The Ultra High-Performance Liquid Chromatograph (UHPLC) Thermo Scientific (Germering, Germany) Dionex UltiMate 3000 RSLC consisted of SRD-3600 solvent degasser, HPG-3400RS high-pressure binary pump, WPS-3000TRS autosampler, and TCC-3000RS thermostatic column compartment. The UHPLC effluent was online connected to a Thermo Scientific (Bremen, Germany) Q Exactive Plus Orbitrap mass spectrometer equipped with a heated electrospray ionization (HESI-II) probe. All solvents were of HPLC or LC/MS grade and were purchased from Fisher Scientific (Pittsburgh, USA). Hyperoside, chlorogenic acid, and mangiferin (≥ 97%, HPLC) were purchased from Sigma-Aldrich (Taufkirchen, Germany) or TCI Deutschland GmbH (Eschborn, Germany).

The above-ground parts of Hypericum aucheri Jaub. et Spach were gathered from a wild population near Momchilgrad (Kardzali District, Bulgaria) in July 2021. The botanical identity was confirmed by P. Nedialkov. A voucher specimen taken from the population (SOM-Co-1344) was deposited in the herbarium of the Institute of Biodiversity and Ecosystem Research (IBER) at the Bulgarian Academy of Sciences (BAS).

The powdered air-dried aerial parts of H. aucheri (250.0 mg) were sonicated at room temperature with ca 20 mL 70% EtOH for 30 min and then were diluted to 25 mL with the same solvent. The resulting extract was centrifuged at 15000 rpm for 15 min. One mL aliquot of the supernatant was evaporated to dryness under N₂, suspended in 500 µL 1% formic acid in water, and further purified by solid-phase extraction over Phenomenex (Torrance, USA) Strata C18-E (55 µm, 70 Å, 200 mg, 3 mL) cartridge. The sorbent was first washed with H₂O (5 × 500 µL), then eluted with 35% MeCN (10 × 500 µL) in 10.0 mL volumetric flask and diluted to the nominal volume with the same solvent. Subsequently, 1 mL of solution was diluted to 25 mL 35% MeCN. The latter solution was used for qualitative and quantitative analysis of phenolic compounds by UHPLC–ESI-MS/MS.

UHPLC separations were performed on a Nouryon (Göteborg, Sweden) Kromasil C18 column (2.1×100 mm, 1.8 μm) coupled with a precolumn Phenomenex SecurityGuard ULTRA UHPLC EVO C18 at 40 °C. Each chromatographic run was carried out with a binary mobile phase consisting of water containing 0.1% (v/v) formic acid (A) and acetonitrile also with 0.1% (v/v) formic acid (B). A gradient program was used as follows: 0–0.5 min, 5% B; 0.5–3 min, from 5 to 8% B; 3–12 min, from 8 to 15% B; 12–15 min, from 15 to 25% B; 15–24 min, from 25 to 55% B, 24–25 min, from 55 to 95% B, 25–27 min, kept 95% B. Before each run the column was equilibrated for 4.5 min with the initial conditions. The flow rate was 0.3 mL.min⁻¹ and the sample injection volume was 2 µL.

The experiments were run in negative mode. The tune parameters of the HESI source were as follows: spray voltage −2.5kV; capillary temperature – 320 °C; sheath gas – 38 arbitrary units (a.u.); auxiliary gas – 12 a.u.; probe heater temperature – 320 °C; S-Lens RF Level – 50. The detection and identification of the metabolites were done using a full scan – data-dependent MS/MS (Top 5) experiment. The full scan parameters: resolution, automatic gain control (AGC) target, max. inject time (IT), and mass range were set to 70000 FWHM, 3×10⁶, 100 ms, and m/z 150 to 1000, respectively. The data-dependent MS/MS (ddMS²) parameters were as follows: resolution 17500 FWHM, AGC target 1×10⁵, max. IT 50 ms, TopN 5, isolation window m/z 2.0, stepped NCE 20, 40, 70. The quantitation of phytochemicals in Hypericum aucheri was done using full MS/SIM scan experiments. The method parameters were set as follows: resolution 70000 FWHM, AGC target 3×10⁶, max IT 200 ms, mass range m/z 200 to 1000. The selected quantification ions for chlorogenic acid, mangiferin, and hyperoside were at m/z 353.0867, 421.0765, and 463.0871, respectively. The mass tolerance was 20 ppm. The data were acquired and processed with Thermo Fisher Scientific Xcalibur ver. 4.1 or FreeStyle ver. 1.8 SP2 QF1.

The quantification of phenolic compounds was carried out using the external standard method. The amount of 44 detected phenolic compounds was calculated relative to external standards of chlorogenic acid, mangiferin, and hyperoside. Each of the external standards (about 5 mg) was dissolved in 20 mL 70 vol. % EtOH (primary solutions). The stock standard solution of the external standards was prepared by combining the aliquots (1 mL) of each primary solution and dilution to 50 mL with 70 vol. % EtOH. The working standard solutions of appropriate concentration were prepared by diluting the stock standard solution with 70 vol. % EtOH. External standard calibrations were established on six data points covering the concentration range of 16.56–530.00 ng/mL for chlorogenic acid, 16.72–535.00 ng/mL for mangiferin, and 17.97–575.00 ng/mL for hyperoside. The procedure and the parameters of validation were previously described in detail elsewhere (Kokanova-Nedialkova and Nedialkov 2021).

In this study, ultra-high performance liquid chromatography–high-resolution mass spectrometry (UHPLC-HRMS) was used to detect the polar phenolic compounds in the EtOH extracts from the aerial parts of Hypericum aucheri Jaub. et Spach. The efficiency of the extraction procedure and optimization of the chromatographic conditions were as given in the literature (Kokanova-Nedialkova and Nedialkov 2021). Briefly, MeOH, EtOH, i-PrOH, and MeCN as well as their mixtures with water were employed as solvents. The best results were obtained with 70% EtOH. Three chromatographic columns, namely Kromasil C18 column (2.1×100 mm, 1.8 μm), Kromasil Eternity XT C18 column (2.1×100 mm, 1.8 μm), and Phenomenex Kinetex EVO C18 (100×2.1 mm, 1.7 μm) were tested for separation of polar phenolic compounds from the titled plant. The former column showed the best results and was selected for developing the method. Quantitative determination of phenolic compound contents in the aerial parts of H. aucheri was performed by the method of the external standard. Chlorogenic acid, mangiferin, and hyperoside were selected as standards for the calculation of the amount of the polar phenolic metabolites. The separation of the standard is given in Fig. 1.

The calibration curves were linear over the concentration range of 16.56–530 ng/mL, 16.72–535 ng/mL, and 17.97–575 ng/mL for chlorogenic acid, mangiferin, and hyperoside, respectively. All calibration curves showed very good linear regressions and the correlation coefficients were R² > 0.999 (Table 1). The method showed that LODs and LOQs were 0.75 ng/mL and 2.26 ng/mL for chlorogenic acid, 1.11 ng/mL and 3.35 ng/mL for mangiferin, and 0.56 ng/mL and 1.70 ng/mL for hyperoside, respectively (Table 1).

The accuracy of the method was checked by the addition of a standard solution mixture at three concentrations (53.0, 106.00, and 159.00 ng/mL for chlorogenic acid; 65.50, 131.00, and 196.50 ng/mL for mangiferin; 57.50, 115.00 and 172.50 ng/mL for hyperoside) close to that expected in the real plant samples. Blank samples from the same un-spiked plant extract were analyzed at the same time as the spiked samples and the measured values were subtracted. Furthermore, the related compounds showed overall recoveries ranging from 96.29% to 103.42% with RSD ranging from 0.24% to 2.18%. The method has acceptable accuracy evidenced by the good correlation of the spiked and determined concentrations (Table 2).

The precision of the retention times was estimated by analyzing the repeated runs during a single day and on three different days, respectively. The RSDs of retention times of the standards were ≤ 0.18 % for intra‐day and ≤ 0.08 % for inter‐day evaluations, respectively (Table 3). For intra‐day and inter‐day precision tests the evaluated analytes exhibited overall recoveries ranging from 97.40% to 100.46% with RSDs from 0.30% to 1.12%. (Table 3).

The developed UHPLC-HRMS method was applied for the quantification of the polar phenolic compounds detected in the EtOH extract from the aerial parts of H. aucheri.

The identified metabolites and their quantities were listed in Table 4 while the chromatogram of the EtOH extract was given in Fig. 2.

Hydroxybenzoic acids derivatives

The deprotonated molecule [M−H]⁻ of compound 1 appeared at m/z 329.0880 in the full MS scans. Its MS/MS spectrum showed a product ion at m/z 167.03 resulting from a neutral loss of 162 Da, indicative of the presence of an O-linked hexose. Subsequently, the decay of the later product ion produced fragments with m/z 123.04, 152.01, and 108.02 that corresponded to a loss of carboxyl (44 Da), methyl (15 Da), and both carboxyl and methyl (59 Da) groups, respectively. This fragmentation was specific to vanillic acid (Barragán-Zarate et al. 2022). Thus, compound 1 was tentatively identified as 1-O-vanilloyl-β-D-glucose and was reported for the first time to occur in H. aucheri. The quantity of this metabolite, calculated as chlorogenic acid, was found to be relatively small (139.89 µg/g D.W.).

Benzophenones

The deprotonated molecule [M−H]⁻ of compound 2 appeared at m/z 423.0935. Its MS/MS spectrum showed a base peak ion at m/z 261.04 indicating a loss of a hexose moiety. The fragment at m/z 151.00 undergoes neutral loss of CO₂ producing an ion at m/z 107.01 that is in conformance to the postulated fragmentation pathway of maclurin (Fig. 3) (Berardini et al. 2004). Thus, compound 2 was tentatively identified as a maclurin-O-hexoside (Kaya et al. 2011; Xu et al. 2011). Compound 37 showed a deprotonated molecule [M−H]⁻ at m/z 527.1189. Its MS/MS spectrum shared a similar fragmentation pattern with that of 2. In addition, it showed product ion at m/z 405.08 indicating a neutral loss of 122 Da, which is typical for esterified benzoic acid. Thus, compound 37 was tentatively identified as a maclurin-O-(benzoyl)-hexoside. The quantities of metabolites 2 and 37, calculated as hyperoside, were 3.36 and 2.27 mg/g D.W., respectively. This class of phenolic compounds was reported here for the first time for the title plant. Furthermore, compound 2 was previously identified only in species of the genus Gentiana and Garcinia (Marinov et al. 2023), while compound 37 was tentatively determined as a new natural product.

Hydroxycinnamic acids derivatives

The deprotonated molecules [M−H]⁻ of compounds 3, 8, and 11 appeared at m/z ranging from 353.0875 to 353.0879, while 6, 12 and 19 at m/z ranging from 337.0934 to 337.0935. The MS/MS spectrum of 3 produced a base peak at m/z 191.06 and secondary peaks at m/z 179.03 and 135.04, while 6 showed a base peak at m/z 163.04 and secondary peaks at m/z 119.05 and 191.06. The product ion at m/z 191.06 was indicative of the presence of quinic acid, while the other fragments in MS/MS spectra of 3 and 6 were due to the presence of hydroxycinnamic acid moiety. The MS/MS spectra of compounds 8, 11, 12, and 19 showed a base peak at m/z 191.06. Metabolites 12 and 19 produced ions with low intensity at m/z 163.04 and 119.05, while 8 and 11 at m/z 179.03 and 135.04. The compounds 3, 6, 8, 11, 12, and 19 showed similar fragmentation patterns typical for hydroxycinnamoyl quinic acids (Clifford et al. 2003). Thus, compounds 3 and 6 were tentatively identified as 3-O-caffeoylquinic acid and 3-O-p-coumaroylquinic acid, respectively. Both metabolites 12 and 19 correspond to 5-O-p-coumaroylquinic acid while 8 and 11 were identified as 5-O-p-caffeoylquinic acid. There was a substantial difference in retention times of compounds 8, 11, 12, and 19 which were 5.79, 8.19, 8.26, and 10.94 min., respectively that phenomenon corroborates with the recently reported data (Ncube et al. 2014). Thus, the metabolites 8, 11, 12, and 19 were tentatively identified as 5-O-trans-p-caffeoylquinic acid, 5-O-cis-p-caffeoylquinic acid, 5-O-trans-p-coumaroylquinic acid, and 5-O-cis-p-coumaroylquinic acid, respectively. Furthermore, compound 17 showed a deprotonated molecule at m/z 367.1039. Its MS/MS spectrum showed a similar fragmentation pattern as compounds 3, 6, 8, 11, 12, and 19 but differed in the presence of a product ion at m/z 193.05 which was typical for ferulic acid derivatives. According to literature data, the metabolite 17 was tentatively identified as 5-O-feruloylquinic acid (Clifford et al. 2003; Masike et al. 2017). The deprotonated molecules [M−H]⁻ of compounds 5 and 10 appeared at m/z 355.1037–355.1038. In the MS/MS spectra of both metabolites, the product ion at m/z 193.05 corresponded to 162 Da neutral loss which is typical for O-hexoses. In addition, the MS/MS spectrum of 5 showed fragments at m/z 134.04, 149.06, and 178.03 typical for ferulic acid (Sinosaki et al. 2020), while in those of 10 appeared product ions at m/z 217.05, 193.05, 175.04, and 160.02. Thus, metabolites 5 and 10 were tentatively identified as ferulic acid 4-O-hexoside and 1-O-feruloyl-β-glucose (Umehara et al. 2018), respectively. The total amount of hydroxycinnamic acid derivatives in Hypericum aucheri was 13.55 mg/g D.W. and was dominated by chlorogenic acid (>85% of the mixture). Excluding chlorogenic acid 8, all other hydroxycinnamic acid derivatives were reported here for the first time for the title plant.

Flavan-3-ols (catechins) and dimers

The MS/MS spectra of the deprotonated molecules [M−H]⁻ (m/z ranging from 289.0718 to 289.0720) of 7 and 14 showed product ions m/z 271.06, 179.03, 109.03, 151.02, 137.02, 125.02, 245.08, and 203.07. The loss of a water molecule (18 Da), catechol group (110 Da) and ring A and C (180 Da) yielded fragment ions at m/z 271.06, 179.03, and 109.03, respectively. The product ions at m/z 151.02 and 137.02 resulted from RDA reactions, while the main fragment from heterocyclic ring fusion had m/z 125.02. The loss of the -CH₂-CHOH- group from the benzofuran skeleton led to the formation of a fragment at m/z 245.08, which further decayed to an ion at m/z 203.07. Thus, metabolites 7 and 14 were tentatively identified as catechin and epicatechin (Zeeb et al. 2000; Verardo et al. 2008; Yuzuak et al. 2018; Mutungi et al. 2021). The deprotonated molecule [M−H]⁻ of 4 at m/z 305.0667 showed a similar fragmentation pattern, but in MS/MS produced a different fragment ion with m/z 287.06 corresponding to a water molecule loss (18 Da), suggesting that the compound is a hydroxy derivative of 7 and 14. According to literature data 4 was tentatively identified as (+)-gallocatechin (Miketova et al. 2000). The deprotonated molecule [M−H]⁻ of 9 appeared at m/z 577.1364. Its MS/MS spectrum showed characteristic product ions at m/z 125.02, 425.09, 407.08, 289.07, 245.08, 161.02 and 137.02. The fragment at m/z 425.09 was a product of the RDA reaction underwent subsequent water loss to give an ion with m/z 407.07. The product ion at m/z 289.07 corresponded to the catechin core derived from the quinone methide cleavage of the inter-flavonoid bond (Rue et al. 2018). Subsequently, the later ion underwent further fragmentations to produce fragments with m/z 245.08, 137.02, and 125.02 which corresponded to neutral loss of 44 Da, 152 Da, and 164 Da, respectively. Besides, the loss of 84 Da from the former ion produced a fragment at m/z 161. According to a recent data report compound 9 was tentatively identified as procyanidin B2 (Mutungi et al. 2021). The amount of the individual catechins was calculated as hyperoside. The total content of flavan-3-ols in the aerial parts of H. aucheri was estimated to be 1.24 mg/g D.W. dominated by epicatechin (59.50 %) and catechin (21.85%). Flavan-3-ols derivatives were reported here for the first time to occur in the title plant.

Xanthones

The deprotonated molecules [M–H]⁻ of metabolites, 13, 15, 16, and 24 appeared at m/z 421.0776, 437.0722, 421.0771, and 405.0825, respectively. In the MS/MS spectrum the isobaric compounds 13 and 16 produced fragments at m/z 403.07, 259.03, 271.03, 301.04, and 331.05 while 24 showed product ions at m/z 387.07, 243.03, 255.03, 285.04, and 315.05. In addition, the MS/MS spectrum of the metabolite 15 showed product ions at m/z 419.06, 275.02, 287.02, 317.03, and 347.04. The above fragmentation pattern (Fig. 4) was characteristic of xanthone-C-glycosides resulted from water loss, C−C glycosidic bond split off, and ^0,1X⁻, ^0,2X⁻, ^0,3X⁻ cleavages of sugar moiety. The comparison of the MS/MS spectra with literature data, the compounds 13, 16, 15, and 24 were tentatively identified as mangiferin, isomangiferin (Trevisan et al. 2016), pentahydroxyxanthone-C-hexoside and lancerin (Ling et al. 2013), respectively. Compounds 18 and 31 showed deprotonated molecules [M−H]⁻ at m/z 421.0768–421.0775. Their MS/MS spectra revealed the presence of a base peak at m/z 259.02, corresponding to the loss of the hexose as a sugar moiety, which is typical of O-glycosides. Subsequently, the later ion gave a fragment at m/z 215.03 indicative of CO₂ loss followed by a cleavage of CO resulted in a fragment at m/z 187.04, which according to the literature data is typical of norathyriol (Heinrich et al. 2017; Islam et al. 2020). Thus, compounds 18 and 31 were tentatively identified as norathyriol-O-hexoses. The deprotonated molecules of [M−H]⁻ 21 and 25 appeared at m/z 421.0775, but their MS/MS spectra considerably differed in fragmentation pattern with product ions at m/z 259.02, 258.02 (base peak), 241.01, 229.01 and 213.02. The former fragment ion resulted from a loss of an O-glycosidic linked hexose (162 Da) and corresponded to the deprotonated form of the aglycone, which further lost a proton and producing the base peak (Wolfender et al. 1998). Subsequent fragmentation of the later ion showed loss of OH followed by CO cleavage giving product ions at m/z 241.01 and 213.02, respectively. Alternatively, the base peak (m/z 258.02) broke down to a fragment ion at m/z 229.01 which was indicative of CHO loss. Thus, compounds 21 and 25 were tentatively identified as 1,3,5,6-tetrahydroxyxanthone-O-hexosides (Tusevski et al. 2013). The deprotonated molecules [M−H]⁻ of compounds 38 and 42 appeared at m/z 525.1041 and 525.1036, respectively. The MS/MS spectra showed product ion at m/z 403.06 which was an indication of a benzoic acid split off from the precursor (neutral loss of 122 Da), while the fragment at m/z 259.02 resulted from the loss of a benzoylated hexose [M−H−C₁₃H₁₄O₆]⁻ and corresponds to the aglycon. Subsequently, the later product ion gave characteristic fragments at m/z 215.03 and 187.04 indicative of CO₂ and C₂O₃ losses, respectively that was typical for norathyriol (Heinrich et al. 2017; Islam et al. 2020). Thus, compounds 38 and 42 were tentatively identified as norathyriol-O-(benzoyl)-hexosides. The total amount of xanthones in H. aucheri was found to be 10.34 mg/g D.W. Mangifern 13 was the main constituent (87.77%) of this mixture. Except for norathyriol, mangiferin, and isomangiferin, all other xanthones were reported here for the first time to occur in the title plant while compounds 15, 38, and 42 were tentatively established as new natural products.

Flavonol aglycones and their glycosides

The deprotonated molecules [M−H]⁻ of thirteen flavonol-O-glycosides, namely myricetin-O-hexosides 20 and 23 (m/z 479.0827 and 479.0828), myricetin-O-hexauronides 22 and 29 (m/z 493.0621 and 493.0636), myricetin-O-deoxyhexoside 26 (m/z 463.0881), quercetin-O-hexosides 27 and 30 (m/z 463.0880 and 463.0881), quercetin-O-hexauronide 28 (m/z 477.0670), quercetin-O-pentoside 32 (m/z 433.0767), quercetin-O-deoxyhexoside 36 (m/z 447.0931), kaempferol-O-hexosides 33 and 35 (m/z 447.0932 and 447.0930), and kaempferol-O-hexauronide 34 (m/z 461.0704), were detected in the full scan MS spectrum. In the MS/MS spectra, the corresponding precursor ions and the neutral losses of 134 Da (for 32), 147 Da (for 26, 36), 163 Da (for 20, 23, 27, 30, 33, and 35), and 176 Da (for 22, 28, 29, and 34) indicated the presence of pentose, deoxyhexose, hexose, and hexauronic acid as sugar moieties, respectively. Moreover, characteristic fragment ions at m/z 317.03 (for 20, 22, 23, 26, and 29), 301.04 (for 27, 28, 30, 32 and 36), and 285.04 (for 33, 34, and 35) corresponded to the deprotonated molecules of myricetin, quercetin, and kaempferol, while fragment ions at m/z 316.02, 300.03, and 284.03 (all being the base peaks), respectively, were derived from homolytic cleavage of the glycosidic bond (Cuyckens and Claeys 2004). Furthermore, the MS/MS spectra of the deprotonated molecules [M−H]⁻ of 39 (m/z 317.0301) and 41 (m/z 301.0353) exhibited similar product ions at m/z 193.01, 151.00, 107.01, and 178.99, resulting from [M−H−ring B]⁻ loss, ^1,3A⁻, ^0,4A⁻ retro Diels-Alder (RDA) fragmentation pathways (Fig. 5), and ^1,2A⁻ retrocyclization, respectively. Furthermore, characteristic fragments at m/z 137.02 and 165.02 for 39 and at m/z 121.03 and 149.02 for 41 were observed due to ^1,2B⁻ retrocyclization and ^1,3B⁻ cleavage. Following the nomenclature applied by Fabre et al. (Fabre et al. 2001), the compounds were identified as myricetin 39 and quercetin 41. The flavonols represent the major group of polar phenolics in the aerial parts of H. aucheri estimating a total of 16.54 mg/g D.W. Quercetin and its glycosides were dominant in the mixture estimating 57.16% of it followed by myricetin and its glycosides (42.18%).

Chromones

In the full MS scans, compound 40 appeared as formate adduct [M+HCOO]⁻ at m/z 427.1248. The MS/MS spectrum showed a product ion at m/z 381.12 corresponded to the deprotonated molecule of 40 and a base peak ion at m/z 219.06 that indicated a loss of a hexose unit. Thus, compound 40 was tentatively identified as a 5-hydroxy-2-isopropylchromone 7-O-glucoside (An et al. 2009). It was reported here for the first time to occur in the studied plant. The amount of 40, expressed as hyperoside, was found to be 175.27 µg/g D.W.

Biflavones

In the full MS spectrum, the deprotonated molecules [M−H]⁻ of the isobaric compounds 43 and 44 appeared at m/z 537.0826 537.0828, respectively. Their MS/MS spectra showed similar product ions at m/z 443.04 and 417.06, resulting from [M−H−C₆H₆O]⁻ and [M−H−C₉H₆O₃]⁻ losses and characteristic fragments [M−H−C₇H₄O₄]⁻ at m/z 385.07 for 43 and [M−H−C₆H₆O]⁻ at m/z 375.05 for 44. Furthermore, the RDA reaction of 44 led to split off a ketene derivative (m/z 162) of 4-hydroxycinnamic acid, whereas the compound 43 showed a cleavage of a phloroglucinol derivative (m/z 151). According to the literature data (Michler et al. 2011; Zhang et al. 2012) the compounds 43 and 44 were identified as 3,8’-biapigenin and 3’,8’’-biapigenin (amentoflavone), respectively. The amount of the former compound 43 was found to be 3.45% (1697.85 µg/g D.W.) of the total phenolic mixture while later 44 was found in very small quantities (50.24 µg/g D.W.). The amentoflavone 44 was reported here for the first time to occur in H. aucheri.