Authors:Eva Kranjc, Alen Albreht, Irena Vovk, Vesna Glavnik, and Damjan Makuc
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.
Authors:Urška Jug, Vesna Glavnik, Eva Kranjc, and Irena Vovk
High-performance thin-layer chromatography (HPTLC) and HPTLC–mass spectrometry (MS)/(MSn) methods for analyses of phenolic acids (chlorogenic acid, rosmarinic acid, protocatechuic acid, gallic acid, syringic acid, ellagic acid, trans-cinnamic acid, o-coumaric acid, m-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid) were developed. Separation was performed on HPTLC silica gel plates with and without fluorescent indicator (F254) in a saturated twin-trough chamber using n-hexane–ethyl acetate–formic acid (12:8:2, v/v) as the developing solvent. The developed HPTLC method is also suitable for the preliminary screening of some flavonoids (flavone, apigenin, luteolin, chrysin, quercetin, myricetin, kaempferide, kaempferol, hesperetin, naringenin, pinocembrin), although some interferences of phenolic acids with flavonoids were observed. The effect of pre-development on the HPTLC analysis of phenolic acids on the detection by densitometry and mass spectrometry was also explored. Pre-development of the plates with chloroform–methanol (1:1, v/v) decreased the intensity of secondary front like dark band that appeared at RF 0.7 on unpre-developed plates and enabled densitometric evaluation of phenolic acids at 280 and 330 nm. To eliminate severe spectral background observed during HPTLC–MS analysis, caused by the presence of an acidic modifier in optimized developing solvent, two pre-developments of the plates (1st methanol–formic acid 10:1, v/v and 2nd methanol) were applied. This resulted in a substantial decrease in the intensity of the background signals of sodium formate clusters and considerably improved the analysis of phenolic compounds. The applicability of the developed HPTLC and HPTLC–MS/(MSn) methods was confirmed by analyses of different complex matrix samples, e.g., propolis, roasted coffee, and rose hip crude extracts.