D diethyl ether were distilled from analytical-grade solvents (Penta, Czech Republic
D diethyl ether had been distilled from analytical-grade solvents (Penta, Czech Republic). Other chemical substances, NaCl (99 , Sigma-Aldrich, St. Louis, MO, USA), di-tert-butyl-4-methylphenol (Fluka, Buchs, Switzerland), Rhodamine 6G (Sigma-Aldrich, St. Louis, MO, USA), and Diazald (99 , Sigma-Aldrich, St. Louis, MO, USA) had been utilized. The requirements of crepenynic acid (99 ) and punicic acid methyl ester (purity 98 ) had been from Larodan (Malm Sweden), and 9-octadecynoic acid methyl ester, 9(E),11(E)-octadecadienoic acid methyl ester, and 9(Z),11(Z)-octadecadienoic acid methyl ester (all 98 ) have been bought from Cayman Europe (Tallinn, Estonia). The requirements were dissolved in chloroform at 1 mg/mL concentrations and stored at -25 C. Bombus pratorum males have been collected within the Czech Republic throughout the spring season and immobilization at -18 C. Cold-pressed pomegranate seed oil (organic, unrefined) was from Biopurus Ltd. (Ashford, England). Seeds of Marrubium vulgare and Santalum album had been bought from a nearby garden center. 3.2. Extraction and Transesterification of Lipids The samples have been treated with organic solvents to get total lipid extracts. Briefly, peripheral fat bodies of 3 B. pratorum males were dissected and extracted with CHCl3 /CH3 OH (1:1, v/v) IQP-0528 References containing di-tert-butyl-4-methylphenol at a concentration of 25 mg/mL (500Molecules 2021, 26,16 ofeach) and sonicated for 15 min. The extract was collected making use of a Pasteur pipette. M. vulgare seeds (approx. 240 pieces; 0.25 g) or S. album seeds (5 pieces; 0.94 g) had been crushed and extracted in methanol/chloroform (2:1 v/v, 10 mL) for 30 min. Immediately after filtration, five mL of 0.9 NaCl was added, shaken for couple of seconds, and also the aqueous (upper) phase was removed. The cleaning step was repeated 3 far more times with 2 mL of 0.9 NaCl option. Total lipid extracts or seed oil were separated by semipreparative TLC to isolate TGs. Pre-cleaned, in-house made silica-gel glass TLC plates (60 mm 76 mm) and hexane/diethyl ether (80:20, by vol.) mobile phase were utilized. TLC zones had been created visible by spraying Rhodamine 6G solution (0.05 in ethanol). A zone corresponding to TGs (B. pratorum Rf = 0.36.55, pomegranate Rf = 0.20.55, M. vulgare Rf = 0.33.55, S. album Rf = 0.30.55) was scraped off the plate and extracted with 10 mL freshly distilled diethyl ether. The solvent was evaporated to dryness below a nitrogen stream. Whilst TGs from B. pratorum, pomegranate seed oil, and M. vulgare seeds have been transesterified in acidic conditions [98], base-catalyzed transesterification [99] was necessary for S. album lipids containing triple bonds. FA requirements were methylated by RP101988 site diazomethane (synthesized in-house from Diazald). Diazomethane in diethyl ether was added dropwise to the FA option in chloroform (ten mg/mL) till the colour with the reaction mixture turned light-yellow. Unreacted diazomethane was deactivated by formic acid. three.three. RP-HPLC/APCI-MS and APCI-MS The liquid chromatograph consisted of a Rheos Allegro UHPLC pump, Accela autosampler with an integrated column oven, and an LCQ Fleet ion-trap mass spectrometer; the method was controlled by Xcalibur computer software (all Thermo Fisher Scientific, San Jose, CA, USA). Develosil RP-Aqueous C30 (250 4.6 mm, particle size: 5 ; Nomura Chemical, Seto, Japan) stainless-steel column and isocratic elution with acetonitrile at 0.7 mL/min flow price [20] have been applied. The chromatography proceeded at laboratory temperature except for B. pratorum sample separated at 40 C. The injecte.