2.2. IRMS

Honey proteins were prepared according to the method described by Bilikova [19] with slight readjustments. A 15 g sample of honey was weighed, and 15 mL of deionized water was added. Semi-permeable SnakeSkin (10K MWCO) dialysis membrane was filled with a homogeneous clear honey solution. After dialysis, the purified protein solution was quantitatively transferred into a beaker and placed in the drying oven at 40 °C for about 48 h until the proteins were dried. Then, proteins were weighed and stored at 4 °C until IRMS analysis.

Continuous flow IRMS (Nu Horizon) coupled with an elemental analyzer (EuroEA3024) was used for the analysis. The complete combustion of the samples and the operation of the element analyzer were verified by performing stability tests on the equipment. Certified reference materials USGS-40 and USGS-41 were used as reference materials. The device conditions were prepared as described in previously published method [20].