Nce procedures displaying acceptable recovery percentages and repeatability have been established, the bees in the experimental groups were analyzed (N = 12) and also the imply concentrations inside the bees have been calculated. Then, the analytical protocols had been fully validated employing eight replicates (person lyophilized and pulverized bees) by spiking an amount corresponding towards the imply concentration measured in the person bees (Table two). Recovery percentages had been evaluated in accordance with the EURACHEM recommendations.29 The limits of detection (LOD) and quantification (LOQ) had been established by determining the standard deviation (SD) from the person compounds within the spiked bees of the replicated recovery experiment. Based on the EURACHEM recommendations, the LOD was determined as three SD, although the LOQ was set to ten SD. The quantification precision was assessed as the relative SD (RSD ) of your eight spiked replicates. Optimized Approaches for Sample Preparation and Extraction of Entire Honey Bees. Twelve individual bees from every single on the experimental groups had been rinsed with water and placed individually in 1.five mL Eppendorf tubes. The bees have been then lyophilized and transferred individually to Falcon tubes for extraction. 3 metal beads had been added, and also the bees were pulverized by vibration for 30 s at 1500 rpm employing a Geno/Grinder (SPEX Sample Prep 2010, Metuchen NJ 08840). The extraction solvents have been added, and also the bees had been extracted by shaking using an Intelli-Mixer for 1 h (ULK2 review Journal of Agricultural and Food ChemistryTable S2. For each compound, two MRM transitions were monitored (Table two); a single transition was employed for quantification, whereas the other was employed as a qualifier MRM to make sure appropriate identification. The identity of the compounds was also confirmed by recording complete MS/MS spectra in chosen bee extracts and comparing these with spectra recorded of authentic analytical standards. Atropine, gelsemine, senkirkine, senecionine, and methyllycaconitine were analyzed collectively in good mode. The eluents had been A: 7 acetonitrile in Milli-Q water with 0.5 formic acid and B: 95 acetonitrile and 5 Milli-Q water with 0.5 formic acid. The compounds had been separated on a Synergy Fusion column (150 mm 2 mm, four m; Phenomenex, V l e, Denmark) with a flow price of 0.four mL/min, and also the gradient was as follows: 0-2 min: 100 A; 2-18 min: 100-30 A; 18-19 min: 30-0 A; 19-22 min: 0 A; 22-23 min: 0-100 A and 23-30 min: one PDE11 Purity & Documentation hundred A. Supply parameters have been as follows: curtain gas (CUR), 45 psi; collision gas (CAD), medium; temperature (TEM), 400 ; ion supply gas 1 (GS 1), 90 psi; ion supply gas two (GS two), 30 psi; and ionspray voltage (IS), 4200 V. Amygdalin was analyzed making use of the identical source parameters and chromatographic approach as described above, but in unfavorable mode. Aucubin was also analyzed in damaging mode working with the exact same solvent program and column as described above, but the gradient was as follows: 0-3 min: 100 A; 3-13 min: 100-75 A; 13-14 min: 75- 0 A; 14-17 min: 0 A; 17-18 min: 0-100 A; and 18-28 min: 100 A. From 1 to 14 min, the flow price was 0.two mL/min, whereas from 14 to 28 min, it was improved to 0.3 mL/min. Supply parameters have been as follows: CUR, 50 psi; CAD, medium; TEM, one hundred , GS 1, 50 psi; GS 2, 50 psi; and IS, -4500 V. Triptolide was analyzed in optimistic mode as its ammonium adduct, as previously reported by Zhuang et al.32 The column was a Hypersil BDS C18 (250 mm two.1 mm, five m; Thermo Fisher, Hvidovre, Denmark), and also the solvent technique consist.