Feathers were trimmed, and the weighed pigmented portions were washed in hexane using Whatman GF/A glass filters and finely ground in 3 mL methanol for 10 min at 20 Hz using a Retsch MM301 mixer mill (Newtown, PA), equipped with ZrO grinding jars and balls. Carotenoids were extracted using a 0.2 µm filter (GHP Arcodisc 13 mm Minispike; Pall Life Sciences, East Hills, NY) and the filtrate was dried under vacuum at 40 °C and reconstituted in 150 µL of HPLC mobile phase (methanol:acetonitrile 50:50, v/v).
Carotenoids were quantified by injecting 50 µL of pigment extract into an HPLC System (Shimadzu Corporation, Pleasanton, CA) fitted with an YMC Carotenoid 5.0 µm column (250 × 4.6 mm) and guard column (YMC America, Allentown, PA). Analytes were eluted at a constant flow rate of 1.1 mL/min using isocratic elution with 42:42:16 (v/v/v) methanol:acetonitrile:dichloromethane for the first 11 min, followed by linear gradient up to 42:23:35 (v/v/v) methanol:acetonitrile:dichloromethane through 21 min, isocratic elution at this condition until 30 min when it returned with step function to the initial isocratic condition at which it was held until 40 min. Carotenoids were detected using a Shimadzu SPD-M10AVP photodiode array detector, and data were collected from 200 to 800 nm. Peak areas were integrated at 450 or 470 nm depending on the absorbance maximum (λ max) for each compound.
Carotenoids were quantified by injecting 50 µL of pigment extract into an HPLC System (Shimadzu Corporation, Pleasanton, CA) fitted with an YMC Carotenoid 5.0 µm column (250 × 4.6 mm) and guard column (YMC America, Allentown, PA). Analytes were eluted at a constant flow rate of 1.1 mL/min using isocratic elution with 42:42:16 (v/v/v) methanol:acetonitrile:dichloromethane for the first 11 min, followed by linear gradient up to 42:23:35 (v/v/v) methanol:acetonitrile:dichloromethane through 21 min, isocratic elution at this condition until 30 min when it returned with step function to the initial isocratic condition at which it was held until 40 min. Carotenoids were detected using a Shimadzu SPD-M10AVP photodiode array detector, and data were collected from 200 to 800 nm. Peak areas were integrated at 450 or 470 nm depending on the absorbance maximum (λ max) for each compound.