The samples of the worker bees and the brood were lyophilized using a Labconco Freezone 2.5 freeze dryer (Labconco, USA) (pressure: 0.024 mbar; temperature: 50 °C, time: 168 h).
Analytical portions of 5 g of the lyophilized animals or honey were shaken with 10 mL of acetonitrile (Chempur, Poland). Then, a mixture of salts containing 4 g of anhydrous magnesium sulfate (VI) (Chempur, Poland), 1 g of sodium chloride (Chempur, Poland), 1 g of trisodium citrate (Chempur, Poland), and 0.5 g of sesquihydrate disodium hydrogen citrate (Chempur, Poland) was added. The contents were shaken for 2 min and centrifuged for 5 min at 4500 rpm at 21 °C. Six millilitres of the acetonitrile phase was transferred to a polypropylene test tube that contained 150 mg of PSA (primary secondary amine) (Agilent, USA) and 900 mg of anhydrous sodium sulfate(VI) (Chempur, Poland). The extract was vigorously shaken for 2 min and centrifuged for 5 min as described above. Four millilitres of the obtained extract was taken and transferred to a glass tube, evaporated to dryness on a Heidolph Efficient Labware 4000 rotary evaporator (Heidolph, Germany), and then dissolved in 4 mL of petroleum ether (Chempur, Poland).
Analytical portions of 5 g of the lyophilized animals or honey were shaken with 10 mL of acetonitrile (Chempur, Poland). Then, a mixture of salts containing 4 g of anhydrous magnesium sulfate (VI) (Chempur, Poland), 1 g of sodium chloride (Chempur, Poland), 1 g of trisodium citrate (Chempur, Poland), and 0.5 g of sesquihydrate disodium hydrogen citrate (Chempur, Poland) was added. The contents were shaken for 2 min and centrifuged for 5 min at 4500 rpm at 21 °C. Six millilitres of the acetonitrile phase was transferred to a polypropylene test tube that contained 150 mg of PSA (primary secondary amine) (Agilent, USA) and 900 mg of anhydrous sodium sulfate(VI) (Chempur, Poland). The extract was vigorously shaken for 2 min and centrifuged for 5 min as described above. Four millilitres of the obtained extract was taken and transferred to a glass tube, evaporated to dryness on a Heidolph Efficient Labware 4000 rotary evaporator (Heidolph, Germany), and then dissolved in 4 mL of petroleum ether (Chempur, Poland).