Volatile Organic Compound Profiling of Traditional Multi-Herbal Prescriptions for Chemical Differentiation and Ethnopharmacological Insights

Traditional herbal prescriptions composed of multiple botanicals remain central to ethnopharmacological practice; however, their chemical basis and classification remain poorly understood. Non-volatile compound analyses of herbal medicines are well established, but comparative studies focusing on volatile organic compounds (VOCs) across multi-herbal prescriptions are scarce. To enhance the chemical understanding of traditional formulations and clarify prescription-level characteristics, this study applied headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME–GC–MS) to characterize VOC-based chemical signatures in 30 prescriptions composed of 76 herbal ingredients. Multivariate analyses such as principal component analysis, partial least squares discriminant analysis (PLS-DA), and orthogonal PLS-DA (OPLS-DA) enabled systematic differentiation of various prescriptions and identified 25 discriminant VOCs, 9 of which were common among multiple therapeutic categories. These shared compounds, such as 5-hydroxymethylfurfural (5-HMF) and 4H-pyran-4-one derivatives, reflect recurrent chemical patterns associated with broad-spectrum applications, whereas category-specific volatiles (including isopsoralen, senkyunolide, and fenipentol) delineated therapeutic boundaries, even among prescriptions with overlapping botanicals. Capturing both shared and distinct volatile signatures clarified ambiguous boundaries between categories such as cold, inflammation, or diabetes versus kidney disorder prescriptions, thereby linking chemical patterns with ethnopharmacological indications. Together, these findings highlight VOC profiling as a valuable diagnostic and interpretive tool that bridges traditional categorization systems with modern chemical analysis, offering a robust framework for future pharmacological and mechanistic investigations. Such an approach not only substantiates traditional categorization but also provides a practical basis for quality control and pharmacological evaluation of multi-herbal formulations.