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.