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  • 1 National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia
  • 2 National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia
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High-performance thin-layer chromatography (HPTLC) is a powerful separation technique which is often overlooked. In this study, we comprehensively assessed the applicability, ease, and performance of HPTLC in combination with densitometry and mass spectrometry (MS) to characterize physalins — relatively polar secondary metabolites from Physalis alkekengi L. HPTLC silica gel plates were evaluated in combination with 14 developing solvents (13 published in the literature). Bonded stationary phases (HPTLC RP-18, RP-18 W, CN F254S) were also tested. Four detection reagents (sulfuric acid, anisaldehyde, 4-dimethylaminocinnamaldehyde (DMACA), and molybdatophosphoric acid) were compared to ascertain which one is the most suitable. For all chromatographic analyses, a commercial standard physalin L and a P. alkekengi L. crude extract were used. In some cases, physalin L standard appeared as two clearly resolved bands on silica plates, but only after derivatization, where sulfuric acid reagent provided the best selectivity and sensitivity. Physalin L standard impurity was found to belong to the physalin family as confirmed by HPTLC–MS/(MS) and nuclear magnetic resonance (NMR) spectroscopy. Compared to high-performance liquid chromatography (HPLC), our HPTLC method showed extremely high sensitivity for standard impurity (ca. 4% determined by NMR) as it was clearly visible on the plate during image analysis after derivatization. Unlike (ultra)-high-performance liquid chromatography ((U)HPLC), HPTLC was also able to separate physalin L standard from its impurity. We show that (HP)TLC is a suitable chromatographic technique for the analysis of physalins and can even surpass the performance of (U)HPLC, namely, due to a wide array of different developing solvents available.

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