4.4. HPLC-MS Analysis of Phospholipids

Lipids were extracted from 1 mg of liver with a method adapted from that described by Bligh and Dyer [26]. Extractions were performed in dichloromethane/methanol (2% acetic acid)/water (2.5:2.5:2 v/v/v), in the presence of six internal standards, including ceramides (Cer, d18:1/15:0, 16 ng); phosphatidylethanolamine (PE 12:0/12:0, 180 ng); phosphatidylcholine (PC, 13:0/13:0, 16 ng); SM (d18:1/12:0, 16 ng); phosphatidylinositol (PI, 16:0/17:0, 30 ng); and phosphatidylserine (PS, 12:0/12:0, 156.25 ng). The solution was centrifuged at 1500 rpm for 3 min. The organic phase was collected and dried under nitrogen, then dissolved in 50 µl methanol. The extract (5 µL) was analyzed with an Agilent 1290 UPLC system coupled to a G6460 triple quadrupole spectrometer (Agilent Technologies, Santa Clara, CA, USA) and equipped with MassHunter software, for data acquisition and analysis. A Kinetex HILIC column (Phenomenex, 50 × 4.6 mm, 2.6 µm) was used for LC separations. The column temperature was controlled at 40 °C. The flow rate of the mobile phase was 0.3 mL/min. The mobile phase contained two parts: A was acetonitrile; and B was 10 mM ammonium formate in water, pH 3.2. The gradient was prepared with the following specifications: from 10% to 30% B in 10 min; 10–12 min in 100% B; and then, at 13 min, back to 10% B for 1-min re-equilibrium, prior to the next injection. An electrospray source was employed in positive ion mode for Cer, PE, PC, and SM analyses and in negative ion mode for PI and PS analyses. The collision gas was nitrogen. The needle voltage was set at +4000 V. Several scan modes were used. First, to obtain the natural masses of different species, we analyzed cellular lipid extracts with precursor ion scans of 184 m/z, 241 m/z, and 264 m/z for PC/SM, PI, and Cer, respectively; and neutral loss scans of 141 and 87 for PE and PS, respectively. The collision energy optimums for Cer, PE, PC, SM, PI, and PS were 25 eV, 20 eV, 30 eV, 25 eV, 45 eV, and 22 eV, respectively. Then, the corresponding SRM transitions were used to quantify different phospholipid species in each class. Two MRM acquisitions were necessary, due to important differences between phospholipid classes. Data were analyzed with QqQ Quantitative (vB.05.00) and Qualitative analysis software (vB.04.00).