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3 protocols using 24 s hydroxycholesterol

1

Quantification of Oxysterols by LC-MS/MS

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24S-Hydroxycholesterol [cholest-5-ene-3-beta,24S-diol], 25-hydroxycholesterol [cholest-5-ene-3-beta,25-diol], 27-hydroxycholesterol [cholest-5-ene-3beta,27-diol], 24(R/S)-hydroxycholesterol-d6 [26, 26, 26, 27, 27, 27-hexadeuterocholest-5-ene-3 beta,24-diol], 25-hydroxycholesterol-d6 [26, 26, 26, 27, 27, 27-hexadeuterocholest-5-ene-3 beta,25-diol] and 27-hydroxycholesterol-d6 [25, 26, 26, 26, 27, 27-hexadeuterocholest-5-ene-3 beta,27-diol] were obtained from Avanti Polar Lipids (Alabama, USA). 2-methyl-6-nitrobenzoic anhydride, 4-dimethylaminopyridine, picolinic acid, pyridine and trimethylamine were purchased from Sigma Aldrich Co. (St. Louis, USA). Phosphoric acid and potassium hydroxide were provided by Junsei (Tokyo, Japan). Distilled water was purified using a Milli-Q purification system (Millipore, Massachusetts, USA). High performance liquid chromatography (HPLC)-grade methanol, ethanol, hexane and 2-propanol were purchased from Burdick & Jackson (Ulsan, Korea).
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2

Quantification of Oxysterol Biomarkers

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Authentic standards (24(S)-hydroxycholesterol, 27-hydroxycholesterol, 25-hydroxycholesterol, 7ß-hydroxycholesterol) and deuterated (24(R/S)-hydroxycholesterol-d7, 25-hydroxycholesterol-d6, 27-hydroxycholesterol-d6, 7ß-hydroxycholesterol-d7, 7-ketocholesterol-d7) were purchased from Avanti polar lipids, Alabama. Authentic standard 7 keto cholesterol was purchased from Cayman chemicals, MI, USA. Butylacetate, hexane, isopropanol, methanol and formic acid (HPLC/MS grade) were purchased from Fisher Scientific, UK. Butylated hydroxytoluene (BHT) and Discovery DSC18 cartridges were from Sigma-Aldrich, UK. Oasis HLB Prime and Oasis HLB cartridges were purchased from Waters.
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3

Quantitative Analysis of Oxysterols

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Authentic standards (24(S)-hydroxycholesterol, 26-hydroxycholesterol, 25-hydroxycholesterol, 7β-hydroxycholesterol) and deuterated (24(R/S)-hydroxycholesterol-d7, 25-hydroxycholesterol-d6, 26-hydroxycholesterol-d6, 7β-hydroxycholesterol-d7, 7-ketocholesterol-d5) were purchased from Avanti polar lipids, Alabama. Authentic standard 7 keto cholesterol was purchased from Cayman chemicals, MI, USA. Butyl acetate, hexane, isopropanol, methanol, and formic acid (HPLC/MS grade) were purchased from Fisher Scientific, UK. Butylated hydroxytoluene (BHT) was from Sigma-Aldrich, UK. Oasis HLB Prime cartridges were purchased from Waters.
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