With the worldwide rapid increasing interest in the use of natural products as dietary supplements, medical remedies and functional foods, it has been accepted that omitting the plant constituents with potential adverse effects was a huge fault of the past. Several countries developed regulations to limit the consumption of such products in the markets. Among these natural products, butterbur (Petasites) has been used for years as herbal supplement for its anti-spasmodic and anti-inflammatory effects. However, its hepatotoxic alkaloid content limits the direct usage. In this study, investigation of sesquiterpenes and pyrrolizidine alkaloids (PAs) together with their N-oxide forms has been conducted with an offline 2D-combination using high-performance liquid chromatography with UV detection (HPLC-UV) and liquid chromatography - multi mode ionization - time-of-flight mass spectrometry (LC-MMI-ToF-MS) for plant screening. The content has been qualitatively investigated to provide information on the constituents of the plant rhizomes extracted using ethanol. Besides the reported hepatotoxic and medically bio-active plant constituents, a strategy has been suggested for estimating the retention order and retention times with respect to calculated logD (distribution coefficient) and hydrophobicity distributions on C18 reversed-phase column when all standard compounds are not available in laboratory. In this sense, the influence of calculated log D and hydrophobicity distributions on retention time has been clarified via available PA and PA-N-oxide standards. The ethanolic extract of Petasites hybridus has been used for examination of the strategy in a real-sample model. Additionally, the advantages of the developed HPLC-UV and LC-MMI-ToF-MS combination have been discussed with respect to the presented results. (C) 2013 Elsevier B.V. All rights reserved.