6550 q tof

An Agilent 1290 UHPLC system (Santa Clara, CA, USA) coupled with an HSS T3 2.1 × 100 mm 1.7 µm column from Waters Corp (Milford, MA, USA) was used to perform UHPLC analysis of the vegetable extracts. Samples were mixed with distilled water, vortexed, and passed through a 0.2 µm filter. Then, a 5 µL portion of the filtrate was injected onto the column followed by elution at a flow rate of 0.5 mL/min using mobile phases A and B (0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively) at 40 °C. The following gradients were used: initial holding time 0.5 min, mobile phase B ramped up to 50% after 5 min, 95% after 6 min, held for 1 min, and re-equilibrated for 1.5 min. Compounds were identified using a diode array detector at a wavelength range of 230–640 nm in 2 nm increments and a frequency of 5 Hz. The mass spec was carried out in an Agilent 6550 QTOF (Santa Clara, CA, USA) at 200 °C, using a drying gas pressure of 18 psi, 40 psi nebulizer and 350 °C sheath gas, a pressure of 12 psi, and 3500 V capillary with a 1000 V nozzle, and ran in positive ion electrospray at a frequency of 3Hz and acquisition from 30–1700 m/z. The MS/MS was performed at a narrow quadruple setting (1.3 atomic mass units), using 10, 20, and 40 eV collision energy and 30–1700 m/z. The compounds were identified using MS/MS fragmentation patterns and quantified based on the MS peak area.

Metabolites were extracted from 10 μl plasma 3 times with 70% ethanol at a temperature > 70°C. Extracts were analysed using flow injection–time‐of‐flight mass spectrometry (Agilent 6550 QTOF) operated in negative mode, as described previously (Fuhrer et al, 2011). Distinct mass‐to‐charge (m/z) ratio could be identified in each batch of samples (typically with 5,000–12,000 ions). Ions were annotated by aligning their measured mass to compounds defined by the KEGG database, allowing a tolerance of 0.001 Da. Only deprotonated ions (without adducts) were considered in the analysis. When multiple matches were identified, such as in the case of structural isomers, all candidates were retained. For enrichment analysis, metabolites with P < 0.05 and log2(fold change) > 0.25 or < −0.25 follow a previously described procedure (Subramanian et al, 2005). Enrichments were considered significant when FDR < 0.05 after multiple testing corrections using the Benjamini–Hochberg post hoc test.

NOD islets were prepared for LC–MS/MS analyses as described elsewhere (5 (link)). In vitro reactions and prepared NOD islets were analyzed by LC–MS/MS using an Agilent 1200 series UHPLC system with a nanoflow adapter and an Agilent 6550 Q-TOF equipped with a nano-ESI source. Online separation was accomplished by reversed-phase liquid chromatography using a Thermo Acclaim PepMap 100 C18 trap column (75 μm × 2 cm; 3 μm particles; 100 Å pores) and Thermo Acclaim PepMap RSLC C18 analytical column (75 μm inner diameter; 2 μm particles; 100 Å pores) in a trap forward-elute configuration using a water/acetonitrile gradient (buffer A: 0.1% formic acid in water; buffer B: 0.1% formic acid and 90% acetonitrile in water). A detailed description of the MS data collection was previously published (12 (link)).

Data were acquired using an Agilent 6550 QToF instrument. Samples were ionized using an Agilent Jet Stream electrospray ionization source operated in negative mode. Gas temperature in the ion source was 150 °C with a flow rate of 14 L/min. Nebulizer pressure was 45 psig; sheath temperature was 325 °C with a gas flow rate of 12 L/min. Voltage for both capillary and nozzle was 2000 V. The funnel DC voltage was −30 V, funnel voltage drops were −100 and −50 V in the high- and low-pressure funnels, respectively. The RF voltages were 110 and 60 V in the high- and low-pressure funnels respectively. Mass spectra were acquired between 50 m/z and 1100 m/z with a rate of 2 spectra per second. Mass lock mixture was used as described in Wan et al.^{63 (link)}.

Samples were analyzed by LC-MS/MS using an Agilent 1200 series UHPLC system with a nanoflow adapter and an Agilent 6550 Q-TOF equipped with a nano-ESI source. Samples were separated online by reversed-phase liquid chromatography using a trap forward-elute configuration (trap column: Thermo Acclaim Pepmap 100, 75 µm x 2 cm, 3 µm particles, 100Å pores; analytical column: Thermo Acclaim Pepmap RSLC C18 analytical column, 75 µm inner diameter, 2 µm particles, 100Å pores). A water/acetonitrile gradient was used (buffer A: 0.1% formic acid in water; buffer B: 0.1% formic acid and 90% acetonitrile in water). Mass spectrometric data was collected in positive ion mode with an MS scan range of 290-1700 m/z, an MS acquisition rate of 5 spectra/sec, an MS/MS scan range of 50-1700 m/z, and a minimum MS/MS scan rate of 3 spectra/sec. Abundance dependent accumulation was enabled with a target of 80,000 counts/spectrum. Using auto-MS/MS mode, the ten most abundant precursors per cycle were selected for fragmentation (absolute threshold: 3000 counts; relative threshold: 0.01%). Singly-charged precursors were excluded.