HILIC-TripleTOF MS/MS analysis

5 μl of resuspended sample was injected onto a Waters Acquity UPLC BEH Amide column (1.7 μm, 150 mm × 2.1 mm) with a Waters Acquity UPLC BEH Amide VanGuard pre-column (1.7 μm, 5 mm × 2.1 mm). Columns are maintained at 40 °C. The mobile phases were prepared with two solutions: (A) 10 mM ammonium formate and 0.125% formic acid (Sigma, St. Louis, MO) in ultrapure water and (B) 10 mM ammonium formate and 0.125% formic acid (Sigma, St. Louis, MO) in 95:5 v/v acetonitrile: ultrapure water. Samples were eluted at 0.4 mL/min with the following gradient: 100% (B) at 0–2 min, 70% (B) at 2–7 min, 40% (B) at 7.7–9 min, 30% (B) at 9.5–10.25 min, 100% (B) at 10.25–12.75 min, 100% (B) until 16.75 min. Mass spectra were collected using an AB Sciex TripleTOF 6600 (SCIEX, Framingham, MA) in ESI (−) mode. Electron ionization voltage was −4.5 kV and spectra were acquired from m/z 60–1200 at 2 spectra/sec. Internal standard mix were injected along with each sample to monitor instrument performance during the data acquisition. A standard HBP metabolite mix, with the amount of each metabolites ranging from 6.25 to 50 ng, was used to generate the standard curve.

Mass spectra were deconvoluted, aligned and identified using MS-DIAL v3.90⁶⁵. The metabolite data were normalized to the sum peak intensity of internal standard mix. Standard curves of the HBP metabolites were generated by plotting the normalized peak intensity against the amount of compound injected on the column. The amount of each HBP metabolites in fly samples was then calculated and converted to concentration (mg/g tissue) based on the injection volume and the input amount of fly tissues. The targeted metabolomics dataset has been deposited to Metabolomics Workbench under the accession no. [ST001452].