Synthesis-Supported GC–MS/Retention-Index Library of Hydroxythymol Diesters for Analysis of Botanical Matrices

Reliable identification of volatile constituents in complex natural matrices by gas chromatography–mass spectrometry (GC–MS) remains challenging when closely related regioisomeric and acyl-homologous compounds display similar mass spectra and inconsistent retention-index (RI) data. This study aimed to develop a synthetic GC–MS/RI reference library of diacylated hydroxythymol derivatives and apply it to the characterization of thymol-derived constituents in the root essential oil of Doronicum columnae (Asteraceae). A total of 49 hydroxythymol diesters bearing isobutyryl, 2-methylbutyryl, and isovaleryl residues were included in the library, covering all accessible regioisomeric combinations within the selected set of six positions of additional oxygenation/acylation on the thymol scaffold (C-2, C-5, C-6, C-7, C-8, and C-9); 44 of these compounds have not previously been described. RI values were determined under unified conditions on non-polar DB-5MS and polar HP-Innowax columns, and the compounds were characterized by GC–MS and, when applicable, nuclear magnetic resonance (NMR) spectroscopy. The dataset revealed clear structure–retention relationships, with positional isomerism exerting a stronger influence on retention than acyl-residue variation. Correlation analysis and a semi-empirical model ester subtraction approach revealed regular structure–retention trends within defined regioisomeric series. Application of the synthetic reference library, supported by dual-column RI data, mass spectral comparison, and co-injection, enabled the identification of 16 diacylated hydroxythymol derivatives in the more polar fraction of D. columnae root essential oil, 12 of which are reported here for the first time as natural constituents. The developed approach provides an analytical framework for distinguishing thymol-derived esters in complex botanical matrices.