HPLC Analysis of Carotenoid Extracts

The carotenoid extracts were suspended in 250 μL of methyl t-butyl ether and 242 μL of MeOH. The carotenoid suspension was filtered through a 0.45-μm syringe filter (SmartPor^®-II NYLON Syringe filter with 13 mm, 0.45 µm, Woongki Science Co., Ltd., Seoul, Korea) and put in an HPLC vial. The carotenoid analysis was performed using a 1260 Infinity series HPLC instrument (Agilent, Santa Clara, CA, USA) and the Chemstation software. The injection volume was 25 μL and the flow rate was 1 mL min⁻¹. The column temperature was maintained at 25 °C for all the analyses. The carotenoids were separated under a polar to nonpolar gradient using mobile phases consisting of 100% methanol with 0.1% ammonium acetate (A) and methyl t-butyl ether (B) through a guard column (YMC Guard Cartridge, YMC Co., Ltd., Kyoto, Japan) and C₃₀ stationary phase (YMC Carotenoid S-5 μm, 250 × 4.6 mm, YMC Co., Ltd., Kyoto, Japan). A gradient elution was performed with 100% of A for 6 min, which was then gradated to 4% of A and 96% of B over 20 min. After 26 min, A was increased to 100% until 36 min. The elution of carotenoids was observed on an online Diode Array Detector (PDA) at five wavelengths (286 nm, 348 nm, 434 nm, 450 nm, and 471 nm). The carotenoids were identified based on their absorption maxima and spectrum [39 (link),41 ].