High-performance liquid chromatographic determination of erdosteine and its optical active metabolite utilizing a fluorescent chiral tagging reagent, R-(-)-4-(N,N-dimethylamiosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2 ,1,3-benzoxadiazole.

Chiral separation of racemic M1 metabolized from erdosteine was investigated by reversed-phase chromatography. The sensitive determination of M1 and erdosteine with UV detection was difficult because of their low absorptivity in the effective wavelength region. To improve the sensitivity and separatability, one thiol and two carboxyl groups in the M1 structure were labelled with DBD-F and R-(-)-DBD-APy, respectively. Non-fluorescent DBD-F quantitatively reacted with thiol in M1 at room temperature for 30 min in borate buffer (pH 9.3) to produce the fluorescent derivative. On the other hand, the labelling of two carboxyls was carried out with a chiral fluorescent reagent, R-(-)-DBD-APy, in acetonitrile containing DPPA. The derivatives corresponding to a pair of the enantiomers were completely separated with water-acetonitrile containing 0.1% TFA as the mobile phase by an ODS column. Erdosteine with a carboxyl group was also labelled with R-(-)-DBD-APy and separated together with M1 derivatives. The detection limits (S/N=3) of erdosteine and M1 were 0.37 and 0.22 pmol, respectively. The proposed derivatization and separation methods were applied to simultaneous determination of racemic M1 and erdosteine in rat plasma after administration of erdosteine. The amounts of both enantiomers of M1 were essentially the same in oral and intravenous administrations. In contrast, total amounts (reduced-form and oxidized-form) of S-(-)-M1 in rat plasma were higher than those of R(+)-M1 in both administrations.