Development and Validation of an HPLC Method for the Analysis of Flowers of Wild-Growing Primula veris from Epirus, Greece.

Abstract

An HPLC-PDA method was developed for the determination of the flavonoids in the flowers of Primula veris from Epirus, Greece. The aim was to investigate the chemical content of the over-harvested P. veris populations of Epirus and to develop and optimize an extraction protocol to allow fast, exhaustive, and repeatable extraction. Qualitative analysis revealed that the P. veris flowers from Epirus were particularly rich in flavonoids, especially flavonol triglycosides including derivatives of quercetin, isorhamnetin, and kaempferol. A phytochemical investigation of a 70% hydromethanolic extract from the flowers afforded a new flavonoid, namely, isorhamnetin-3-Ο-β-glucopyranosyl-(1 → 2)-β-glucopyranosyl-(1 → 6)-β-glucopyranoside, which is also the main constituent of the flower extracts. Its structure elucidation was carried out by means of 1D and 2D NMR and mass spectrometry analyses. The HPLC-PDA method was developed and validated according to the International Council for Harmonisation guidelines. Since the main flavonol glycoside of the plant is not commercially available, rutin was used as a secondary standard and the response correction factor was determined. Finally, the overall method was validated for precision (% relative standard deviation ranging between 1.58 and 4.85) and accuracy at three concentration levels. The recovery ranged between 93.5 and 102.1% with relative standard deviation values < 5%, within the acceptable limits. The developed assay is fast and simple and will allow for the quality control of the herbal drug.