Agilent 7000d triple quadrupole mass spectrometer

Prior to analysis, 100 mg frozen stool sample was mechanically homogenized using 10% isobutanol and ceramic beads. Formate, acetate, propionate, butyrate, isobutyrate, and valerate, in addition to the amino acids alanine, L-arginine, L-cystine, L-glutamic acid, L-leucine, L-lysine, L-serine, L-threonine, L-tyrosine, L-valine, and L-histidine, were obtained from Sigma-Aldrich (St. Louis, MO, USA) for generation of calibration curves. Samples and calibration standards were derivatized with chloroformate isobutyl. Metabolites were extracted and derivatized as previously described (Kulecka et al., 2023 (link)).
Gas chromatographic analysis was performed using an Agilent 7000D Triple Quadrupole mass spectrometer coupled with a 7890 GC System with G4513A autosampler (Agilent Technologies, Santa Clara, CA, USA). MS data were analyzed using MassHunter software (Agilent Technologies, Santa Clara, CA, USA).

Total juvenile hormone content was quantified for whole bodies of day 0 flies and day 2 flies treated with ± 1 µM CHX for 2 days post-eclosion. For each group, 25 samples were collected, each consisting of 75 flies homogenized in 1.5 ml 90% HPLC grade methanol. Samples stored at −80 °C until analysis. JH was extracted with increasingly polar solutions of hexane, column purified, and converted to a d3-methoxyhydrin derivative. Total JH was measured using an established GC-MS method^{71 (link),72 (link)}, conducted on an Agilent 8890 Series GC, equipped with a 30 m x 0.25 mm ZB-Wax GC column (Phenomenex), and coupled Agilent 7000D triple quadrupole mass spectrometer. Sample peaks were analyzed monitoring for m/z 76 and 225 to insure specificity to JHIII. Total abundance of JHIII was calculated against a standard curve and normalized to wet masses.