studied for all variables that affect populations and intrinsic spectra of the diastereomeric solvates. Optimization of the experimental conditions in terms of probe temperature, substrate concentration and solvating agent to substrate molar equivalents provided resolved enantiomeric signals suitable not only for chiral recognition but also for quantification. Enantiomeric impurity was determined on the basis of relative intensities of the tert-butyl methyl protons resonances; the assignment of enantiomeric configuration was based on the relative field positions of these resonances. The analysis of synthetic mixtures of the enantiomers by the proposed NMR method resulted in assay values which agreed closely with the known quantities of each enantiomer in mixtures tested. The mean +/-SD recovery values for the (R)-(+)-enantiomer was 100.0+/-1.6% of added antipode (n = 8). The optically pure enantiomers were used to establish the minimum detection limits of0.1%. The developed methodology represents a rapid and powerful tool for regulatory analysis.