Synthese (Genay, France). Triptolide was purchased from BioNordika (Herlev, Denmark), whereas methyllycaconitine was supplied by Sigma-Aldrich. Sucrose (99 ) for the feeding options was bought from Sigma-Aldrich.https://dx.doi.org/10.1021/acs.jafc.0c03584 J. Agric. Food Chem. 2021, 69, 627-within the range naturally identified in nectar and pollen and under toxicity thresholds.Chemical compounds. Organic solvents employed for extractions have been HPLC grade obtained from Rathburn (Mikrolab, Aarhus, Denmark), except for ethanol (96 ), which was obtained from Kemetyl AB (Haninge, Sweden). Acetonitrile and methanol for LC-MS analysis was LC- MS grade purchased from Fisher Scientific (Roskilde, Denmark). Analytical grade formic acid and LC-MS grade ammonium formateMATERIALS AND METHODSJournal of Agricultural and Meals Chemistrypubs.acs.org/JAFCArticleTable two. Validation of your Analytical Strategy and MRM Transitions (Q1/Q3) Monitored for the Eight PhytochemicalsMRM transitiona phytochemical atropine senecionine senkirkine gelsemine methyllycaconitine MGAT2 list amygdalin aucubin triptolidea bquantifier (m/z) 290/124 336/120 366/168 323/70 683/216 456/323 345/183 378/qualifier (m/z) 290/77 336/308 366/150 323/236 683/651 456/59 345/165 378/retention time (min) eight.9 9.1 9.9 7.6 12.eight 9.0 five.1 four.spike level (ng/bee) 240 4.9 7.4 510 0.three 240 16,400 four.recoveryb ( ) 108 78 72 74 99 67 76 82 6 7 5 3 12 3 10RSD ( ) six 9 8 5 12 4 13LODc (ng/bee) 45 1.1 1.2 56 0.1 21 4809 0.LOQc (ng/bee) 151 three.five 4 185 0.4 70.two 16,030 2.The quantifier MRM transition was employed for quantitation, whereas the qualifier MRM transition was utilised to aid compound identification. Recovery percentages are listed because the SD. cLOD and LOQ were calculated as three and ten times the SD, respectively, from the eight replicates ready for system validation. rpm). The extraction solvents have been as follows: aucubin: 20 mL methanol; methyllycaconitine: three mL 1:1 methanol/water; triptolide: three mL methanol; senecionine: eight mL 7:3 methanol/water; senkirkine: 8 mL 1:1 ethanol/water; amygdalin: 8 mL 7:1 methanol/water + 0.5 acetic acid; gelsemine: eight mL 1:1 ethanol/water, and atropine: eight mL 7:three methanol/water + 0.5 acetic acid. After extraction, the samples were centrifuged (12 min, 4 , 4500 rpm). Extracts of bees fed on aucubin, methyllycaconitine, senecionine, senkirkine, amygdalin, gelsemine, and atropine were diluted to 10 organic solvent with Milli-Q water containing 0.5 acetic acid, filtered utilizing a syringe filter (Kinesis KX PTFE syringe filter 13 mm, 0.22 m, Mikrolab, Aarhus, Denmark), and analyzed by HPLC-MS/MS as described under. Extracts of bees fed on triptolide were additional purified by strong phase extraction (SPE) utilizing a protocol modified from Wang et al.30 1 milliliter on the methanol extracts was diluted to 10 mL with Milli-Q water, one hundred L of formic acid was added, and the diluted extracts had been loaded onto 30 mg Oasis HLB prime SPE (Waters, Hedehusene, Denmark) cartridges without the need of prior column conditioning. The SPE cartridges had been initially washed with 1 mL of 2 ammonium hydroxide in 1:9 methanol/water and then with 1 mL of 2 acetic acid in 3:7 methanol/water, and triptolide was eluted in the cartridges with 1 mL of 4:1 methanol/water. Prior to HPLC-MS/MS analyses, 225 L on the SPE eluates was diluted with 275 L of 5 mM ammonium TLR3 drug formate and filtered working with a syringe filter (Kinesis KX PTFE syringe filter 13 mm, 0.22 m, Mikrolab, Aarhus, Denmark). Sample Preparation and Extraction of Dissected Honey Bees. Six bees fr.