Analytical Protocol for Sterols and Bile Acids

Neutral sterols and bile acids were analyzed as described by Anita Fechner et al. [21 (link)] and Sylvia Keller [22 (link)] using an Agilent GC 7890B-5977B MSD GC-MS system (Agilent Technologies) equipped with a DB-5MS column (0.25 µm, 30 m × 0.25 mm). Total sterols were defined as the sum of cholesterol, coprostanol (Steraloids Inc., Newport, RI, USA), coprostanone (Carbosynth Ltd., Newbury, UK), cholestanol, cholestanone, and cholestenone, and 5α-cholestane was used as the internal standard. All reagents except for coprostanol and coprostanone were purchased from Sigma Aldrich Corporation
Sterol extracts were saponified by the addition of NaOH for bile acid analysis. The primary external standards were cholic acid and chenodeoxycholic acid, and the secondary external standards were iso-lithocholic acid (Steraloids Inc.), lithocholic acid, iso-deoxycholic acid (Steraloids Inc.), and deoxycholic acid; hyodeoxycholic acid was used as the internal standard. All reagents were purchased from Sigma-Aldrich Corporation unless otherwise indicated.