Identification of Iridoids in Edible Honeysuckle Berries (Lonicera caerulea L. var. kamtschatica Sevast.) by UPLC-ESI-qTOF-MS/MS

Iridoid profiles of honeysuckle berry were studied. Compounds were identified by ultra-performance liquid chromatography coupled with electrospray ionization mass spectrometry UPLC-ESI-qTOF-MS/MS in positive and negative ions mode. The MS fragmentation pathways of detected iridoid glycosides were also studied in both modes. In the negative ESI mass spectra, iridoids with a methyl ester or lactone structure have preferentially produced adduct [M + HCOOH − H]− ions. However, protonated ions of molecular fragments, which were released by glycosidic bond cleavage and following fragmentation of aglycone rings, were more usable for iridoid structure analysis. In addition, the neutral losses of H2O, CO, CO2, CH3OH, acetylene, ethenone and cyclopropynone have provided data confirming the presence of functional substituents in the aglycone. Among the 13 iridoids, 11 were identified in honeysuckle berries for the first time: pentosides of loganic acid (two isomers), pentosides of loganin (three isomers), pentosyl sweroside, and additionally 7-epi-loganic acid, 7-epi-loganin, sweroside, secologanin, and secoxyloganin. The five pentoside derivatives of loganic acid and loganin have not been previously detected in the analyzed species. Honeysuckle berries are a source of iridoids with different structures, compounds that are rarely present in fruits.


Introduction
Lonicera caerulea L. var. kamtschatica Sevast. (Caprifoliaceae) is a fruit bush originating from the Far East of Russia (Kamchatka Peninsula). Edible honeysuckle berries are gaining popularity in many European countries, Japan, China, Canada and others. These berries are good fresh fruit for consumption and good raw material for the production of juices, snacks, dry fruits, and jams [1][2][3]. Anthocyanins, flavan-3-ols, flavonols, phenolic acids, and vitamin C are the main compositional groups in honeysuckle berries that exhibit antioxidant, antibacterial, antiviral and anti-inflammatory effects [1,[3][4][5][6][7][8]. Honeysuckle berries' health benefits have been known and used for a long time in traditional medicine in Russia and China.
Iridoids are a large group of secondary metabolites found both in a variety of plant and selected animal species. They belong to the monoterpenes with a cyclopentanopyran skeleton and occur in plant materials naturally as glucoside forms. Iridoids, depending on the chemical structure, exhibit different pharmacological properties, such as antibiotic, anti-inflammatory or hypertensive activities [9]. They have varying degrees of bitterness. Especially known in this respect are secoiridoids, which also

Results and Discussion
The acidified methanolic extract of honeysuckle berries was analyzed by the UPLC-qTOF-MS/MS method. The compounds were identified by their UPLC retention times, elution order, UV-Vis (200-600 nm) and MS spectra, and by comparison with available standards and literature data. The UPLC chromatogram of honeysuckle berry extract, obtained in the UV spectral region (254 nm), is shown in Figure 1. UV-Vis spectra of 13 compounds indicate that they are iridoids. In our previous initial studies, we determined five iridoids (loganic acid, loganin and their three derivatives) from honeysuckle berries [14,22], but we could not completely prove their structures. In this study, 13 iridoids were exactly identified by LC-MS/MS; among these, eight compounds were discovered for the first time.
Iridoids with methyl ester or lactone structure showed a strong tendency for associated product ion formation in negative ionizations; therefore MS data of ions of these and other compounds, in both negative and positive ionization modes, were investigated (Table 1). In the literature, those associated product ions are commonly called adducts [10,11,19]. Depending on the acid applied in the mobile phase, formic acid [11] or acetic acid [19] adducts may appear. Under our analysis conditions, possible associations of loganin, secologanin and sweroside with formic acid connected by hydrogen bonds are shown in Figure 2. However, in the case of secoxyloganin, which possesses a methyl ester and additionally a free carboxyl group, we did not observe a similar phenomenon. The reason for this difference may be mutual influence of the discussed groups resulting in internal weak hydrogen bonds, preventing formation of an association with formic acid.
The results of LC-MS/MS analysis of four compounds (peaks 1, 2, 7, and 10) were compared to the results of the available iridoid standards. The compounds 1 and 2 (t R 3.73 min, and t  Table 1.  Table 1.      These data, UV-Vis absorption spectra and retention times show that compounds 1 and 7 are loganic acid and loganin, respectively. Identification of these compounds was consistent with our previous studies [14,22]. Compounds 2 and 10 displayed the same pseudomolecular and fragment ions in negative and positive mode as compounds 1 and 7, respectively, but they differed in Loganin Secologanin Sweroside Figure 2. Loganin, secologanin, and sweroside with formic acid possible adducts. These data, UV-Vis absorption spectra and retention times show that compounds 1 and 7 are loganic acid and loganin, respectively. Identification of these compounds was consistent with our previous studies [14,22]. Compounds 2 and 10 displayed the same pseudomolecular and fragment ions in negative and positive mode as compounds 1 and 7, respectively, but they differed in abundance of the major fragment ions [ (Figure 3). This indicates that compound 2 is 7-epi-loganic acid. Similar differences were observed in the case of compounds 10 and 7, but only in positive mode. In compound 7 and in loganin standard, higher abundance was detected for the ion at m/z 229.1062 [M − 162 + H] + and in compound 10 for the ion at m/z 211.0961 [M − 162 − 18 + H] + (Figure 4). This indicates that compound 10 is 7-epi-loganin. Ion [M − 162 + H] + is more stable when the -OH group in C-7 is above the molecule plane. Conversely, when the -OH group is below the plane, this ion is less stable, or its stabilization is not possible; therefore the ion appears after loss of water (18 Da). Similar observations were made by Madhusudanan et al. [23] for the epimers of loganin after fragmentation in positive mode.  Figure 4). This indicates that compound 10 is 7-epi-loganin. Ion [M − 162 + H] + is more stable when the -OH group in C-7 is above the molecule plane. Conversely, when the -OH group is below the plane, this ion is less stable, or its stabilization is not possible; therefore the ion appears after loss of water (18 Da). Similar observations were made by Madhusudanan et al. [23] for the epimers of loganin after fragmentation in positive mode.    (Table 1). These ions corresponded to the molecular ion of the loganic acid after loss of 132 Da. The difference between these compounds, in addition to the retention times, is in MS2.   (Table 1). These ions corresponded to the molecular ion of the loganic acid after loss of 132 Da. The difference between these compounds, in addition to the retention times, is in MS2. show that the pentose is not attached to glucose, and does not form with it a disaccharide. Loss of glucose, while preserving the glycosidic bond of the pentose, indicates that the only possible position to attach pentose to aglycone is at the -OH group in C-7 (Tables 2 and 3, Figure 5). This proves that compounds 3 and 4 are loganic acid 7-O-pentoside and 7-epi-loganic acid 7-O-pentoside, respectively. The fragmentation pathways of loganic acid and its derivatives in positive and negative mode are shown in Figure 5. The molecular ion at m/z 509.1876 had two fragmentation pathways: one through preceding loss of water followed by that of the sugars, and conversely the second primarily through loss of sugars followed by that of water. In addition, the neutral losses of CO, CO 2 , acetylene and cyclopropynone have provided data for confirming the presence of functional substituents in the aglycone. All possible secondary structures of monoisotopic ions after negative and positive fragmentations of 7-O-pentoside loganic acid, 7-O-pentoside epi-loganic acid, loganic acid, and epi-loganic acid are shown in Tables 2 and 3. disaccharide. Loss of glucose, while preserving the glycosidic bond of the pentose, indicates that the only possible position to attach pentose to aglycone is at the -OH group in C-7 (Tables 2 and 3, Figure 5). This proves that compounds 3 and 4 are loganic acid 7-O-pentoside and 7-epi-loganic acid 7-O-pentoside, respectively. The fragmentation pathways of loganic acid and its derivatives in positive and negative mode are shown in Figure 5. The molecular ion at m/z 509.1876 had two fragmentation pathways: one through preceding loss of water followed by that of the sugars, and conversely the second primarily through loss of sugars followed by that of water. In addition, the neutral losses of CO, CO2, acetylene and cyclopropynone have provided data for confirming the presence of functional substituents in the aglycone. All possible secondary structures of monoisotopic ions after negative and positive fragmentations of 7-O-pentoside loganic acid, 7-O-pentoside epi-loganic acid, loganic acid, and epi-loganic acid are shown in Tables 2 and 3.                                    Compounds 5, 6, 8, 9, 12, and 13, like loganin (7) and epi-loganin (10), all displayed the formic acid adduct [M + 46 − H] − in negative mode, suggesting that their structures may contain for example the methyl ester functional group or lactone [11]. Similarly, a strong tendency for formation of an acetic adduct was observed by Zhang et al. (2015) for iridoids without a carboxyl group at the mobile phase with acetic acid [19]. Compound 5 (t R 5.68 min) showed the same formic acid adduct as compounds 9 (t R 6.15 min) and 12 (t R 6. According to these data, in compounds 9 and 12, pentose is attached to aglycone at the -OH group in C-7, whereas in compound 5, pentose is attached to glucose producing disaccharide (Tables 4 and 5). Compounds 5 and 9 exhibited a stable fragment ion at m/z 229.1062 [M − 132 − 162 + H] + , whereas compound 12 exhibited a stable ion after loss of water at m/z 211.0961 [M − 132 − 162 − 18 + H] + , which is comparable to compounds 7 and 10 ( Figure 4). Further characteristic protonated fragment ions, which were produced after neutral loss of methanol (32 Da) at m/z 197.0802 and 179.0701 were also detected. Thus, they were pentosyl loganin (5), loganin 7-O-pentoside (9), and 7-epi-loganin 7-O-pentoside (12). The pentose derivatives of loganin, as derivatives of loganic acid, had two fragmentation pathways ( Figure 6). In negative ion mode, iridoids with the structure of methyl ester showed weaker ionization, and delivered only a few fragmentation ions. In the case of loganin and 7-epi-loganin glycosides, relatively high abundance of ions resulted from free aglycone (m/z 227.0748) and from two fragments produced as a result of opening the pyran ring from their basic skeleton (m/z 127.0748 and 101.0252, Table 4).

MS
The most possible structures of monoisotopic ions after negative and positive fragmentations of pentosyl-loganin, loganin 7-O-pentoside, 7-epi-loganin 7-O-pentoside, loganin, and 7-epi-loganin are shown in Tables 4 and 5. The pentose derivatives of both loganic acid and loganin epimers were identified in edible honeysuckle berries for the first time. Additionally, to our knowledge, those compounds were not described in raw materials (plants). In the literature, only apiofuranosyl attached to iridoids, such as sweroside and mussaenosidic acid, has been described [24,25]. Complete elucidation of structures of detected iridoid pentosides, especially glycone (sugar) moieties, will be possible after further studies, including separation of individual compounds from plant material and their spectroscopic analysis. after loss of water at m/z 211.0961 [M − 132 − 162 − 18 + H] + , which is comparable to compounds 7 and 10 ( Figures 4). Further characteristic protonated fragment ions, which were produced after neutral loss of methanol (32 Da) at m/z 197.0802 and 179.0701 were also detected. Thus, they were pentosyl loganin (5), loganin 7-O-pentoside (9), and 7-epi-loganin 7-O-pentoside (12). The pentose derivatives of loganin, as derivatives of loganic acid, had two fragmentation pathways ( Figure 6). In negative ion mode, iridoids with the structure of methyl ester showed weaker ionization, and delivered only a few fragmentation ions. In the case of loganin and 7-epi-loganin glycosides, relatively high abundance of ions resulted from free aglycone (m/z 227.0748) and from two fragments produced as a result of opening the pyran ring from their basic skeleton (m/z 127.0748 and 101.0252, Table 4).    The most possible structures of monoisotopic ions after negative and positive fragmentations of pentosyl-loganin, loganin 7-O-pentoside, 7-epi-loganin 7-O-pentoside, loganin, and 7-epi-loganin are shown in Tables 4 and 5. The pentose derivatives of both loganic acid and loganin epimers were identified in edible honeysuckle berries for the first time. Additionally, to our knowledge, those compounds were not described in raw materials (plants). In the literature, only apiofuranosyl attached to iridoids, such as sweroside and mussaenosidic acid, has been described [24,25]. Complete elucidation of structures of detected iridoid pentosides, especially glycone (sugar) moieties, will be possible after further studies, including separation of individual compounds from plant material and their spectroscopic analysis. Table 4. Characteristics of monoisotopic ion in negative mode and chemical structures of pentosyl loganin, loganin 7-O-pentoside, 7-epi-loganin 7-O-pentoside, loganin, and 7-epi-loganin, and their possible fragments. The most possible structures of monoisotopic ions after negative and positive fragmentations of pentosyl-loganin, loganin 7-O-pentoside, 7-epi-loganin 7-O-pentoside, loganin, and 7-epi-loganin are shown in Tables 4 and 5. The pentose derivatives of both loganic acid and loganin epimers were identified in edible honeysuckle berries for the first time. Additionally, to our knowledge, those compounds were not described in raw materials (plants). In the literature, only apiofuranosyl attached to iridoids, such as sweroside and mussaenosidic acid, has been described [24,25]. Complete elucidation of structures of detected iridoid pentosides, especially glycone (sugar) moieties, will be possible after further studies, including separation of individual compounds from plant material and their spectroscopic analysis. The most possible structures of monoisotopic ions after negative and positive fragmentations of pentosyl-loganin, loganin 7-O-pentoside, 7-epi-loganin 7-O-pentoside, loganin, and 7-epi-loganin are shown in Tables 4 and 5. The pentose derivatives of both loganic acid and loganin epimers were identified in edible honeysuckle berries for the first time. Additionally, to our knowledge, those compounds were not described in raw materials (plants). In the literature, only apiofuranosyl attached to iridoids, such as sweroside and mussaenosidic acid, has been described [24,25]. Complete elucidation of structures of detected iridoid pentosides, especially glycone (sugar) moieties, will be possible after further studies, including separation of individual compounds from plant material and their spectroscopic analysis.

Plant Material
Honeysuckle berries (Lonicera caerulea L. var. kamtschatica Sevast.) of "Atut" cultivars were used for this study. Fruits were collected from the Research Station for Cultivar Testing in Masłowice and hand-harvested at the stage of consumption during the growing season of May 2014. Before analysis, fruits were frozen and stored at −20 • C.

Extraction of Compounds for Qualitative Analysis
Frozen fruits of honeysuckle were homogenized and 5 g of the homogenate was extracted with 50 mL of 80% aqueous methanol (v/v) acidified with 1% HCl by ultrasonication for 20 min. The extract was centrifuged and diluted (re-distilled water with the ratio 1:1, v/v). For UPLC-MS analysis the supernatant was filtered through a Hydrophilic PTFE 0.22 µm membrane (Millex Samplicity Filter, Merck, Darmstadt, Germany) and used for analysis.

Identification of Iridoids by UPLC-qTOF-MS/MS
The method was described previously [27]. Identification of compounds was performed on the Acquity ultra-performance liquid chromatography (UPLC) system, coupled with a quadrupole-time of flight (q-TOF) MS instrument (UPLC/Synapt q-TOF MS, Waters Corp., Milford, MA, USA), with an electrospray ionization (ESI) source. Separation was achieved on the Acquity TM BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm; Waters). The mobile phase was a mixture of 4.5% aq. formic acid v/v (A) and acetonitrile (B). The gradient program was as follows: initial conditions-1% B in A, 12 min-25% B in A, 12.5 min-100% B, 13.5 min-1% B in A. The flow rate was 0.45 mL/min and the injection volume was 5 µL. The column was operated at 30 • C. UV-vis absorption spectra were recorded on-line during UPLC analysis, and the spectral measurements were made in the wavelength range of 200-600 nm, in steps of 2 nm. The major operating parameters for the q-TOF MS were set as follows: capillary voltage 2.0 kV, cone voltage 40 V, cone gas flow of 11 L/h, collision energy 28-30 eV, source temperature 100 • C, desolvation temperature 250 • C, collision gas, argon; desolvation gas (nitrogen) flow rate, 600 L/h; data acquisition range, m/z 100-2000 Da; ionization mode, negative and positive. The data were collected with Mass-Lynx V 4.1 software. The runs were monitored at the wavelength of 254 nm.

Conclusions
In this study, 13 iridoids in the methanolic extract from honeysuckle berries were characterized using an UPLC-ESI-qTOF-MS/MS method in positive and negative mode, which can be complementary. The study shows that in honeysuckle berries there is a range of different iridoids, which are rarely present in edible fruits. The use of both ionization modes enabled the elucidation of the structure of analyzed iridoid glycosides, especially the position of pentose substitution and the aglycone stereochemistry. The presence of fragment ions deriving from the secondary pentosides, after the cleavage of glucose, confirmed additional glycosylation of aglycone at the C-7 position (at the -OH group). For diagnostic purposes, more useful data were derived from the MS + spectra, which come from the fragment structures formed after the successive loss of monosaccharide residues and neutral molecules as water (18 Da), CO (28 Da), CO 2 (44 Da), acetylene (26 Da), methanol (32 Da), ethenone (42 Da) and cyclopropynone (52 Da). It was also observed that the iridoids with the methyl ester structure poorly ionized in the negative ion current, which makes the identification of their epimer pairs particularly difficult. Among the 13 iridoids, 11 were identified for the first time. The identified compounds included pentoside derivatives of loganic acid (two isomers), loganin (three isomers), and sweroside (one compound) and additionally epimeric pairs of loganic acid and loganin, sweroside, secologanin and secoxyloganin. The five pentoside derivatives of loganic acid and loganin have not previously been detected in the analyzed species.