The aim of this study was to develop and validate a method for reliable discrimination of bee pollen from
different floral origins via simultaneous analysis of three flavonoid glycosides. A simple sample preparation
procedure consisted of an ultrasonic-assisted extraction followed by high performance liquid
chromatographyeelectrospray ionizationetandem mass spectrometry (HPLCeESIeMS/MS) technique to
effectively identify and differentiate ten kinds of bee pollens has been developed and optimized in this
study. For sample extraction, several parameters were optimized including extraction solvents, extraction
patterns, solvent volume, extraction duration and temperature. The analytical method was validated
in terms of linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, matrix effect and
carryover test using quality control sample. This method can consistently distinguish bee pollen by
analyzing three flavonoid glycosides (quercetin-3-O-b-D-glucosyl-(2/l)-b-glucoside, kaempferol-3, 40
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di-O-b-D-glucoside and kaempferol-3-O-b-D-glucosyl-(2/l)-b-D-glucoside). Therefore, this technique
will be a suitable to be used as a discrimination method to identify the floral origin of bee pollen via the
proposed marker components.