Chromatographic techniques for metabolite fingerprinting of propolis polyphenols and volatile compounds

This study was aimed at developing advanced chromatographic methods for a complete phytochemical characterization of raw propolis polyphenols and volatile compounds, which are responsible for the biological activity of the extracts widely used in phytotherapy. The study of polyphenols was based on RP-HPLC coupled with DAD and MS detection, using the new fused-core column technology, which allowed a sensitive reduction in the total analysis time in comparison with conventional particulate stationary phases. The HPLC analyses were carried out on an Ascentis Express C18 column (150 mm × 3.0 mm I.D., 2.7 µm), with a mobile phase composed by 0.1% aqueous formic acid and ACN, under gradient elution. Different extraction methods were compared, including sonication and microwave extraction, in order to obtain a high recovery of polyphenols from raw propolis. The study of volatile compounds was based on GC coupled with MS detection. A suitable HS-SPME extraction procedure, whose experimental parameters were properly optimized, was developed using a 1 cm PDMS fiber. The GC analyses were carried out on an HP-5 MS cross-linked 5% diphenyl-95% dimethyl polysiloxane capillary column (30 m × 0.25 mm I.D., 1.00 µm film thickness), under programmed-temperature elution. The volatile compounds extracted from raw propolis were identified by comparing the MS fragmentation patterns with those of pure compounds and by mass spectrum database search using NIST. The chromatographic methods developed in this study were applied to samples of raw propolis collected in different Italian regions and allowed us to determine their metabolite fingerprinting, thus providing new and reliable tools for the complete phytochemical characterization of this material.