Agilent 6490 qqq mass spectrometer

Liquid chromatography–tandem mass spectrometry (LC-MS/MS) was performed according to previously published methods, with slight modification for cultured cells (45 , 46 (link), 47 (link)). Cellular extracts were analyzed using either (i) a 4000 Qtrap mass spectrometer (AB Sciex) with an Agilent 1290 series HPLC and a ZORBAX eclipse plus C18 column (2.1 × 100 mm 1.8 μm, Agilent) with the thermostat set at 60 °C to analyze the cell extracts; or (ii) an Agilent 6490 QQQ mass spectrometer with an Agilent 1290 series HPLC system and a ZORBAX eclipse plus C18 column (2.1 × 100 mm 1.8 μm, Agilent) with the thermostat set at 45 °C. Mass spectrometry analysis was performed using dynamic scheduled multiple reaction monitoring (MRM) in positive ion mode; transitions, internal standards, and conditions have been previously reported (47 (link)). Lipids were identified based on their retention time, precursor, and product ions. For the experiments using nonlabeled fatty acids, data was analyzed in MultiQuant 2.1.1 (AB Sciex) software. Lipid abundances were determined by normalizing the area under the chromatogram for each lipid species against the corresponding internal standard. Lipid abundance was normalized to the total PC of the respective sample. Total PCs were not different between groups.

LC-MS/MS was performed in negative electrospray ionization (ESI^-) mode on a tandem quadrupole mass spectrometer (Agilent Technologies) with an electrospray interface. Multiple reaction monitoring (MRM) was selected to quantify test substances and the IS using a slight modification of a previously described method [24 (link)]. In detail, the LC-MS/MS system consisted of an Agilent HPLC system (1290 Infinity, Agilent Technologies, Santa Clara, CA, USA) and an Agilent 6490 QQQ mass spectrometer with an ESI^- Agilent Jet Stream ion source (Agilent Technologies, Santa Clara, CA, USA). Carbohydrates were separated using a Luna amino column (150 mm × 2 mm ID, 3 μm particle size; Phenomenex, Torrance, CA, USA) at a flow rate of 0.2 mL/min. The mobile phase consisted of ACN and water (80:20). Analytical data was acquired using Mass Hunter software (version A.02.00; Agilent Technologies, Santa Clara, CA, USA). Concentrations of glucose and fructose were determined before and after incubating D-allulose in PBS, SGF, FaSSIF, and hepatocytes.