HPLC STUDY OF CHEMICAL COMPOSITION OF HONEYBEE (Apis mellifera L.) VENOM

The aim of the study was to the development of the method for the separation, identification and determination quantitatively of the major constituents of honeybee (Apis mellifera L.) venom by high performance liquid chromatography (HLPC) and determination of the chemical composition of the domestic product by running routine tests. In the study to develop an HPLC method to assay the major venom compounds the following elements were tested: chromatographic columns with C18 packing materials of different pore size (100, 180 and 300 ), separation temperature (25, 30 35°C), flow rate (1.0; 1.5; 2.0 ml/min.), conditions of gradient elution (0%B 45%B, 60 min and 0%B 60%B, 60 min, 5%B 80%B, 40 min). Chromatographic separation was performed using the following mobile phases: A 0.1% trifluoroacetic acid (TFA) in water, B 0.1% TFA in acetonitrile : water (80:20). The assays of the separated venom compounds were made using UV detector at 220 nm wavelength. The best separation of the bee venom protein fraction was obtained on 180 and 300 pore size columns at a temperature of 25°C. The routine assays of bee venom chemical composition involved 29 samples collected from the apiary of the Apiculture Division, Institute of Pomology and Floriculture in Pu3awy over three consecutive beekeeping season (2002-2004). Bee venom samples were analyzed for the following major protein fraction compounds: melittin, phospholipase A2 and apamine. Melittin content varied from 61.15 to 70.15 and averaged 64.40%. Phospholipase A2 content came within a range of 11.24 to 15.05, and averaged 13.00%, and apamine content was between 2.09 to 4.18, averaging 3.10%. Statistically significant year-to-year differences were found for melittin.