Optimization of an UHPLC method for flavonoids from Hypericum species
The broad spectrum of pharmacological activities of Herba Hyperici preparations is determined by the potential additive effect, synergism or even possible antagonism of this multicompound system.
The in silico assisted development of an analytical chromatographic method for acquisition of a well separated fingerprint of flavonoids, suitable for bioprocess control of conventionally and biotechnologically derived plant material of Hypericum species is presented here. Drylab® software is based on modelling of physicochemical phenomena in an LC system and supports efficient method development by experimental design models. H. perforatum, as well as species with indigenously high (H. richeri, H. rumeliacum) and lacking hypericin production (H. calycinum) were selected. Separation was performed on ACQUITY UPLC (Waters) system with PDA Detector. DryLab® software (Molnár Institute) was used to model and predict experiments for the optimization of UHPLC conditions establishing an appropriate method for separation of flavonoid compounds. The fingerprint and the identification of the well described lead flavonoids rutin and hyperoside for the quantification of flavonoids as a sum parameter are suitable for comparison of Hypericum samples from different accessions or bioprocess conditions. It was confirmed that the predicted chromatogram matched the peak order in the fingerprint analysis of a real sample from H. calycinum extract. The analysis of the samples with the in silico optimized method revealed that ex situ sample of H. perforatum has content of around 9 – 11 ug/100ug of total flavonoids calculated as hyperoside of all species tested. Among the different in vitro samples, the flavonoid content varied in the order of magnitude of 2 – 7 ug/100ug. It was demonstrated that the in silico assisted optimized UHPLC method is suitable for bioprocess control of flavonoids in in vitro and ex situ biomass.
Acknowledgements: We acknowledge BSRP, grant No. IZEBZ0, 142989; DO2 – 1153.