Development of an LC-MS/MS Method for Quantitation of Western Honeybee (<i>Apis mellifera</i>) α-Glucosidase III as a Potential Honey Authenticity Marker

Western honeybee (<i>Apis mellifera</i>) α-glucosidase III (HBG-III), which is secreted from the hypopharyngeal glands of honeybees, plays a role in converting nectar into honey. Consequently, hypothesizing that HBG-III is a suitable marker of honey authenticity, we developed an analytical method to determine the HBG-III content and investigated its applicability to various commercial products. Following extraction from honey using phosphate-buffered saline, HBG-III was concentrated using an ultrafiltration membrane and subsequently fragmented with trypsin and lysyl endopeptidase mixture. The specific peptide fragments were used for quantitation by liquid chromatography-tandem mass spectrometry. The established method was validated for linearity, accuracy, precision, and the limit of quantitation (LOQ). As a result, the calibration curve was linear in the range of 0.01-0.3 μM, the mean recovery ranged from 73.8 to 89.2 %, the within-laboratory reproducibility (RSD_wr) ranged from 3.9 to 6.5 %, and the LOQ was 1.9 mg/kg. An investigation of HBG-III concentrations in 65 honey products available on the Japanese market revealed that the HBG-III content of 15 low-priced honey products was below the LOQ. This suggested that these products may be adulterated with non-honey syrups. Therefore, this method can serve as an effective tool to verify the authenticity of honey products.