Sensitive enantioseparation and determination of isoprenaline in human plasma and pharmaceutical formulations.

A simple, sensitive and fast RPHPLC method was developed and validated for the enantioselective determination of (RS)-isoprenaline (Ipn) in human plasma. The enantiomers were converted to diastereomeric derivatives using s-triazine (cyanuric chloride) based chiral derivatizing reagents. l-isoleucine and l-methionine were introduced as chiral auxiliary in s-triazine and two new monochloro-s-triazine reagents were synthesized. These reagents were characterized and used for synthesis of diastereomeric derivatives of (RS)-Ipn spiked in human plasma. (RS)-Ipn was isolated (purified and characterized) from a commercial pharmaceutical formulation and was used as the standard racemic sample. Structures of the two diastereomeric derivatives were optimized for lowest energy using the Gaussian 09 Rev A. 02 program and hybrid density functional B3LYP with 6-31G* basis set which showed the spatial orientation of hydrophobic groups on stereogenic centers in the diastereomeric derivatives. The results were correlated with the mechanism of separation and elution order. Limit of detection values were found to be 24.6 and 26.8 ng mL-1 for the first and second eluting diastereomeric derivatives, respectively.