Generic extraction of polar metabolites from bee hemolymph

Since there are no protocols available for bumblebee hemolymph extraction of polar metabolites, two different solvent systems were tested in a preliminary experiment, i.e. methanol and methanol-ethyl acetate (v/v, 1/1), whereby the latter proved more efficient in achieving high metabolome coverage (35% higher with methanol-ethyl acetate). As such, 40 μL of methanol-ethyl acetate mixture was used for the extraction of polar metabolites. To remove proteins, all hemolymph samples were precipitated with extraction solvent, and 5 μL internal standard valine-d₈ (ISTD, 25 ng/μL) was pre-added. Subsequently, samples were incubated at 4 °C for 30 min to enhance protein precipitation and centrifugated at 15,000 g for 15 min at 4 °C to remove the resulting precipitate. Ultimately, the supernatant was transferred to a 1.5 mL microfuge tube, and consecutively dried using the Speed-Vac. All dried samples were suspended in 100 μL ultrapure water and transferred to an LC-MS vial with glass insert. Solvents used for extraction of hemolymph metabolites were of LC-MS grade, and obtained from Fisher Scientific (Loughborough, UK) and VWR International (Merck, Darmstadt, Germany).