Adult male Drosophila hemolymph aqueous metabolite profiling on 7-day and 14-day High sugar diets with normal diet controls.

Metabolomics: The samples were three replicates of Hemolymph collected from adult males flies subjected to 7- and 14- day normal or High Sugar (30% more sucrose in standard lab food). Per replicate we collected hemolymph from 30 male flies. Targeted LC-MS metabolite analysis was performed on a duplex-LC-MS system composed of two Shimadzu UPLC pumps, CTC Analytics PAL HTC-xt temperature-controlled auto-sampler, and AB Sciex 6500+ Triple Quadrupole MS equipped with ESI ionization source. UPLC pumps were connected to the auto-sampler in parallel and could perform two chromatography separations independently from each other. Each sample was injected twice on two identical analytical columns (Waters XBridge BEH Amide XP), where separations were performed in hydrophilic interaction liquid chromatography (HILIC) mode. While one column was performing separation and MS data acquisition in ESI+ ionization mode, the other column was getting equilibrated for sample injection, chromatography separation, and MS data acquisition in ESI- mode. Each chromatography separation was 18 minutes (total analysis time per sample was 36 minutes). MS data acquisition was performed in multiple-reaction-monitoring (MRM) mode. LC-MS system was controlled using AB Sciex Analyst 1.6.3 software. Measured MS peaks were integrated using AB Sciex MultiQuant 3.0.3 software. Up to 158 metabolites (plus 4 spiked standards) were measured across the fly samples study set, and up to 148 metabolites (plus 34 SILISs) were measured in the hemolymph sample set. For the hemolymph set, absolute concentrations of 30 metabolites were determined. In addition to the two study samples, two sets of quality control (QC) samples were used to monitor the assay performance and data reproducibility. One QC [QC(I)] was a pooled human serum sample used to monitor system performance, and the other QC [QC(S)] was pooled study samples. This QC was used to monitor data reproducibility. Each QC sample was injected per every 10 study samples. The median CV for the fly set was 2.8%, while for the blood samples was 11.1%.