PhD Scientific Days 2024

Budapest, 9-10 July 2024

Pharmaceutical Sciences and Health Technologies II.

Elucidating the Enantioseparation Mechanism of Laudanosine Derivatives: CE, HPLC and NMR Spectroscopic Study


Erzsébet Várnagy1, Gergő Tóth2, Sándor Hosztafi2, Milo Malanga3, Ida Fejős1, Szabolcs Béni4
1: Department of Pharmacognosy, Semmelweis University, Budapest, Hungary
2: Department of Pharmaceutical Chemistry, Semmelweis University, Budapest, Hungary
3: CarboHyde Ltd, Budapest, Hungary
4: Department of Analytical Chemistry, Institute of Chemistry, Eötvös Loránd University, Budapest, Hungary

Text of the abstract

Introduction: Chiral separations pose a significant challenge in contemporary drug analytics, given the distinct biological effects of the enantiomers. Among the applied analytical methods capillary electrophoresis (CE) stands out as particularly efficient, while HPLC remains robust. Essential for CE enantioseparations are the chiral selectors, predominantly cyclodextrins (CDs), which distinguish enantiomers via diastereomeric complexation. The diverse sized apolar cavities of CDs and the ad-hoc selection of functional groups enable customized separations, thereby enhancing resolution effectively.
Aims: Our aim was to achieve the enantiomeric separation of tetrahydrobenzylisoquinoline alkaloid laudanosine (LAU) and its four synthetic derivatives (6'-bromo-laudanosine, norlaudanosine, N-formyl-norlaudanosine, N-propyl-norlaudanosine) and a tetrahydroprotoberberine alkaloid, xylopinine. Additionally, we sought to identify the structures of the analyte-CD complexes to gain insight into the mechanism of complex formation.
Methods: CE screening involved over 20 CDs at various concentrations. In chiral HPLC, we utilized 3 CD-based and 5 polysaccharide-based columns using MeOH:DEA 100:0.1 eluent. For structural studies, we conducted 1H NMR and conventional 2D NMR techniques.
Results: Subetadex (SBX) and sugammadex (SGX) resulted in baseline separations for LAU derivatives (Rs 2.2-8.7). Xylopinine enantiomers could be separated with outstanding resolution (Rs 9.3) applying SBX. In chiral HPLC the Chiralpak AD and Chiracel OD columns displayed chiral recognition capabilities towards all racemates. In NMR studies, enantiorecognition was observed for the LAU-SGX complex, the resonances of LAU enantiomers were resolved due to the formation of diastereomeric complexes. In the 2D ROESY NMR spectrum, the aromatic protons of LAU exhibited cross-peaks with SGX inner cavity protons of H3 and H5.
Conclusion: Our findings showed that γ-CD derivatives are optimal for enantioseparation of LAU derivatives, while β-CD derivatives are suitable hosts for xylopinine in CE. The chiral HPLC separations resulted in semi-preparative scale isolation of individual enantiomers, thereby enabling the determination of enantiomer migration order in CE and HPLC elution. The NMR results confirm the chiral recognition and the formation of inclusion complexes of LAU-SGX.