Reversed-phase high-performance liquid chromatographic enantioresolution of six beta-blockers using dinitrophenyl-L-Pro-N-hydroxysuccinimide ester, N-succinimidyl-(S)-2-(6-methoxynaphth-2-yl) propionate and twelve variants of Sanger's reagent as chiral derivatizing reagents.

Twelve chiral derivatizing reagents (CDRs) were synthesized by substituting one of the fluorine atoms in 1,5-difluoro-2,4-dinitrobenzene (DFDNB) with three optically pure amines [(R)-(-)-1-cyclohexylethylamine, (+)-dehydroabietylamine and (S)-(-)-alpha,4-dimethylbenzylamine], six amino acid amides [L-Ala-NH(2), L-Phe-NH(2), L-Val-NH(2), L-Leu-NH(2), L-Met-NH(2) and D-Phg-NH(2)] and three amino acids [L-Ala, L-Val and L-Leu]. In addition, dinitrophenyl-L-Pro-N-hydroxysuccinimide ester and N-succinimidyl-(S)-2-(6-methoxynaphth-2-yl) propionate were also synthesized and used as CDR. Keeping in view the presence of an amino group, diastereomers of six beta-blockers (atenolol, propranolol, bisoprolol, metoprolol, salbutamol, and carvedilol) were synthesized by reaction with these 14 CDRs. The diastereomers were separated by RP-HPLC. The method was validated for linearity, accuracy, limit of detection and limit of quantification.