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 (MS n ) 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‒MS n 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‒MS n 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.