The reference standards of 18 analytes examined in this study (Table 1) were obtained from Sigma (St. Louis, MO). The deuterium-labeled internal standards (ISs) used were: 2,2,3,4,4,6-d6-cholesterol and 2,2,3,4,4,6-d6-cholesteryl stearate (C/D/N Isotopes, Pointe-Claire, Quebec, Canada) for cholesterol and CEs, respectively; and 25,26,26,26,27,27,27-d7-4β-OHC and 25,26,26,26,27,27-d6-27-OHC (Avanti Polar Lipids, Alabaster, AL) for three cholesterol precursors and eight OHCs. The trimethylsilylating (TMS) agents, N-methyl-N-trifluorotrimethylsilyl acetamide (MSTFA), ammonium iodide (NH4I), and dithioerythritol (DTE), were purchased from Sigma. The hybrid SPE-precipitation cartridge (H-PPT) (1 ml, 30 mg) was supplied by Supelco (Bellefonte, PA). All organic solvents were of analytical and HPLC grade and were purchased from Burdick and Jackson (Muskegon, MI).
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