Development of liquid chromatographic methods for enantioseparation and sensitive detection of β-adrenolytics/β2-agonists in human plasma using a single enantiomer reagent

•TLC and RPHPLC enantioseparation of four β-adrenolytics as diastereoisomers after derivatization.•(S)-Naproxen based new chiral reagent was Synthesized and characterized.•Fast synthesis of diastereomeric derivatives under microwave irradiation.•Enantiomers isolated from plasma and derivatized with single enantiomer reagent.•Established configuration and elution order of diastereomers using 1HNMR.

Enantioseparation of four commonly used β-adrenolytics (bisoprolol, salbutamol, and carvedilol, marketed as racemic mixtures) has been achieved by both TLC and RPHPLC via an indirect approach. A new chiral reagent, (S)-naproxen benzotriazole ester, was synthesized and it was characterized by UV, IR, 1HNMR, elemental analysis and polarimetry. It was used to synthesize diastereomeric derivatives of the three β-adrenolytics under microwave irradiation. TLC separation of diastereomeric derivatives was achieved which were then isolated by preparative approach; these were characterized and were used as standard reference for determining absolute configuration of diastereomeric derivatives and for establishing validated HPLC method for enantioseparation and sensitive detection of the three β-adrenolytics in human plasma. Mobile phase in gradient mode containing methanol and aqueous triethylaminephosphate (TEAP) was successful for HPLC separation; conditions with respect to pH, flow rate, and buffer concentration were optimized. The method is capable to accurately quantitate β-adrenolytics in human plasma with minimal sample clean-up and rapid separation by TLC and RPHPLC. The limit of detection values were 0.97 and 0.87 ng/mL for diastereomeric derivatives of (S)- and (R)-bisoprolol, respectively, which are very low in comparison to literature reports.