Ultra high performance liquid chromatography single and tandem mass spectrometry (UHPLC-QTOF-MS/MS) data were obtained from an Agilent 6530B Accurate Mass QTOF LC–MS system coupled to an Agilent 1260 Infinity II UHPLC system (Agilent, Cheshire, UK). Mobile phases consisted of methanol (0.1% formic acid) and 0.1% formic acid in water. The samples were dissolved in methanol at a concentration of 1 μg/mL and a 5 μL injection volume was injected onto a stainless steel back pressure sample loop. The flow rate was isocratic, set to 50% methanol at 0.4 mL/min, the column controller was held at 25°C, and data were acquired for 3 min. The QTOF-MS data were acquired in positive mode scanning from m/z 100–1000 for MS and m/z 50–500 for MS/MS analysis. Ionization was achieved with an Agilent JetStream electrospray source and infused internal reference masses. QTOF parameters: gas temperature 325°C, drying gas 10 L/min, nebulizer gas 45 L/min, sheath gas temperature 400°C, sheath gas flow 11 L/min. Scan source parameters: VCap 3500, nozzle voltage 500 V, fragmentor 130, skimmer1 65, and octapole RF peak 750. Targeted MS/MS was obtained for m/z 392.2333 with a fixed 25 CeV collision energy. Internal reference ions at m/z 121.05087 and m/z 922.00980 were used for calibration purposes.
Agilent 1260 infinity 2 uhplc system
Manufactured by Agilent Technologies
Sourced in United Kingdom
The Agilent 1260 Infinity II UHPLC system is a high-performance liquid chromatography (HPLC) instrument designed for ultra-high-performance applications. It features advanced hardware and software components to deliver reliable and efficient separation of complex samples.
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2 protocols using agilent 1260 infinity 2 uhplc system
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UHPLC-QTOF-MS/MS Protocol for Metabolite Analysis
2
High-throughput HPLC-MS/MS Analysis
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The positive reactions of the combinatorial high-throughput screen were analyzed by performing HPLC-MS/MS analysis using an Acquity BEH C18 column (2.1 × 50 mm, 1.7 μm) connected to an Agilent 1260 Infinity II UHPLC system (Agilent Technologies) equipped with a Thermo-Finnigan 7 T LTQ-FT mass spectrometer. A volume of 5 μl of the diluted sample (1:1 using distilled water) was injected, and chromatographic separation was performed at a flow rate of 0.3 ml/min with a gradient of 5% to 80% buffer B over 5 min, followed by two isocratic steps with 90% buffer B over 0.5 min, and 5% buffer B over 2.5 min where buffer A was water with 0.1% formic acid and buffer B was acetonitrile with 0.1% formic acid. The analytes were detected using mass spectrometry in negative mode, and MS/MS fragmentation was done for selected m/z ratios at an isolation window of 2.5 Da, activation time of 30 s, and using 15 and 20 normalized collision energies (cid).
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