Fragmentation patterns of the differential plasma metabolites between patients with a CASH event in the prior year and non-CASH patients were validated in an independent propensity matched validation cohort using a supervised LC-MS/MS approach.
Metabolites were extracted using a 1:8 dilution (i.e., one volume of plasma for eight volumes of extraction solvent composed of 100% methanol spiked with internal standards). Plasma samples were extracted at −80 °C for 1 h. Samples were centrifuged at 20,000g for 15 min at −10 °C. Two-hundred µL of the supernatant was dried down under nitrogen stream at 1 L/min (bottom)−30 L/min (top) at 30 °C using the SPE Dry 96 Dual (Biotage, Uppsala, Sweden). The samples were then resuspended in 200 µL of 1:1 water:methanol diluent, and mixed at 1000 rpm for 15 min at 4 °C using a thermomixer (Eppendorf). Samples were then centrifuged at 20,000g for 15 min at 4 °C to remove insoluble debris, and 150 µL of extracted supernatant was taken through subsequent analysis on an Agilent 1290 infinity II liquid-chromatography system coupled to an Agilent 6546 QTOF mass spectrometer, equipped with an Agilent Jet Stream Electrospray Ionization source. The detection window was set to 100–1700 m/z with continuous infusion of a reference mass (Agilent ESI TOF Biopolymer Analysis Reference Mix) for mass calibration.
Bile acids were analyzed in negative mode and 5 µL of extracted supernatant was injected onto an XBridge BEH C18 Column (Waters Corporation, Milford, MA, USA) fitted with an XBridge BEH C18 guard (Waters Corporation) at 45 °C. The mobile phase A was water with 0.1% formic acid, while the mobile phase B was acetone with 0.1% formic acid. Gradient elution started with 28% of the mobile phase B with a flow rate of 0.4 mL/min for 1 min, and linearly increased to 33% over 5 min, then to 65% over 14 min. The flow rate was then increased to 0.6 mL/min, while the mobile phase B was increased to 98% over 0.5 min. These conditions were held constant for 3.5 min. Then, re-equilibration was performed for 3 min at a flow rate of 0.4 mL/min with 28% of the mobile phase B. The electrospray ionization conditions were set with the capillary voltage at 3.5 kilovolts (kVs), nozzle voltage at 2 kVsA ten-point calibration curve of glycodeoxycholic acid was generated, starting at a concentration of 100 µg/mL diluted in 1:1 water:methanol followed by nine 1:3 serial dilutions.
Metabolites were extracted using a 1:8 dilution (i.e., one volume of plasma for eight volumes of extraction solvent composed of 100% methanol spiked with internal standards). Plasma samples were extracted at −80 °C for 1 h. Samples were centrifuged at 20,000g for 15 min at −10 °C. Two-hundred µL of the supernatant was dried down under nitrogen stream at 1 L/min (bottom)−30 L/min (top) at 30 °C using the SPE Dry 96 Dual (Biotage, Uppsala, Sweden). The samples were then resuspended in 200 µL of 1:1 water:methanol diluent, and mixed at 1000 rpm for 15 min at 4 °C using a thermomixer (Eppendorf). Samples were then centrifuged at 20,000g for 15 min at 4 °C to remove insoluble debris, and 150 µL of extracted supernatant was taken through subsequent analysis on an Agilent 1290 infinity II liquid-chromatography system coupled to an Agilent 6546 QTOF mass spectrometer, equipped with an Agilent Jet Stream Electrospray Ionization source. The detection window was set to 100–1700 m/z with continuous infusion of a reference mass (Agilent ESI TOF Biopolymer Analysis Reference Mix) for mass calibration.
Bile acids were analyzed in negative mode and 5 µL of extracted supernatant was injected onto an XBridge BEH C18 Column (Waters Corporation, Milford, MA, USA) fitted with an XBridge BEH C18 guard (Waters Corporation) at 45 °C. The mobile phase A was water with 0.1% formic acid, while the mobile phase B was acetone with 0.1% formic acid. Gradient elution started with 28% of the mobile phase B with a flow rate of 0.4 mL/min for 1 min, and linearly increased to 33% over 5 min, then to 65% over 14 min. The flow rate was then increased to 0.6 mL/min, while the mobile phase B was increased to 98% over 0.5 min. These conditions were held constant for 3.5 min. Then, re-equilibration was performed for 3 min at a flow rate of 0.4 mL/min with 28% of the mobile phase B. The electrospray ionization conditions were set with the capillary voltage at 3.5 kilovolts (kVs), nozzle voltage at 2 kVsA ten-point calibration curve of glycodeoxycholic acid was generated, starting at a concentration of 100 µg/mL diluted in 1:1 water:methanol followed by nine 1:3 serial dilutions.
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