Quantifying Metabolites using HPLC

Sample tissues and cells were extracted with 416 μL methanol and 84 μL 1,8-dihydroxyanthraquinone (internal standard, IS, 200 μg/mL) in a centrifuge tube. The tissue homogenates were centrifuged at 12,000 rpm for 10 min at 4 °C and the supernatant was transferred to a clean tube. The combined organic solvent of the supernatants was evaporated to a final volume of 400 μL, and subsequently placed in a sealed amber vial for HPLC analysis [23 (link)]. To improve the sensitivity and precision of quantification, we purified the samples and cited 1,8-dihydroxyanthraquinone (Sigma-Aldrich, St. Louis, MO, USA) as the internal standard and trans-resveratrol (Sigma-Aldrich) as the standard for drawing a standard curve by the methods described elsewhere [24 (link)].