A fast, adaptable piecewise gradient method for high-throughput quantification of leaf carotenoids using RP-HPLC-PDA

Abstract

Lutein and β-carotene are the major carotenoids in plant leaves, playing crucial roles in photosynthesis. These pigments also have important applications in human and animal health; therefore, it is essential to accurately determine their quantities in crops. A high-throughput RP-HPLC technique was developed for quantifying the levels of these carotenoids in leaf tissue using a piecewise gradient system. The analysis time was reduced to only 10 min, including column re-equilibration, through the use of a short, efficient Poroshell column with an HPLC-PDA instrument. The method was successfully validated, resulting in sub-μg mL⁻¹ quantification limits for both analytes. To demonstrate the adaptability of the method, application to both saponified and unsaponified leaf samples was performed. The piecewise gradient allows for the customizability of the mobile phase selectivity towards specific sample components and can potentially be adapted to screen multiple tissues of plant varieties and mutants with more extensive carotenoid profiles.