Cp sil 88 for fame column

Fecal lipids were extracted as described by Folch and Lees [28 (link)]. Methyl esterification of fatty acids (palmitic acid, stearic acid, oleic acid, and linoleic acid) was carried out using a Fatty Acid Methylation Kit (Nacalai Tesque, Kyoto, Japan), and fatty acids were analyzed on an Agilent 7890 GC-MS with CP-Sil 88 for FAME column (Agilent, Palo Alto, CA, USA). The initial oven temperature of 100 °C was increased to 240 °C (3 °C/min) and maintained for 15 min. One microliter of sample was injected into the GC/MS system. Fecal total bile acids were also determined using a Total Bile Acids Test Wako Kit (Fujifilm Wako Pure Chemical Corporation) after ethanol extraction.

Dried lipid extracts were redissolved in chloroform/methanol (2:1, v/v) and a solution containing nonadecanoic acid (19:0) as the internal standard was added. A detailed sample preparation protocol for fatty acid (FA) derivatization was previously published [24 (link)]. FA analysis was performed using capillary GC with a CP-Sil 88 for FAME column (100 m × 0.25 mm, Agilent, Santa Clara, CA, USA) and a PerkinElmer gas chromatograph CLARUS 680 with a flame ionization detector and split injection (PerkinElmer Instruments, Waltham, MA, USA). The detailed GC conditions were described only recently [25 (link)]. The calibration was performed with the help of the reference standard mixture ‘Sigma FAME’ (Sigma-Aldrich, Deisenhofen, Germany) and the methyl esters of 18:1cis-11, 22:5n-3 and 18:2cis-9, trans-11 (Matreya LLC, State College, PA, USA), 20:3n-9, 22:3n-3 and 22:4n-6 (Sigma-Aldrich), and 18:4n-3 (Larodan, Limhamn, Sweden). The five-point calibration of single FAs ranged between 16 and 415 µg/mL and was checked after GC analysis of five samples.