Evaluating secondary metabolites from citrus peel waste by supercritical fluid chromatography: Part 1 - stationary and mobile phase study.

Citrus peel waste is a valuable source of bioactive compounds, including terpenoids (TERP), coumarins and psoralens (COUM), polymethoxylated flavones (PMF), and phenolic flavonoids (FLAV). However, comprehensive analytical methods capable of simultaneously separating these chemically diverse metabolites remain scarce. Supercritical fluid chromatography (SFC) has emerged as a sustainable alternative for the analysis of natural products. This study systematically investigated the influence of stationary-phase chemistry and mobile-phase composition on the simultaneous separation of citrus secondary metabolites by SFC. Twelve stationary phases with different chemistries were evaluated, followed by optimization of the modifier composition using a two-level full factorial design investigating the effects of acetonitrile, water, and methanesulfonic acid (MSA) in methanol. The influence of mobile-phase density was also assessed by comparing high-density (25 °C, 150 bar) and low-density (50 °C, 103 bar) operating conditions. The Torus 1-aminoanthracene stationary phase combined with CO₂ modified with methanol/acetonitrile (1:1, v/v) containing 0.1% (v/v) MSA, without water, provided the best overall separation of COUM, PMF, and FLAV in citrus peel extracts and commercial essential oils from different Citrus species. In contrast, terpenoids were not retained under any of the conditions investigated. These findings provide practical guidelines for stationary-phase and mobile-phase selection in SFC analysis of citrus secondary metabolites and establish a robust basis for future targeted method optimization and validation, which will be shown in a future publication.