2.6. Adipose tissue Acyl-CoAs extraction and quantification

Acyl-CoAs were extracted and analyzed using a modified version of a previously described method [34]. Briefly, gWAT was ground to a homogenous powder using a liquid nitrogen-chilled mortar and pestle. Approximately 30–40 mg (wet mass) of each pulverized tissue sample was rapidly transferred to a pre-weighed chilled Eppendorf tube before the addition of extraction solvent and the internal standard. For acyl-CoAs, 1 ml of extraction solvent was used; solvent was composed of 1:1 of 100 mM KH₂PO₄ and organic mixture (3:1:1 ACN, IPA: Methanol). The internal standard was 20 ng/ml of C17-CoA. The samples were sonicated on ice, followed by centrifugation at 4 °C. Clean extract was recovered and dried under a gentle stream of nitrogen. Dried sample was reconstituted with cold 1:1 (methanol: water) and 10 μl was injected on the LC-MS. Chromatographic separations were performed with an Agilent Technologies (Santa Clara, CA) 1200 HPLC system equipped with a Waters (MA, USA) Acquity UPLC® BEH C18 column (2.1 × 100 mm, 1.7 μm) and a 2.1 × 5 mm VanGuard™ pre-column using the following conditions: mobile phase A was 10% acetonitrile, 90% MS water, mobile phase B was acetonitrile. Both mobile phases contained 15 mM ammonium hydroxide. The gradient program for LC-CoA started at 0% B which was held for 3 min while LC eluent was directed to the waste during this period. %B was ramped linearly to 30% B over 3 min, then ramped to 40% B over 6 more minutes, then held at 100% B for 4 min, after which the column was reconditioned using 100% A for 6 min. The total run time was 22 min and the flow rate was 0.25 ml/min. Metabolites were quantified by measuring the metabolite peak area as a ratio to its ¹³C internal standard analog and was normalized to the tissue weight.