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.