2.5. Lipid Extraction and LC-MS/MS Analysis

Sterols and cholesteryl esters from human plasma were extracted using the Matyash method with some modifications [22]. The tube used for lipid extraction was Eppendorf tube (Eppendorf safe-lock tube 2.0 mL, product number: 0030 120 094). Briefly, 100 μL of human plasma was added to an Eppendorf tube containing cholesterol-d7 (internal standard, 500 ng), followed by the addition of 400 μL of ice-cold 75% methanol with 0.1% BHT and 1 mL MTBE, and shaken for 1 h at room temperature with 2000 rpm speed (using the Multi Reax shakers and mixers). For phase separation, 250 μL of water was added, and the sample was centrifuged at 14,000 g at 4 °C for 15 min. The upper phase was transferred to a new tube and dried under vacuum. Lastly, the sample was reconstituted in 100 μL of chloroform/methanol (1:9, v/v). Quantitative analysis of sterols was performed using a Nexera2 LC system (Shimadzu Corporation, Kyoto, Japan). The LC system was connected to a triple quadrupole mass spectrometer (LC-MS 8060; Shimadzu, Kyoto, Japan) with a reversed phase Kinetex C18 column (100 × 2.1 mm, 2.6 μm, Phenomenex, Torrance, CA, USA) for chromatographic separation of sterols. Mobile phase A consisted of water/methanol (1:9, v/v) containing 10 mM ammonium acetate, and mobile phase B consisted of isopropanol/methanol (5:5, v/v) containing 10 mM ammonium acetate. The gradient elution program was as follows: 0 min with 30% B, 0–15 min with 95% B, 15–20 min with 95% B, and 20–25 min with 30% B. The total run time was 25 min, and the flow rate was 0.2 mL/min. For each run, 5 μL of sample was injected. An electrospray ionization interface was used as the ionization source in the positive mode. The optimum operating conditions were as follows: capillary temperature, 350 °C; vaporizer temperature, 300 °C; collision gas (argon) pressure, 1.5 mTorr. To determine the concentration of each target sterol, the calculated ratio of target analyte and internal standard (IS) was multiplied by the concentration of the IS [23,24]. The selective reaction monitoring transitions and collision energies determined for each sterol are listed in Table S1. The concentration of stigmasterol was not determined in this study.