Development of a Simplified Heart-Cutting Two-Dimensional Liquid Chromatography Method for Green Analysis of Four Polyphenols in Aerides Plants

Thai traditional medicine utilizes herbs to treat various ailments, but robust quality control measures are crucial to ensuring their safety and efficacy. Quantifying the active compounds is a key approach for achieving this. Aerides plants, distributed in southeast Asia, are epiphytic orchids containing various bioactive compounds, including alkaloids, coumarins, flavonoids, terpenoids, and polyphenols. Various polyphenols such as coelonin, imbricatin, 6-methoxycoelonin, and gigantol are present in Aerides species and can act as analytical markers for orchid plants. This study proposes an innovative strategy using single heart-cutting 2D-LC to quantify phytochemicals in Aerides plants. In addition, the developed method emphasizes the importance of integrating green analytical chemistry metrics into analytical method development, thereby offering insights for potential enhancements. The method utilized BetaSil (C18, 4.6 × 100 mm, 3.0 μm) and HALO (C18, 4.6 × 100 mm, 5.0 μm) columns for chromatographic separation employing gradient elution, with a mobile phase comprising 0.1% formic acid in water and 0.1% formic acid in acetonitrile. These chromatographic conditions gave excellent peak shape and resolution. This method was validated following the ICH Q2(R1) and AOAC guidelines. The method demonstrated linearity within the range of 0.12–2.40 μg/mL, with a coefficient of determination (r²) exceeding 0.995. The method’s accuracy, indicated by % recovery, varied between 90% and 110%. Its precision, represented by % CV, was less than 7.4%. Furthermore, the environmental sustainability of the proposed single heart-cutting technique in 2D-LC was evaluated by using in-silico tools such as GAPI, AGREE, and BAGI software. This developed method also emphasizes environmental sustainability to reduce adverse environmental impacts.