25 hydroxycholesterol d6

All chemicals used for metabolite extraction and LC-TripleTOF experiments were ultra-pure LC-MS grade. Acetonitrile and methanol were procured from Honeywell Research Chemicals (Muskegon, MI, USA). Formic acid and 1N HCl were obtained from Sigma-Aldrich (St. Louis, MO, USA). Demineralized water (ddH2O) was generated by Millipore Advantage Milli-Q system (EMD Millipore, Billerica, MA, USA). Internal standards including: glucose-D7, glucose-6-phosphate-13C6, octanoic acid-D15, lauric acid-D23, tyrosine-D2, glutamic acid-D3, citric acid-D4, and chenodeoxycholic acid-D5 were purchased from Cambridge Isotopes Ltd. (Tewksbury, MA, USA). 25-hydroxycholesterol-D6 and lauroyl-l-carnitine-D3-(chloride) were obtained from Cayman Chemicals (Ann Arbor, MI, USA). Valine D5, tryptophan D5, and cytosine-D2 were from CDN isotopes (Quebec, Canada). 1-oleoy-2-hydroxy-sn-glycero-3-phosphocholine-D7 was from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), and caffeine-13C2 was from Toronto Research Chemicals (Toronto, Canada). Mass calibration solutions (APCI -Positive and Negative) were purchased from AB Sciex (P/N: 4460131 and 4460134; Concord, CA, USA).

All solvents were HPLC/MS grade. Methanol was from Fluka (Buchs, Switzerland), acetonitrile was from Sigma-Aldrich (Steinheim, Germany). K₂HPO₄·3H2O, KH₂PO₄ and NaCl were from Merck (Darmstadt, Germany), sodium cholate and Triton X-100 from Sigma-Aldrich (Steinheim, Germany), and ammonium acetate from Carlo Erba (Milan, Italy). Water for eluents and sample preparation was of HPLC grade, prepared in-house (Nanopure Diamond, Barnstead, Switzerland and MILLI-RO PLUS 90, MILLIPORE, Molsheim, France). Standards of 22(R)-hydroxycholesterol, 22(S)-hydroxycholesterol and 24(R,S)-hydroxycholesterol-D7 were from Avanti Polar Lipids (Spectra2000, Rome, Italy). Standards of 24(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25-hydroxycholesterol-D6, 25(R)−27-hydroxycholesterol and 25(S)−27-hydroxycholesterol were from Cayman Chemical (Vinci Biochem, Vinci, Italy).