High-performance thin-layer chromatography‒multi-stage mass spectrometry methods for analyses of bee pollen botanically originating from sweet chestnut (Castanea sativa Mill.)

High-performance thin-layer chromatographic (HPTLC) silica gel and amino plates in combination with developing solvents containing formic and acetic acid were examined for HPTLC‒multi-stage mass spectrometry (MSⁿ) analyses of chestnut bee pollen samples from Slovenia and Türkiye. Ethyl acetate‒formic acid‒acetic acid‒water (10:1.1:1.1:2.6, V/V) and ethyl acetate‒dichloromethane‒formic acid‒acetic acid (10:2.5:1:1.1, V/V) were used for development of silica gel and amino plates, respectively. Twofold pre-development was required for the developed HPTLC‒MSⁿ methods. The first pre-development was performed with methanol‒formic acid (10:3, V/V) for silica gel plates and methanol‒formic acid (10:5, V/V) for amino plates. The second pre-development with methanol was equal for both types of the plates. Using the developed HPTLC‒MSⁿ methods, five phenylamides (spermidines), six isorhamnetin glycosides and gluconic acid were identified in both chestnut bee pollen samples. Glycosylated phenolic acid (caffeic acid-hexoside) was detected only in the Turkish bee pollen sample. To the best of our knowledge, this is the first report on isorhamnetin-(hexosyl)hexoside, isorhamnetin-acetylhexoside, isorhamnetin-(pentosyl-deoxyhexosyl)hexoside and caffeic acid-hexoside in chestnut bee pollen. This is also the first report on isorhamnetin-(pentosyl-deoxyhexosyl)hexoside and caffeic acid-hexoside in any bee products.