Simultaneous Determination of Major Constituents of Honeybee Venom by LC-DAD |
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Authors: | Zenon J Kokot and Jan Matysiak |
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Institution: | (1) Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6 Street, 60-780 Poznan, Poland |
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Abstract: | The aim of the study was to develop an LC method for honeybee venom analysis, using cytochrome c as an internal standard. The SynChropack C8 6.5 μm, 4.6 × 100 mm column was applied. The bee venom was separated by linear
gradient 5–80% B at 30 min (eluent A—0.1% TFA in water, eluent B—0.1% TFA in acetonitrile:water (80:20)). The flow rate of
mobile phase was maintained at 1 mL min−1, injection volume: 40 μL, separation temperature: 25 °C. The analysis was monitored at 220 nm. Several honeybee venom constituents
were separated and the content of four of them (apamine, mast cell degranulating peptide, phospholipase A2 and melittin) were determined. By applying this methodology differences in chemical composition of honeybee venom were evaluated.
In order to confirm the data obtained, the following steps and parameters were taken into account for the validation of the
method: selectivity, precision (injection repeatability, analysis repeatability), accuracy (recovery), linearity and operating
range, limit of detection and limit of quantitation. All steps of validation proved that the developed analytical procedure
was suitable for its intended purpose (standardization). Due to its simplicity, the developed method can be easily automated
and incorporated into routine operations both in the bee venom identification, quality control and assay tests. |
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