Directly coupled chiral HPLC-NMR and HPLC-CD spectroscopy as complementarymethods for structural and enantiomeric isomer identification: Applicationto atracurium besylate

Directly coupled HPLC-NMR spectroscopy has now become a standard, commercia lly available technique for mixture characterization. Here the extension of the technique to chiral HPLC separation is reported and it is shown that H PLC-NMR together with HPLC-CD provide complementary approaches for the iden tification of structural isomers and enantiomers, The general approach has been exemplified using the neuromuscular blocking agent atracurium besylate , which comprises a mixture of 10 isomers in various proportions as four ra cemic pairs and two meso compounds. Diagnostic reporter resonances in the H -1 NMR spectrum of atracurium besylate were assigned using a combination of one-dimensional and two-dimensional NMR experiments at 750 MHz. Stop-now 7 50-MHz H-1 NMR spectroscopy was used on-line after chiral column HPLC separ ation to identify the enantiomeric pairs, to distinguish the meso compounds , and to identify key configurational features of the isomers, The parallel HPLC-CD experiments served to assign the enantiomers based upon the known CD and absolute stereochemistry of (R)-laudanosine hydrochloride, an analog ue with the same tetrahydroisoquinoline structural unit as atracurium, It i s thereby demonstrated that high-field HPLC-NMR and HPLC-CD is a powerful c ombination of techniques which could be combined on-line for mixture charac terization.