In this study, HPTLC silica gel plates were developed with the developing solvent ethyl acetate–formic acid–acetic acid–water (10:1.1:1.1:2.6, V/V). This developing solvent contained formic and acetic acid, which caused intense background signals of sodium formate clusters (with ∆m/z 68) and sodium acetate clusters (with ∆m/z 82) in the MS spectrum (Fig. 1a). The same problem was observed on NH2 plates (modified silica gel) developed with ethyl acetate–dichloromethane–formic acid–acetic acid (10:2.5:1:1.1, V/V), which also contained formic and acetic acid that caused intense background signals of sodium formate clusters (with ∆m/z 68) (Fig. 2a). Based on previous HPTLC–MS and HPTLC–MSn studies [14, 16,17,18,19,20,21,22,23], in which the developing solvent mixtures contained only formic acid, it was predicted that methanol–formic acid (10:1, V/V, in the first pre-development step) would not be sufficient to eliminate sodium formate and sodium acetate clusters. Therefore, the following ratios of methanol–formic acid: 10:1.5, 10:3, 10:5 (V/V) were tested on silica gel and NH2 plates. It was observed that increasing the concentration of formic acid in the pre-developing solvent resulted in improved background with less pronounced ions of acid clusters. Acid clusters were successfully removed from the layer of silica gel plates by pre-development with methanol–formic acid 10:3 (V/V) (Fig. 1b). In the case of NH2 plates, acid clusters were observed in the MS spectrum even after pre-development with methanol–formic acid 10:5 (V/V) (Fig. 2b), but their intensity had dropped significantly. Based on previous studies [14, 16,17,18,19,20,21,22,23], the second pre-development of silica gel and NH2 plates was performed with methanol that was intended for use as an elution solvent in HPTLC–MSn experiments. The developed pre-development procedures enabled HPTLC–MSn analyses of chestnut bee pollen samples on silica gel and NH2 plates without any problem related to ion suppression of the desired analytes.

MS spectra of backgrounds obtained at RF = 0.71 from not pre-developed (a) and twofold pre-developed (b; first: methanol–formic acid (10:3, V/V); second: methanol) HPTLC silica gel plates developed with ethyl acetate–formic acid–acetic acid–water (10:1.1:1.1:2.6, V/V). Sets of background signals of sodium acetate clusters with Δm/z 82 are in bold, with m/z numbers in black, blue and green

MS spectra of backgrounds obtained at RF = 0.71 from not pre-developed (a) and twofold pre-developed (b; first: methanol–formic acid (10:5, V/V); second: methanol) HPTLC NH2 plates developed with ethyl acetate–dichloromethane–formic acid–acetic acid (10:2.5:1:1.1, V/V). Sets of background signals of sodium formate clusters with Δm/z 68

Possible influence of the pre-developing solvents on the separation on silica gel plates and NH2 plates was examined before HPTLC–MSn analyses of STSs. This examination was performed by HPTLC analyses of STS–SI on silica gel (Fig. 3a–d) and NH2 (Fig. 3e–h) plates that were not pre-developed (Fig. 3a, b, e, f) or were twofold pre-developed (Fig. 3c, d, g, h). Silica gel plates were pre-developed with methanol–formic acid (10:3, V/V) and methanol, while NH2 plates were pre-developed with methanol–formic acid (10:5, V/V) and methanol. Silica gel plates were developed with ethyl acetate–formic acid–acetic acid–water (10:1.1:1.1:2.6, V/V) (Fig. 3a–d) and NH2 plates with ethyl acetate–dichloromethane–formic acid–acetic acid (10:2.5:1:1.1, V/V) (Fig. 3e–h). Both plates were derivatised with NP reagent while enhancement and stabilisation of the fluorescent chromatographic zones were achieved with PEG reagent. Pre-development influenced the separation resulting in (1) a different number of the separated chromatographic zones and (2) different resolutions (Fig. 3). The effect of plate pre-development was more pronounced on NH2 than on silica gel plates. A lower number of blue fluorescent and green fluorescent zones were observed on twofold pre-developed (Fig. 3c, d) than on not pre-developed (Fig. 3a, b) HPTLC silica gel plates. A lower number of green fluorescent zones and a higher number of blue fluorescent zones were observed on twofold pre-developed (Fig. 3g, h) than on not pre-developed (Fig. 3e, f) HPTLC NH2 plates. Changes between RF values for particular chromatographic zones on not pre-developed (Fig. 3e, f) and on twofold pre-developed (Fig. 3g, h) NH2 plates were much larger than changes between RF values on not pre-developed (Fig. 3a, b) and on twofold pre-developed (Fig. 3c, d) silica gel plates.

Chromatograms of STS–SI (100 mg/mL, 1 µL) on not pre-developed (a, b, e, f) and twofold pre-developed (c, d, g, h) HPTLC silica gel (a–d) and NH2 (e–h) plates developed with ethyl acetate–formic acid–acetic acid–water (10:1.1:1.1:2.6, V/V) (a–d) and ethyl acetate–dichloromethane—formic acid–acetic acid (10:2.5:1:1.1, V/V) (e–h), respectively. Documentation was performed at 366 nm after derivatisation with NP reagent and after enhancement and stabilisation of fluorescent zones with PEG reagent

Non-targeted HPTLC–MSn analyses of chestnut bee pollen test solutions from Slovenia (STS–SI) and Türkiye (STS–TR) were performed on twofold pre-developed silica gel plates. Compounds were tentatively identified by comparing fragmentation patterns of MS2 (Fig. 4, Table 1), MS3, MS4 and MS5 (Table 1) spectra with those published in the literature. The molecular ion at m/z 582 [M–H]– with MS2 fragments at m/z 462, m/z 342 and m/z 436 with neutral losses of 120 u, 240 u and 146 u, respectively, which corresponded to coumaroyl moiety at positions N1, N5 and N10 [29], was assigned as N1,N5,N10-tricoumaroylspermidine (Fig. 5) based on the literature [11, 13, 29, 30]. The molecular ion at m/z 598 was previously assigned as [M−H]− of N1,N5-dicoumaroyl-N10-caffeoylspermidine in the extracts of chestnut bee pollen [11] and chestnut bee bread [30] or as N1,N10-dicoumaroyl-N5-caffeoylspermidine in the extracts of chestnut bee pollen [11]. Fragmentation of the molecular ion at m/z 598 can yield base ions at 478 m/z [11] or at 462 m/z [11, 30]. In this study only a base ion at 478 m/z was detected. Similarly, as in other studies [11, 30], a base ion at 478 m/z was also observed in MS2 experiments after fragmentation of spermidines bonding caffeoyl moiety at position N5. Three other spermidine derivatives with caffeoyl moiety at position N5 also had similar fragmentation patterns in MS4 and MS5 experiments as the molecular ion at 598 m/z. Therefore, the molecular ion at 598 m/z was assigned as [M−H]− of N1,N10-dicoumaroyl-N5-caffeoylspermidine [11, 13] (Fig. 5).

HPTLC–MS analyses of STS–SI (a, b) and STS–TR (c, d) on twofold pre-developed [first methanol–formic acid, 10:3 (V/V), second methanol] HPTLC silica gel plates developed with ethyl acetate–formic acid–acetic acid–water (10:1.1:1.1:2.6, V/V) and documented at 366 nm (a, c) and white light (b, d) after derivatisation of the left-most part with NP and PEG reagents. MS spectra with bolded signals of [M−H]−: N1,N5,N10-tricoumaroylspermidine (m/z 582), N1,N10-dicoumaroyl-N5-caffeoylspermidine (m/z 598), N1,N5,N10-tricaffeoylspermidine ([M−H]− at m/z 630), N1-coumaroyl-N5,N10-dicaffeoylspermidine (m/z 614), N1-feruloyl-N5,N10-dicaffeoylspermidine (m/z 644), isorhamnetin-acetylhexoside (m/z 519), isorhamnetin-hexoside (m/z 477), isorhamnetin-(pentosyl)hexoside (m/z 609), isorhamnetin-(hexosyl)deoxyhexoside (m/z 623), isorhamnetin-(hexosyl)hexoside (m/z 639) gluconic acid (m/z 195), isorhamnetin-(pentosyl-deoxyhexosyl)hexoside (m/z 755), caffeic acid-hexoside ([M−2H2O]− at m/z 377)

The chemical structures of phenylamides identified in chestnut bee pollen samples

Fragmentation of the molecular ion at m/z 614 [M–H]– resulted in the ions at m/z 478, 452 and 494, with neutral losses of 136 u, 162 u and 120 u, respectively. Fragmentation ions at m/z 478 and 452 corresponded to caffeoyl residue at position N10 and N5, respectively [29], while fragmentation ions at m/z 494 corresponded to p-coumaroyl residue at position N1. Therefore, the molecular ion at m/z 614 [M–H]– was assigned as N1-coumaroyl-N5,N10-dicaffeoylspermidine (Fig. 5) based on the literature [11, 13, 30]. The molecular ion at m/z 644 [M–H]– fragmented in ions at m/z 508 and 482 with neutral losses of 136 u and 162 u, respectively, which corresponded to caffeoyl residue at position N10 and N5, respectively [29].

An additional MS2 ion at m/z 494 with neutral loss of 150 u suggested the feruloyl residue at position N1 [29]. Therefore, the molecular ion at m/z 644 [M−H]− was assigned as [M−H]− of N1-feruloyl-N5,N10-dicaffeoylspermidine [11, 30, 31] (Fig. 5). The molecular ion at m/z 630 [M−H]− with MS2 fragments at m/z 468, m/z 494 and m/z 358 with neutral losses of 162 u, 136 u and 272 u, respectively, which corresponded to caffeoyl moiety at positions N1, N5 and N10 [29], was assigned as N1,N5,N10-tricoumaroylspermidine [11, 29, 30] (Fig. 5).

Out of the group of flavonols, only glycosides of isorhamnetin (with signals at m/z 314/315 [M−2H]−) [12] were identified (Fig. 4, Table 1). The molecular ion at m/z 477 with neutral loss of 162 u, which corresponded to hexose residue, was assigned as [M−H]− of isorhamnetin hexoside [11, 30]. Fragmentation of the molecular ion at m/z 519 resulted in signals at m/z 459, 315 and 477. Neutral loss of 204 u of the molecular ion at m/z 519 corresponded to hexosyl and acetyl moieties, while the signal at m/z 477 corresponded to isorhamnetin hexoside. Therefore, the signal at m/z 519 was assigned as [M−H]− of isorhamnetin acetylhexoside [32]. The molecular ion at m/z 639 [M–H]– [33] with neutral loss of 324 u corresponded to dimer of hexoses, and was tentatively identified as isorhamnetin-(hexosyl)hexoside. The molecular ion at m/z 609 with neutral loss of 294 u, which corresponded to pentosylhexosyl moiety, was assigned as [M−H]− of isorhamnetin-(pentosyl)hexoside [11, 30, 34]. The molecular ion at m/z 623 [M−H]− fragmented into ions at m/z 314/315 (corresponding to isorhamnetin) with neutral loss of 308 u, which corresponded to hexosylpentosyl [12] or coumaroylhexosyl moiety [35]. Additional ions obtained in MS2 spectra were also at m/z 459 and 477, which corresponded to the neutral loss of hexosyl and deoxyhexosyl residues, respectively. Therefore, the molecular ion at m/z 623 was assigned as [M−H]− of isorhamnetin-(deoxyhexosyl)hexoside [11, 30]. The molecular ion at m/z 755 [M−H]− fragmented into the ions at m/z 314/315 (corresponding to isorhamnetin) with neutral loss of 440 u. Two characteristic ions at m/z 609 and 623 in the MS2 spectrum corresponded to isorhamnetin-(pentosyl)-hexoside and isorhamnetin-(deoxyhexosyl)hexoside; therefore, the molecular ion at m/z 755 [M−H]− can be isorhamnetin-(pentosyl-deoxyhexosyl)hexoside.

After the MS2 fragmentation, the molecular ion at m/z 377 [M−H]− lost two molecules of water. The base ion obtained at m/z 341 further fragmented with a neutral loss of 162 u (corresponding to hexosyl moiety) [36] into a base ion at m/z 179, characteristic for caffeic acid [37]. Therefore, the molecular ion at m/z 377 was tentatively assigned as [M−2H2O]− of caffeic acid-hexoside. The fragmentation pattern of the molecular ion at m/z 195 [M−H]− was similar as previously reported in honey [38] and rose hip [16]; therefore, the signal at m/z 195 was tentatively assigned as [M−H]− of gluconic acid.

The applicability of the pre-developed NH2 plates for on-line HPTLC–MSn analyses of bee pollen samples was examined by the analyses of STS–SI. It was observed that the same phenylamides and glycosylated flavonols were identified in STS–SI using NH2 and silica gel plates. Differences between NH2 and silica gel plates were found in some of the unidentified compounds (Table 2). Some of those compounds (at m/z 114, 292, 315) were only detected when using NH2 plates, and other compounds (at m/z 165, 188, 279, 355, 439, 461, 745, 833) only when using silica gel plates. Comparing both types of the plates, more interferences were observed between compounds detected on NH2 plates (Tables 1, 2).

Five phenylamides, six glycosylated flavonols and one organic acid were identified in the STSs of both chestnut bee pollen samples (Fig. 4, Table 1). One glycosylated phenolic acid was detected only in the STS–TR (Fig. 4, Table 1). Isorhamnetin hexoside, isorhamnetin-(pentosyl)hexoside, isorhamnetin-(hexosyl)deoxyhexoside and all phenolspermidines identified in this study (Fig. 4, Table 1) were recently also found in chestnut bee pollen samples [11]. Isorhamnetin-(hexosyl)hexoside (Fig. 4, Table 1) was previously identified in multi-floral bee pollen samples [12] and in honey collected by sugarbag bee (Tetragonula carbonaria Smith) [33]. Isorhamnetin-acetylhexoside (Fig. 4, Table 1) was previously identified in chestnut flowers [