6550 ifunnel q tof ms

Tandem mass spectrometry (MS/MS) was utilized in this study for structural elucidation and tentative identification of unknown placental metabolites that significantly differed between sexes. All targeted MS/MS experiments were performed on an Agilent G7100A CE system (Agilent Technologies Inc., Mississauga, ON, Canada) equipped with a coaxial electrospray ionization (ESI) source coupled to an Agilent 6550 iFunnel QTOF-MS. A pooled placenta tissue extract was injected hydrodynamically at 100 bar for 20 s followed by a BGE spacer at 100 mbar for 5 s. Precursor ions were selected for collisional induced dissociation (CID) experiments at 10, 20 and 40 V. The ESI conditions were Vcap = 3500 V, nozzle voltage = 2000 V, nebulizer gas = 8 psi, drying gas 14 L/min at 225 °C, whereas, the MS voltage settings were fragmentor = 380 V and Oct1 RF = 750 V. For structural elucidation, the METLIN database^{59 (link)} accessed through the Agilent MassHunter Personal Compound Database and Library (PDCL) manager was used. Since no authentic standards were used to confirm via co-migration with spiking, in silico fragmentation using MetFragWeb was employed for MS/MS spectral comparison^{29 (link)}.

Following the extraction with MeOH:ACN, plasma and CSF sample extracts were subjected to LC-MS analysis using the 6550 iFunnel Q-TOF MS interfaced with 1290 UHPLC (Agilent Technologies, Basel, CH) as previously described [23 (link)]. The data were processed using XCMS Online [24 (link)] and signal drift correction was applied and metabolite features showing analytical variability > 30% were removed. Putative identification was done in XCMS Online linked to METLIN metabolite database [25 (link)], and metabolite identities were further validated with tandem MS experiments as previously described [23 (link), 26 (link)].

An Agilent 1100 HPLC equipped with DAD and FLD was employed in the preliminary studies; the LC-MS analyses were performed on a 1290 UHPLC coupled to a 6550 iFunnel QTOF MS with a Jet Stream ESI source (Agilent, Santa Clara, CA, USA). The mobile phase gradient and flow rates are depicted in Table 9.
An Agilent Dual Jet Stream electrospray (ESI) source was used in positive ionization mode with nitrogen as drying, nebulizer and sheath gas. Parameters such as the sheath gas temperature, ESI source and nozzle voltages, TOF fragmentor voltage, collision energies and mass acquisition rates were optimized to achieve the best analyte responses in the desired concentration ranges. The following final conditions were applied: drying gas temperature 200 °C; drying gas flow 14 L/min; nebulizer pressure 35 psi; sheath gas flow 11 L/min. TOF mass calibration was performed daily in the extended dynamic range (2 GHz) and low mass range (1700 m/z). The reference masses used for the within-run mass correction were m/z 121.0509 and 922.0098.
Data were collected using an Agilent MassHunter Workstation software Qualitative analysis 10.0 and Data Acquisition for 6200 series TOF/6500 series QTOF 10.1 (Santa Clara, CA, USA). Microsoft Office 365 Excel (Redmond, WA, USA) with the PK solver extension and GraphPad Prism 8 (San Diego, CA, USA) were used for data analysis.