2.4. Saponification

A previously described fast saponification protocol [11] was used to release hydrolysed xanthophyll fatty acid esters. Briefly, 400 μL of the extracted carotenoids from the faeces were added to a test tube. The same quantity (400 μL) of pyrogallic acid in ethanol (0.1 M) and KOH in MeOH (30%) was then added and placed in an ultrasonic bath in darkness for 7 min. Then, 800 μL of distilled water and 1600 μL of extraction solvent were added. After vortex for 1 min and centrifuge for 3 min at 3500 rpm, the organic phase (supernatant) was transferred to another test tube. This process (from the point at which distilled water and extraction solvent were added) was repeated 2 more times. The organic phase collected was dried under nitrogen and dissolved in 150 μL of MeOH:MTBE (50:50) in preparation for HPLC analysis.