Evaluation of allergens in propolis by ultra-performance liquid chromatography/tandem mass spectrometry

The purified extract of propolis is used as a traditional remedy for the treatment of several diseases. Its beneficial activities are mainly attributed to the polyphenolic fraction. Nevertheless, propolis can cause allergic dermatitis and the sensitization rate in humans is increasing significantly mainly in younger subjects. The aim of this study was to develop and validate a selective and sensitive ultra-performance liquid chromatography tandem mass spectrometry analysis (UPLC/MS/MS) for the evaluation of the amount of caffeic acid and its esters with allergenic action in raw propolis samples and commercial formulations. The separation was carried out on a 1.7 μm C(18) BEH Shield column and the detection performed by means of electrospray ionization in negative ion mode with multiple reaction monitoring. The confirmation of formulae of the precursor and product ions was accomplished by injection into a high-resolution system (FTICR-MS) using accurate mass measurements. The error was below 1.4 ppm.The range of the standard curves was 0.5-10 μg/mL and dihydrocaffeic acid was used as internal standard (IS). The lower limit of detection (LLOD) for 3-methyl-2-butenyl-(3M2B), 3-methyl-3-butenyl-(3M3B), 2-methyl-2-butenyl-(2M2B), benzyl-(CABE), phenylethylcaffeic acid (CAPE) and for caffeic acid (CA) and the IS was 0.1 and 0.3 μg/mL, respectively. The recoveries were in the range 96-104% and the intra- and inter-day precisions were within 6.2%. In the European (n=8) and Asiatic (n=3) propolis the most abundant allergens were CABE>3M2B>CAPE>3M3B>CA>2M2B. These compounds were not found in the red (n=1) and green (n=1) Brazilian propolis. Hydroalcoholic extracts (n=6) and tablets (n=6) were analyzed by the proposed UPLC/MS/MS method. The results showed that in the commercial products CABE, 3M2B, CAPE and 3M3B were also the most abundant.