Capsanthin

Carotenoid pigments were extracted from 96 LA RILs and 176 FC RILs as described by Lee et al. (2021) (link) with some modifications. Pepper powder was pooled from three individual plants and 0.05 g of pooled powder was used to extract carotenoids. Samples were dissolved in 500 μl of HPLC-grade acetone (Honeywell, Charlotte, NC, United States) and filtered using a 0.2-μm syringe filter (Acrodisc LC 13-mm syringe filter, PVDF membrane, Pall, NY, United States). The HPLC was performed using Ultimate3000 HPLC (Thermo Dionex, Sunnyvale, CA, United States) at the National Instrumentation Center for Environmental Management (Seoul, South Korea). We analyzed seven carotenoids, capsanthin, capsorubin, lutein, zeaxanthin, β-cryptoxanthin, β-carotene, and α-carotene (CaroteNature, Münsingen, Switzerland), as standards.
Carotenoid pigments were extracted from 176 FC RILs. The HPLC was performed using Ultimate3000 HPLC (Thermo Dionex, Sunnyvale, CA, United States) in the Molecular Marker and Food Ingredients Analysis Center of Agricultural Science Research Institute at Chungnam National University.

Carotenoids were determined in a saponified sample of paprika extract using the internal standard by HPLC analysis. This methodology was described in the previous paper [51 (link)]. After saponification, carotenoids were analyzed using a Waters Liquid chromatograph equipped with DAD 2996 detector. Separation of carotenoids was carried out using 4.6 × 150 mm chromatographic column YMC^TM Carotenoid with granulation of 3 μm additionally equipped with a protective column YMC^TM Carotenoid S-3. The mobile phase was a mixture of methanol, acetonitrile, and water (75:10:15, v:v:v) in gradient elution with dichloromethane. The flow rate was 1 mL/min and the column temperature was set at 25 °C. The injection volume was 20 μL.
Capsanthin of 96% purity, capsorubin of 98% purity, β-carotene of 99% purity, β-cryptoxanthin of 97% purity, violaxanthin of 95% purity, and zeaxanthin of 97% purity standards purchased from CaroteNature GmbH (Münsingen, Switzerland) were used for identification and quantification based on a calibration curve. β-Apo-8′-carotenal of ≥96% purity obtained from Sigma-Aldrich (Buchs, Switzerland) was used as internal standards. The qualitative analysis was based on a comparison of the retention time of peak with an appropriate carotenoid standard.