High-performance liquid chromatoghraphy/tandem mass spectrometry methods in the phytochemical analysis of hydro-alcoholic propolis extracts.

Polyphenolic compounds are the major active constituents of propolis extracts. Due to its phytochemical composition, propolis extracts are widely used in phytotherapy, particularly as anti-bacterial, anti-inflammatory and antioxidant.The interest in propolis compounds and their biological activity is still increasing. In this ambit, the development and validation of suitable analytical methods aimed at the phytochemical analysis and quality control of propolis is very important.In this study, a detailed characterization of propolis hydro-alcoholic extracts was carried out by developing advanced chromatographic techniques, based on RP-HPLC coupled with diode array and mass spectrometry detection and analyzing commercially available hydro-alcoholic propolis extracts. In the context of mass spectrometry detection, the performance of two mass analyzers, such as an ion trap and a triple quadrupole, were compared. The HPLC analyses were carried out on an Ascentis C18 column (250 mm × 4.6 mm I.D., 5 um), with a mobile phase composed by 0.1% aqueous formic acid and ACN, under gradient elution. The flow rate was 1.2 mL/min and the column temperature was set at 30°C. In view of UV spectra, MS and MS/MS data, forty polyphenolic compounds, including phenolic acids and flavonoids, were identified as typical components of the analyzed samples. The comparison of the performance of the selected mass analyzers indicated that the triple quadrupole system provided more structural information on target analytes than the ion trap.The HPLC method was then fully validated in agreement with the guidelines of the International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use and then applied to real samples.Although the chromatographic profile of the analyzed samples was similar, the quantitative analysis indicated that there is a great variability in the amount of the active compounds: the content of total phenolic acids ranged from 0.17 to 16.72 mg/mL and the level of total flavonoids from 2.51 to 42.72 mg/mL. In the analyzed samples, the most abundant flavonoids were found to be chrysin, galangin, pinocembrin and pinobanksin (and its esters). Regarding phenolic acids, the most representative compounds were found to be caffeic acid derivatives.The chemical standardization of propolis extracts is necessary to guarantee its quality, efficacy and safety. In this context the proposed method, allowing the determination of the complete phenolic profile characterized by both phenolic acids and flavonoids, can be considered as a useful tool for metabolite profiling and quality control of propolis natural products.