Masshunter metabolomics dmrm database and method

Ion-pair LC-MS/MS analysis was performed by LC separation on a Zorbax RRHD Extend-C18 column (150 mm × 2.1 mm, 1.8 µm particle size, Agilent Technologies) and using a gradient of solvent A (10 mM tributylamine and 15 mM acetic acid in 97:3 water: methanol) and solvent B (10 mM tributylamine and 15 mM acetic acid in methanol) according to the manufacturer’s instructions (MassHunter Metabolomics dMRM Database and Method, Agilent Technologies).

After collection and washing, sperm pellets containing ten million purified sperm were treated with 700 ml of cold 40% acetonitrile, 40% methanol, and 20% water. Then samples were vortexed vigorously for 5 min at 4 °C and spun at 10,000g for 10 min at 4 °C. The supernatant was removed and placed in a SpeedVac until dry. To resuspend, 20 ml of methanol was added, followed by vortexing, and finally, 80 ml of water was added along with a final vigorous vortex. For LC–MS analysis, 4 ml of sample volume was injected. Analysis method was described previously (20 (link)). Briefly, cell metabolite extractions were analyzed with an Agilent 6470A triple quadrupole mass spectrometer, operating in negative mode, connected to an Agilent 1290 ultra-high-performance liquid chromatography (UHPLC) system (Agilent Technologies). The analytical column used was an Agilent ZORBAX RRHD Extend-C18 1.8 mm 2.1 × 150 mm coupled with a ZORBAX Extend Fast Guard column for ultra-high-performance liquid chromatography Extend-C18, 2.1 mm, 1.8 mm. The MassHunter Metabolomics dMRM database and method was used to scan for polar metabolites within each sample (Agilent Technologies). The resulting chromatograms were visualized in Agilent MassHunter Quantitative Analysis for QQQ (Agilent Technologies). The final peak areas were manually checked for consistent and proper integration.

Ion pair LC-MS/MS analysis was performed with LC separation on a Zorbax RRHD Extend-C18 column (150 mm × 2.1 mm, 1.8 μm particle size, Agilent Technologies), and using gradient of solvent A (10 mM tributylamine and 15 mM acetic acid in 97:3 water:methanol) and solvent B (10 mM tributylamine and 15 mM acetic acid in methanol) according to the manufacturer’s instructions (MassHunter Metabolomics dMRM Database and Method, Agilent Technologies).