Agilent 6495 qqq

400 μL MeOH and 400 μL ACN were added to 100 μL serum. Incubated for 1 h at −20 °C, centrifuged for 15 min at 13,000 rpm and 4 °C. Took the supernatant and evaporated to dryness at 4 °C. Reconstituted with 100 μL ACN/H2O (volume/volume, 1:1), vortexed for 30 s, sonicated for 10 min (4 °C water bath). Centrifuged for 15 min at 13,000 rpm and 4 °C. Supernatants were analyzed by LC-MS. The LC–MS analysis was performed using an UHPLC system (1,290 series, Agilent Technologies) coupled to a triple quadrupole mass spectrometer (Agilent 6,495 QqQ, Agilent Technologies). Chromatographic separation was performed on the Merck ZIC-pHILIC column (100 × 2.1 mm, 5 μm). The column was maintained at room temperature, and the flow rate was 0.2 ml/min. Mobile phase A was 100% H2O, 25 mM CH3COONH4, and 25 mM NH4OH, and mobile phase B was 100% acetonitrile. The column was eluted with a linear gradient system (B %): 0 min, 80%; 1 min, 80%; 3 min, 65%; 7 min, 50%; 7.1, 20%; 9.5 min, 20%; 9.6 min, 80%; 13 min, 80%. Optimized MRM transition 123.1/53.1 in positive mode was utilized for quantifying NAM.

All lipid species were analyzed at the brutto (sum composition) or medio (fatty acyl chain) level of identification [29 ].
For analysis of bile acids, sterols, steroids, fatty acids, carnitines, and sphingolipids, an Agilent 1290 UHPLC system coupled to an Agilent 6495 QQQ (Agilent, Santa Clara, CA, USA) or a Sciex 4000 QTRAP (Sciex, Framingham, MA, USA) mass spectrometer equipped with an electrospray ion (ESI) source was used. The MS instruments were operated in multiple-reaction monitoring (MRM) with negative-ion (-) detection for analysis of fatty acids and bile acids, and with positive-ion (+) detection for analysis of carnitines, sphingolipids, sterols and steroids.