Skip to main content
NCBI home page
Food Chemistry: X logoLink to Food Chemistry: X
. 2021 Oct 16;12:100143. doi: 10.1016/j.fochx.2021.100143

Chemical characterization of flavonoids and alkaloids in safflower (Carthamus tinctorius L.) by comprehensive two-dimensional hydrophilic interaction chromatography coupled with hybrid linear ion trap Orbitrap mass spectrometry

Songsong Wang a,1, Jiliang Cao b,1, Jiagang Deng c, Xiaotao Hou c, Erwei Hao c, Lei Zhang d, Hua Yu a,⁎, Peng Li a,⁎
PMCID: PMC8529507  PMID: 34712950

Graphical abstract

graphic file with name ga1.jpg

Open in a new tab

Keywords: Online HILIC×HILIC, LTQ-Orbitrap mass spectrometry, Flavonoids, Alkaloids, Carthamus tinctorius L

Highlights

  • •

    HILIC × HILIC-ESI/HRMS/MSn system was constructed for flavonoids and alkaloids analysis in safflower.

  • •

    The established system exhibited high practical peak capacity and orthogonality.

  • •

    213 peaks were detected from Carthamus tinctorius L.

  • •

    5 constituents were discovered from Carthamus tinctorius L. for the first time.

Abstract

Safflower (Carthamus tinctorius L.) is a famous food additive and herbal medicine in China. In the present research, an online comprehensive two-dimensional hydrophilic interaction chromatography coupled to a diode array detector and a hybrid linear ion trap-Orbitrap mass spectrometry (HILIC × HILIC-DAD-ESI/HRMS/MSn) platform was developed to analyze the flavonoids and alkaloids in safflower. By combining with an XBridge Amide column (150 mm × 4.6 mm, 3.5 μm) and an Ultimate amide column (50 mm × 4.6 mm, 5 μm), the system orthogonality reached 88% and a total of 231 peaks were detected. Altogether 93 compounds, including 75 flavonoids and their glycosides and 10 alkaloids were unambiguously or tentatively identified in both negative and positive ion modes, using accurate mass and MS fragment data. Among them, 5 compounds were discovered and reported from safflower for the first time. The established HILIC × HILIC platform should be a powerful tool for the separation and characterization of complicated matrices in natural herbs.

Introduction

Recently, two-dimensional liquid chromatography (2D-LC) has been widely applied to the separation and analyses of complicated samples due to its excellent separation capability, high resolution, and large peak capacity (Liang et al., 2012, Paola Dugo et al., 2004). The elution from the first dimension (1D) can be collected as fractions that will be individually transferred and separated in the second dimension (2D), providing a feasible tool for separation, identification, and quality control of complex chemicals (Donato et al., 2011, Wang et al., 2008). To achieve high orthogonality, the two dimensions usually employ columns with different separation mechanisms, for example, reversed-phase (RP), normal phase (NP), hydrophilic interaction liquid chromatography (HILIC), size exclusion chromatography (SEC), affinity chromatography (AC), or ion-exchange chromatography (IEX), presenting different selectivities (Li et al., 2014, Li et al., 2015, Sommella et al., 2017, Uliyanchenko et al., 2012). Once coupled to mass spectrometry (MS), 2D-LC can be a more powerful platform with unparalleled capability for chemical separation and identification in samples with complex matrices.

Safflower (Carthamus tinctorius L.), known as Honghua in China, is a famous natural pigment, food additive, and cosmetic (Dai et al., 2014). Red and yellow pigments (e.g. carthamin) can be found in the petals of safflower, and are usually applied to dyeing agents in textile and food industries. Also, safflower is considered an emergent oilseed crop (Villa et al., 2017). Moreover, safflower is a popular herbal medicine widely applied for the treatment of coronary heart disease, stroke, and angina pectoris (Delshad,et al., 2018). Nowadays, several derived products are used to treat cardiovascular and cerebrovascular diseases (Cao et al., 2014, He et al., 2012). Therefore, to scientifically assess the quality of safflower and related products, it is necessary to uncover its chemical compositions, especially the potential bioactive compounds. Flavonoids and their glycosides (O- and C-glycosides) are a group of vital phytochemicals that are related to the therapeutic effects of safflower as a result of their diverse bioactivity (Xiao et al., 2016, Zhang et al., 2016). In addition, alkaloids are active ingredients widely present in safflower and have shown antioxidant, neuroprotective, and hepatoprotective activities both in vitro and in vivo (Zhang et al., 2016).

In most previous studies, the identification of chemical compounds in safflower was mainly based on one-dimensional LC (1D-LC) or offline 2D-LC/MS analyses (Lu et al., 2019, Yao et al., 2017). Although large number of compounds were characterized in the offline 2D-LC/MS analyses, time consumption and sample loss are the biggest concerns. To our knowledge, no reports about the online HILIC × HILIC 2D-LC systems were recorded for the chemical analysis of safflower. Our research group has developed online 2D-LC (HILIC × RP and RP × RP) systems to characterize diterpenoids and phenolic compounds in Salvia Miltiorrhiza, exhibiting remarkable separation power for complex samples (Cao et al., 2017, Cao et al., 2016). Therefore, in the present work, we developed an online comprehensive HILIC × HILIC system coupled with DAD detector and hybrid linear ion trap (LTQ)-Orbitrap mass spectrometry (HILIC × HILIC-DAD-ESI/HRMS/MSn) to systematically analyze the chemical constituents from safflower. The established HILIC × HILIC system showed good orthogonality and peak capacity, and no solvent effect occurred. Besides, chemical components including flavonoid C- and O-glycosides and alkaloids were characterized. This work will generate crucial knowledge for developing HILIC × HILIC system to analyze natural products with complex compounds.

Materials and methods

Chemicals and materials

Methanol, formic acid, and LC-grade acetonitrile were provided by Merck (Darmstadt, Germany). A Milli-Q purification system (Millipore, Bedford, MA, USA) was used to produce deionized water. Chemical reference standards, including quercetin (>98%), rutin (>98%), hyperoside (>98%), isoquercitrin (>98%), hydroxysafflor yellow A (>98%), kaempferol (>98%), myricetin (>96%), nicotiflorin (>96%), quercimeritrin (>98%), 6-methoxykaempferol-3-O-rutinoside (>96%), luteolin (>98%), apigenin (>98%), 6-hydroxykaempferol-3,6-diglucoside (>98%),6-hydroxykaempferol-3,6,7-triglucoside (>98%), and baicalin (>96%) were provided by Nanjing Jingzhu Bio-Tech Co., Ltd (Nanjing, China), and their chemical structures are shown in Fig. 1. Before use, all reference standards were determined by HPLC-DAD-HRMS/MSn and confirmed with UV spectra, accurate mass, and MSn data. Raw samples of Safflower were provided by Tong Ren Tang of Beijing (Macau, China). The voucher specimens were preserved in the Institute of Chinese Medical Sciences, University of Macau, Macao.

Fig. 1.

Fig. 1

Open in a new tab

The chemical structures of the 14 reference compounds.

Sample preparation and standard solutions

The safflower samples were dried with air circulation at room temperature. The safflower samples were extracted following previous reported methods with minor modifications (Wang et al., 2015). An aliquot of 1 g sample powder was accurately weighed and extracted with 8 mL of 25% ethanol (v/v) by an ultrasonic cleaner (44 kHz, Branson Ultrasonic Corp., Danbury, CT, USA) at around 25 °C for 15 min. After centrifugation at 3,000 × g for 10 min, the supernatant was filtered through a 0.22 μm membrane (PVDF Millex-GV, 13 mm, Millipore) before 2D-LC analysis.

Next, 25% ethanol (v/v) was used to the dissolve the reference standards to obtain individual stock standard solutions with concentrations ranging from 0.8 to 3.2 mg/mL. Appropriate amounts of the individual stock solutions were mixed to obtain a mixed standard solution and then diluted to about 20 μg/mL.

Online HILIC × HILIC-DAD-ESI/HRMS/MSn analyses

A Dionex UltiMate 3000 × 2 Dual RSLC system (Dionex, Thermo Fisher Scientific Inc., USA) was used for HILIC × HILIC analyses, including a WPS-3000TRS autosampler, an SRD-3600 degasser, a DGP-3600RS dual-ternary pump (right and left pump), a TCC-3000RS column thermostat, and a DAD-3000RS detector. An electronically controlled two-position ten-port switching valve equipped with two sample loops (200 μL, Rheodyne, CA, USA) was configured to connect the 1D and 2D. The 2D-LC system was connected to a hybrid LTQ-Orbitrap XL mass spectrometry (Thermo Fisher Scientific, Bremen, Germany) equipped with electrospray ionization (ESI) source by a flow splitter (Analytical Scientific Instruments, CA, USA) using a fixed ratio at 1:4. The online HILIC × HILIC-LTQ-Orbitrap platform was controlled by the Xcalibur 2.1 software (Thermo Fisher Scientific, Bremen, Germany).

In the 1D separation, the column flow rate (XBridge Amide column, 150 mm × 4.6 mm, 3.5 μm, Waters) was 0.08 mL/min. The mobile phases were 0.1% aqueous formic acid (v/v) solution (A) and acetonitrile (B). The following gradient program was employed: 0 min, 95% B; 10 min, 95% B; 20 min, 92% B; 30 min, 92% B; 140 min, 82% B; 150 min, 60% B; 172 min, 50% B.

In the 2D separation, a high flow rate of 3 mL/min was applied to the short 2D column (Ultimate amide column, 50 mm × 4.6 mm, 5 μm, Welch Materials). Water containing 0.2 μM ammonium formate (A) and acetonitrile (B) were used as mobile phases. The following segment gradient program with six different segments was applied: i) 0–55 min (0 min, 96% B; 0.25 min, 96% B; 1.7 min, 85% B; 1.75 min, 96% B, 2 min, 96% B), ii) 56–73 min (0 min, 93% B; 0.25 min, 93% B; 1.7 min, 85% B; 1.75 min, 93% B, 2 min, 93% B), iii) 74–91 min (0 min, 90% B; 0.25 min, 90% B; 1.7 min, 82% B; 1.75 min, 90% B, 2 min,90% B) , iv) 92–130 min (0 min, 90% B; 0.25 min, 90% B; 1.7 min, 80% B; 1.75 min, 90% B, 2 min, 90% B), v) 131–140 min (0 min, 85% B; 0.25 min, 85% B; 1.7 min, 75% B; 1.75 min, 85% B, 2 min, 85% B), vi) 141–170 min (0 min, 85% B; 0.25 min, 85% B; 1.7 min, 70% B; 1.75 min, 85% B, 2 min, 85% B). The other parameters were set as follows: DAD sampling rate, 50 Hz; detection wavelength, 281 nm; column temperature, 30 °C; injection volume, 3 μL; and modulation cycle, 2 min.

For the ESI/HRMS/MS2 analyses, samples introduced from the flow splitter were analyzed in the positive ion mode. The MS parameters were employed as below: capillary temperature, 350 °C; spray voltage, 4 kV; capillary voltage, 35 V; and tube lens voltage, 120 V. Both auxiliary and sheath gases were nitrogen (N2), set at flow rates of 8 and 10 arbitrary units, respectively. In the linear ion trap, the collision gas was high-purity helium (He).

A full scan event followed by two data-dependent acquisition (DDA) events were present in the scan cycle. Accurate mass data from m/z 100 to 1200 was recorded by the Orbitrap mass analyzer in the full scan event, and its resolution was set at 30,000 (FWHM as defined at m/z 400). In DDA events, MS2 fragment data were acquired in the linear ion trap triggered by the top two intension ions from the full scan event. Collision-induced dissociation (CID) activation was optimized to a normalized collision energy of 35% with the isolation width set at m/z 2.0. The dynamic exclusion function was enabled for DDA events. The Orbitrap mass analyzer was weekly calibrated to maintain high mass accuracy using the calibration solution according to the manufacturer’s guidelines.

Data analyses

Raw HRMS and MS2 data were visualized and processed by the Xcalibur 2.1 software. The 2D contour plot was built using the LC Image LC × LC-HRMS Edition software (version 2.6, GC Image, LLC., Lincoln, NE, USA). The LC Image software was also used for 2D peaks recognition and location. Effective peak capacity, theoretical peak capacity, orthogonality, and practical peak capacity were computed as previously described (Filgueira et al., 2011, Qiao et al., 2014, Qiao et al., 2015).

Results and discussion

Establishment of the HILIC × HILIC system

Two offline HILIC-HILIC methods were carried out as previously reported to analyze polar components (Liu et al., 2009) and flavonoid O-glycosides (Yao et al., 2017) in the safflower, respectively. However, offline techniques are more time-consuming and may result in sample loss. Therefore, an online 2D-LC strategy was considered and optimized in the current study. Since the main chemicals in safflower were polar ingredients, columns with HILIC mode were preferred for 2D separations. In HILIC, analytes exhibited similar retention behaviors to those of NPLC, and an RP solvent system can be used for separation, which is suitable for polar components analyses. Thus, after a series of optimizations including column types, mobile phases, gradient elution programs, and flow rates, good separation with evenly distributed peaks was finally achieved on an XBridge Amide column (150 mm × 4.6 mm, 3.5 μm, Waters), employed as the 1D column (Fig. S1). It should be particularly noted that, in order to reduce the injection band broadening in 2D separation, the long and narrow columns (e.g., 1 or 2.1 mm i.d.) were preferred as 1D columns. In the current study, although the column with amide stationary phase was demonstrated to be the best choice for 1D separation, a 4.6 mm i.d. column was the only amide option in this study, which was limited by our column resources. Therefore, to minimize injection volume in 2D separation and also provide enough 1D peak samplings, the 1D flow should be operated under the suboptimal linear flow velocities, which was 0.08 mL/min in this study.

To achieve high orthogonality, the combination of HILIC and RPLC was considered first to configure the 2D-LC system. Three RP columns with different stationary phases, including Hypersil gold PFP (150 mm × 2.1 mm, 3 μm), Accucore aQ (50 mm × 4.6 mm, 2.6 μm) and Accucore Polar Premium (50 mm × 3.0 mm, 2.6 μm) were tested for the 2D separation. The best combination was determined by evaluating the correlation coefficient (R2) between the selected 1D column and the tested 2D columns. Results showed that the combinations of XBridge Amide with Hypersil gold PFP and Accucore Polar Premium provided similar low R2 values as 0.03 and 0.01, respectively, being both suitable for 2D separation according to the evaluation index. Thus, these two combinations were applied to analyze the 14 references. Unfortunately, the severe solvent incompatible effect was observed under both column combinations, and each reference compound was splitted into two independent peaks with different 2D RTs (Fig. 2A and 2B). The peaks width in the second dimension were about 80 s in Fig. 2A and 2B. The weaker elution solvent (high acetonitrile ratio) in the 1D HILIC analysis is the stronger one in the 2D RP analysis, which might have led to the loss of separation resolution and efficiency.

Fig. 2.

Fig. 2

Open in a new tab

2D-TIC contour plots of different 2D-LC systems for separation of 14 reference compounds standards. (A) Combination of HILIC with Hypersil gold PFP column, (B) Combination of HILIC with Accucore Polar Premium column, (C) Combination of HILIC with Accucore Polar Premium column configured with online dilution module.

To solve the solvent incompatible effect problem, an online dilution module was integrated into the 2D-LC system. Briefly, another LC pump was connected to the outlet of the 1D HILIC column to introduce a continuous water phase for the organic phase dilution from 1D fractions (Cao et al., 2017). References with retention time (RT) lower than 110 min in the 1D analysis were well separated without peak splitting (Fig. 2C). However, for the references with larger polarities (1D RT > 110 min), peak splitting was still observed despite the peaks width in the second dimension had reduced to about 65 s. Due to the large polarities of these compounds in safflower, it is difficult to build an online HILIC × RP system with high efficiency and resolution, even using an online dilution strategy. Hence, we considered a combination of HILIC × HILIC to reconfigure the 2D-LC system. According to the literature, the HILIC × HILIC employing stationary phases with different functional groups can also offer different selectivity and possible high orthogonality (Wang et al., 2008).

Thus, different HILIC columns were tested for the 2D separation. Samples were separated on short broad-bore columns and eluted at a relatively high flow rate. Therefore, we finally applied a HILIC column named Ultimate amide column (50 mm × 4.6 mm, 5 μm) for the 2D separation in this study. A high flow rate of 3 mL/min was employed to reduce the large volume influence of sample solvent and maintained the system pressure in a reasonable range. The cycle duration should guarantee that the 1D eluent will not exceed the 2D 200-μL sample loops. Thus, the valve switched every 2 min, and 160 μL (80 μL/min × 2 min) fractions eluted from the 1D separation was re-injected to the 2D column for each modulation cycle.

Segment gradient programs were optimized with six different segments as described above. A high flow rate of 3 mL/min was also selected to provide efficient and fast 2D separation. Finally, the safflower sample was well separated on the 2D contour plots under the optimized parameters in 170 min without any visible peak splitting (Fig. 3A), which reduced the possibility of false identification of polar components.

Fig. 3.

Fig. 3

Open in a new tab

(A) The separation of safflower sample on 2D-TIC contour plots by using the optimal online HILIC × HILIC-ESI/HRMS/MSn system, (B) The separation comparison of 1D-HILIC and HILIC × HILIC in safflower.

HILIC × HILIC system evaluation

The established HILIC × HILIC system was evaluated by separating a real safflower sample. On the 2D-TIC contour plot, 213 peaks were recognized by the LC Image software (blob filter settings: minimum volume = 0, minimum area = 3, minimum peak = 5, relative values). The effective gradient time for 1D was 175 min and for 2D was 2 min. The average 4σ peak widths were 2 min and 0.117 min in 1D and 2D chromatograms, respectively. Hence, the theoretical peak capacities were 87 and 17.1 for 1D and 2D analysis, respectively. Then, the effective and theoretical peak capacity was calculated as 817 and 1487 for the optimized HILIC × HILIC system, indicating a much higher separation capability compared to 1D-LC analyses (Li and Schmitz, 2015, Wang et al., 2008).

According to reported equations (Filgueira et al., 2011, Li et al., 2009), the practical peak capacity and orthogonality were evaluated by dividing the separation space into 14 × 14 rectangular bins, which was close to Pmax (2 1 3) and superimposed with the data points (Fig. S2). Our results showed that about 62.75% of the separation space was covered by bins containing data points. Finally, the practical peak capacity and orthogonality were calculated as 932 and 88.27%, respectively (Filgueira et al., 2011, Qiao et al., 2015).

Separation and characterization of flavonoids and alkaloids in safflower

The developed HILIC × HILIC system was used to analyze the flavonoids and alkaloids in safflower. On the 2D-TIC contour plot, the compounds were separated and detected as blobs. In contrast with conventional 1D-LC analyses, the established 2D-LC system exhibited a remarkable separation power. For example, the EIC m/z 395 in safflower got sufficient sampling of three times in the 2D separation with high resolution and excellent peak shape (Fig. 3B). Besides, some undetectable components in 1D-HILIC, such as m/z 489, were successfully detected with high sensitivity by using the established HILIC × HILIC system, indicating its high resolving power and feasibility (Fig. 3).

By analyzing their accurate mass and fragmentation data, and comparing with reference standards, 93 constituents were identified or tentatively identified, including 5 flavonoids, 24 flavonoid C-glycosides, 46 flavonoid O-glycosides and 10 alkaloids (summarized in Table 1). Among them, 5 new constituents were found in safflower for the first time. Herein, the structural elucidation of several representative compounds and potentially new ones were discussed as examples.

Table 1.

Characterization of polar constituents by HILIC × HILIC-LTQ-Orbitrap from safflower in positive ion mode.

Peak No. Retention time Measured+
(m/z)		 Error (ppm) Molecular
formula		 (+) LC/ESI-MS2m/z
(% base peak)		 Identification Ref.
1st dim.
(min)		 2nd dim.(sec)
1S 6 18 271.05933 −2.8 C15H10O5 Apigenin (Yang et al., 2016)
2 6 37.1 584.27661 2.0 C34H37N3O6 438.29,420.26,275.28 N1,N5,N10-(Z)-tri-p-coumaroylspermidine (Zhou et al., 2014)
3 10 41.8 584.27621 1.58 C34H37N3 O6 438.34,420.33 N1,N5,N10-(E)-tri-p-coumaroylspermidine (Zhou et al., 2014)
4 14 32 584.27651 −4.6 C34H37N3O6 438.4,420.9 Safflospermidine A (Zhou et al., 2014)
5 14 46 584.27635 −4.6 C34H37N3O6 438.37,420.37 Safflospermidine B (Zhou et al., 2014)
6 14 90.5 361.0917 −1.7 C18H16O8 Rosmarinic acid (Zhou et al., 2014)
7 16 60.3 373.11194(M + Na) −3.8 C20H18N2O4 329.28,270.95,227.08 Serotobenine (Sato et al., 2014)
8 16 116 439.12247 −2.81 C20H22O11 395.30,353.26,292.31,204.07 Y-Glc-2C (Yao et al., 2017)
9 20 76.6 409.1843(M + Na) 0.82 C19H30O8 391.18,381.41,247.15,203.17, Roseoside (Zhou et al., 2014)
10 20 76 481.12955 −2.89 C22H24O12 437.15,395.17 3,5,7,4′-tetrahydroxy-6-methoxyflavanonol-O-Glc (Yao et al., 2017)
11 22 76 409.18417 0.98 C19H30O8 390.48,365.31,289.16,202.95 Roseoside (Yang et al., 2016)
12 22 76 289.07153 0.86 C15H12O6 271.07,168.88,146.94 Eriodictiol (Yang et al., 2016)
13 26 2.3 343.13742 −1.29 C16H22O8 347.06,305.09,275.21,203.03,185.06 Methyl-3-(4-O-β-d-glucopyranosylphenyl) propionate or Bidenoside D (Zhou et al., 2008)
14 26 90.5 395.13223(M + Na) 0.7 C17H24O9 364.09,233.09,185.12 Methyl-3-(4-O-β-d-glucopyranosyl-3-methoxyphenyl) propionate (Zhou et al., 2014)
15S 28 80 449.10783 −2.0 C21H20O11 287.08 Quercitrin
16 32 95 136.06177 0.3 C5 H5 N5 135.87,118.17,108.08, Adenine (Zhou et al., 2014)
17 32 92.9 119.0355 1.6 C4H6O4 91.05 Succinic acid (Zhou et al., 2014)
18 38 78 385.14783 1.4 C18H24O9 223.16,207.12,203.10 (-)-4-Hydroxybenzoic acid-4-O-[6́-O-(2 “- methylbutyryl)-β-d-glucopyranoside] (Jiang et al., 2013)
19S 42 113 465.10275 1.0 C21H20O12 446.23,303.08,279.27 Hyperoside (Zhou et al., 2014)
20S 44 104 465.10159 1.2 C21H20O12 447.32,303.07 Isoquercitrin (Zhou et al., 2014)
21S 52 111 625.17444 1.9 C28H32O16 479.15,317.06,301.98 6-Methoxykaempferol 3-O-Rutinoside (Yang et al., 2016)
22 52 111 317.06516 2.7 C16H12O7 302.12 5-Methoxyquercetin (Yang et al., 2016)
23 54 100 311.1492 0.9 C16H22O6 293.27,191.13 2Z-Decaene-4,6-diyn-1-O-

-β-glucopyranoside		 (Hong et al., 2015)
24 54 102 625.17847 0.2 C28H32O16 479.15,317.06,301.98 Isorhamnetin

methylpentosyl hexoside isomer		 (Yang et al., 2016)
25S 54 104 595.16498 2.8 C27 H30O15 449.21,287.04 Kaempferol-3-O-rutinoside (Yue et al., 2013)
26 54 102 1151.31848(2 M + H) 4.5 C27H29NO13 Cartormin (Yin & He, 2000)
27 54 102 317.0655 2.7 C16H12O7 302.08 5-Methoxyquercetin isomer (Yao et al., 2017)
28 54 104.5 449.10889 1.2 C21H20O11 453.27,435.27,417.28,387.27 Kaempferol-3-O-β-d-glucoside (Zhou et al., 2014)
29 56 78.9 615.17041 −0.4 C30H30O14 597.37,571.31,501.15,451.20,289.18 Safflomin C (Si et al., 2016)
30 56 88 595.16523 1.5 C27H30O15 599.31,573.61,471.36,331.17 Saffloquinoside C isomer (Zhou et al., 2014)
31 56 88 449.10907 1.2 C21H20O11 453.27,435.27,417.28 Luteolin-7-O-β-d-glucopyranoside (Zhou et al., 2014)
32 62 85 463.0871 −0.5 C21H18O12 445.32, 287.10 Scutellarin (Yang et al., 2016)
33 64 113.8 244.09352 0.9 C9H13N3O5 226.11,148.08 Cytidine (Yao et al., 2016)
34 66 4 595.16547 −0.4 C27H30O15 577.30,433.13 Saffloquinoside C (Zhou et al., 2014)
35S 66 104 465.10275 0.9 C21H20O12 447.34,380.35,345.02,303.04 Quercimeritrin (Zhou et al., 2014)
36 66 62.7 595.16553 −0.3 C27 H30O15 577.42,551.46,449.21,433.24,287.16 Safflor yellow A (Yue et al., 2013)
37 66 104 487.17722 −3.9 C22H30O12 469.41,420.38,403.18 (8Z)-decaene-4,6-diyn-1-ol-1-O-β-d-glucuronyl-(100–20)-β-Dglucopyranoside (He et al., 2011)
38 70 4.6 284.09894 1.1 C10H13N5O5 Guanosine (Fan et al., 2009)
39 70 48 633.1394(M + Na) −5.0 C27H30O16 615.40,487.32,347.19,337.1 6-MeO-Kae-O-Glc-Xyl (Yang et al., 2016)
40 70 4.6 649.11884(M + K) 3.2 C27 H30O16 631.22,529.14,487.37,363.04 Kaempferol 3-O-β-sophoroside (Kazuma et al., 2000)
41 70 116 491.11172 7.5 C23H22O12 495.17,469.25,367.24,331.22 Luteolin-7-O-(6′'-O-acetyl)-β-d-glucoside (Yang et al., 2016)
42 72 67 465.10263 1.3 C21H20O12 447.19,363.20,345.08,303.01 Quercimeritrin isomer (Zhou et al., 2014)
43 72 111.5 633.14636(M + Na) 3.1 C27 H30O16 631.19,621.27,605.36,473.50 Quercetin 3-O-rutinoside (Kazuma et al., 2000)
44 72 4 611.15576 −8.0 C27H30O16 465.25,303.04 Y-2Glc—CH2 isomer (Yang et al., 2016)
45 72 109 611.15717 −5.7 C27H30O16 593.36,449.22,303.07 Y-2Glc—CH2 isomer (Yang et al., 2016)
46 74 78 611.15723 −5.6 C27H30O16 567.22,435.29,391.19 Y-2Glc—CH2 (Yang et al., 2016)
47S 76 67 611.15723 −5.6 C27H30O16 593.41,567.49, 465.22, 303.02 rutin (Yang et al., 2016)
48 76 13 697.1593 −2.5 C30H32O19 679.48,653.37,535.30,287.06 Kae-3-O-diglucoside-Mal. (Yao et al., 2017)
49 78 74 433.11292 −0.9 C21H20O10 415.15,397.10,313.17,301.07 Saffochalconeside (Hong et al., 2015)
50 80 58 633.1394(M + Na) −1.4 C27H30O16 615.32,487.16,331.19 Que-3,6 or 3,7-di-O-Xyl-GluA (Yang et al., 2016)
51 82 25 713.15375 −3.1 C30H32O20 551.33,303.13 6-OH-Kae-3,6 or 3,7-di-O-diglucoside-Mal. (Yao et al., 2017)
52 84 4 611.15686 −6.2 C27H30O16 465.27,303.09 Y-2Glc—CH2 isomer (Yang et al., 2016)
53 86 95 625.17596 −0.5 C28H32O16 479.09,317.16 5,7,4′-trihydroxy-6-methoxy-flavonol-3-O-rutinoside (Yao et al., 2017)
54 88 92 461.10342 9.0 C22H20O11 439.24,397.22,235.11 Acacetin-7-O-β-d-glucuronide (Yao et al., 2017)
55 90 16.2 123.04441 0.3 C7H6O2 99.95,81.81 p-hydroxybenzaldehyde (Yang et al., 2016)
56 92 78 451.1524 −2.0 C21H22O11 433.23,304.17,289.16 Neocarthamin (Hong et al., 2015)
57 92 20 697.15881 −3.2 C30H32O19 535.26,287.05 6-OCH3-Kae-3-O-GlC-Rha-Oxa (Yang et al., 2016)
58S 98 102 627.15771 2.4 C27H30O17 465.16,303.09 6-Hydroxykaempferol 3,6-diglucoside (Zhou et al., 2014)
59 102 109.1 629.17358 2.2 C27H32O17 611.41,403.17 Methylisosafflomin C (Zhou et al., 2014)
60 106 18 576.16858 −4.4 C27H29NO13 Cartormin isomer (Yang et al., 2016)
61 116 76 595.16574 1.5 C27H30O15 577.54,433.18,415.09,397.20,313.23,301.19,277.19 Saffloquinoside A (Hong et al., 2015, Zhou et al., 2014)
62S 120 90 625.13992 3.2 C27 H28O17 606.50,463.03,449.24,340.34,311.33,287.17 6-hydroxyapigenin-6-O-β-d-glucoside-7-O-β-d-glucuronide (Kazuma et al., 2000)
63 N 130 111 409.13150 2.0 C14H26O12 277.10,317.18 Propanetriol-Glu
64 N 130 72 409.13068 −5.5 C14H26O12 277.10 Propanetriol-Glu isomer
65 132 116 365.10678 −3.1 C12H22O11 275.06,203.00,184.95 Sucrose or its isomers (Kubica et al., 2012)
66S 136 90.5 773.21716 3.4 C33H40O21 754.42,627.24,610.45,465.18,303.03 6-Hydroxykaempferol 3-Rutinoside−6-glucoside (Zhou et al., 2008)
67 N 136 104 409.13330 2.2 C14H26O12 277.14,317.12, Propanetriol-Glu isomer
68 144 11 957.22687 −3.6 C44H44O24 938.48,902.45,825.59,794.37 Precarthamin (Yang et al., 2016)
69 144 9.2 911.22113 −4.2 C43H42O22 Carthamin isomer (Yang et al., 2016)
70 146 71 957.2264 −3.0 C44H44O24 Precarthamin isomer (Yang et al., 2016)
71 150 11 911.22113 −4.3 C43H42O22 Carthamin isomer (Yang et al., 2016)
72 152 116.1 1045.281958 4.8 C48H52O26 1027.55,868.64,788.42 Anhydrosafflor Yellow B isomer (Yang et al., 2016)
73 154 39 415.10034 2.5 C21H18O9 397.29, 367.23, 295.15 Saffloflavonesides A isomer (Yang et al., 2016)
74 N 154 4 501.17966 −4.2 C19H32O15 482.45,339.10,193.07 Quinic acid-Glu-Rha (Yang et al., 2016)
75 N 156 88 501.17926 −5.8 C19H32O15 482.32,339.21,285.09,193.06 Quinic acid- Glu-Rha isomer (Yang et al., 2016)
76 156 18 541.17578 −4.9 C19H34O16 409.21,317.12,277.22 Propanetriol-Glu-Xyl-Xyl (Yang et al., 2016)
77S 158 13.9 613.1763 −5.3 C27H32O16 594.36,577.07,549.51,451.20,433.22,331.06,317.27,301.07 Hydroxysafflor yellow A (Yue et al., 2013)
78 158 16 415.1007 0.9 C21H18O9 397.23,367.21,355.14 Saffloflavonesides A isomer (Yang et al., 2016)
79 160 37 415.10129 3.9 C21H18O9 Saffloflavonesides A (Yang et al., 2016)
80 160 76 629.17035 −4.8 C27H32O17 633.27, 531.40,489.19 3,5,6,7,4′-Pentahydroxyflavanonol-O-Glc-Glc (Yao et al., 2017)
81 162 62.7 1045.281958 7.9 C48H52O26 1027.44,1009.62,955.66,822.85,720.67 Anhydrosafflor yellow B (Fan et al., 2009)
82 162 16.2 1045.281958 0.8 C48H52O26 1026.60,883.44,721.28 Anhydrosafflor yellow B isomer (Fan et al., 2009)
83S 162 111 789.21167 7.3 C33H40O22 6-Hydroxykaempferol-3,6,7-triglucoside (Kazuma et al., 2000)
84 162 65 629.19305 0.4 C31H32O14 633.33,531.40,489.21 Methylsafflomin C or methylisosafflomin (Yoon, 2008)
85S 164 109.1 803.20355 4.2 C33H38O23 784.45,641.33,479.32,341.21,303.13 6-Hydroxyl kaempherol-3,6-O-diglucosyl-7-O-Glucuronic acid (Kazuma et al., 2000)
86 164 16 1063.2925 −2.0 C48 H54O27 1045.50,1022.25,697.42 Safflomin B isomer (Yue et al., 2013)
87 164 16 1063.2925 −1.9 C48H54O27 1622.32,1467.74,833.92 Safflor yellow B (Yang et al., 2016)
88 164 60 641.14673 −5.1 C27H28O18 645.42,619.62 Quercetin-3-O-α-l-rhamnoside-7-O-β-d-glucuronide (Yao et al., 2017)
89 166 76 629.18648 3.2 C31 H32O14 Methylisosafflomin C (Yue et al., 2013)
90 166 85 1063.29028 −2.1 C48 H54O27 1039.21,907.82,578.38 Safflomin B (Yao et al., 2017)
91 166 51 911.22113 −3.2 C43H42O22 Carthamin (Sato et al., 2003)
92 168 9 935.26355 −2.9 C39H50O26 6-OH-Kae-3,6 or 3,7-di-O-Glc-Glc-Rha-Glc (Yang et al., 2016)
93 168 116 309.12915 −4.1 C16H20O6 291.16,273.16,225.07 (2E,8Z)-Decadiene-4,6-diyne-1-ol-1-O-β-d-glucopyranoside. (He et al., 2011)
Open in a new tab
S

, indicates the compound was confirmed by reference compounds. N, indicates the compound was identified in safflower for the first time. Y, represents the quinochalcone C-glycoside skeletons with the elemental compositions of C16H12O6.

Compound 2 gave the accurate mass at m/z 584.27661[M + H]+ and its formula was calculated to be C34H37N3O6. In the MS2 spectra, ions at m/z 438 and m/z 420 were observed resulting from the loss of C9H6O2 and C9H10O2N, respectively. Further, these data were further compared with the literature (Zhao et al., 2009) and compound 2 was finally identified as N1,N5,N10-(Z)-tri-p-coumaroylspermidine. Its MS2 spectra and proposed fragmentation are shown in Fig. 4A.

Fig. 4.

Fig. 4

Open in a new tab

The MS/MS spectra and proposed fragmentation pathway for compound 2 (A), and the MS/MS spectra and proposed fragmentation pathway for compound 25 (B) and 61 (D), and compound 65 under positive ion mode (C) and negative ion mode (E).

Compounds 61 and 25 presented the same [M + H]+ ion at m/z 595.16547. Their formula was calculated as C27H30O15. However, their MS2 spectra different. Compound 25 showed fragments at m/z 576, 449, and 287. The characteristic neutral loss of 162 Da and 146 Da indicated it was a bioside (Fig. 4B and 4D). The MS2 spectra of compound 61, showed ions at m/z 577, 433, 415, 397, 385, 313, 301, and 277. The calculated elimination of 162 Da indicated glucose (glc) disaccharide moiety. According to the literature (Li et al., 2013, Lu et al., 2019), the element composition and fragmentation behaviors of compound 61 were consistent with that of saffloquinoside A, reported from Pueraria lobata. Therefore, compound 61 was tentatively characterized as saffloquinoside A.

Compound 65 showed an adduct ion [M + Na]+ at m/z 365.10684 and a deprotonated molecular ion [M-H]- at m/z 341.10712 with a calculated empirical molecular formula of C12H22O11. In its MS2 spectra, the neutral loss of 18, 60, and 162 Da were observed with the production of fragment ions at m/z 347, 305, 203 in the positive ion mode, and m/z 323, 281, 179 in the negative ion mode, indicating the loss of H2O and glucose (Fig. 4C and 4E). By comparing with literature (Kubica, et al., 2012), the calculated molecular formula and MS2 data were consistent with sucrose or its isomers. Thus, compound 65 was tentatively identified as sucrose or its isomers (C12H22O11).

In the current study, we tentatively characterized 5 new constituents in safflower for the first time. To reduce false chemical identifications, a negative ion mode was also applied to provide the supplementary data for verification. Compound 63 was presented the [M + Na]+ ion at m/z 409.13290 and [M-H]- ion at m/z 385.13284 with the calculated element composition as C14H26O12. This formula was consistent with Propanetriol-α-l-arabinofuranosyl (1 → 4)-β-d-glucopyranoside, a safflower compound recorded in the TCM Database@Taiwan (http://tcm.cmu.edu.tw/zh-tw/chemical.php?compoundid = 37322). For compound 63, the MS2 spectra showed fragment ions at m/z 317 and 277 in positive ion mode, and m/z 253 in negative ion mode, indicating the loss of 92 Da and pentoses (Fig. 5A and 5C). Additionally, in the MS3 spectra of m/z 277, a loss of 92 Da and glucose was observed (Fig. 5B). Comparing these results with the chemical structure of Propanetriol-α-l-arabinofuranosyl (1 → 4)-β-d-glucopyranoside, we deduced that the loss of 92 Da was produced by the elimination of the propanetriol group. Besides, loss of H2O and CO were observed, indicating a polyhydric aglycone. However, the glycogen linkage can not be deduced only by mass spectrometry. Therefore, compound 63 was tentatively identified as Propanetriol-Glu. The MS data of compounds 64 and 67 were similar to compound 63 and they were both tentatively identified as isomers.

Fig. 5.

Fig. 5

Open in a new tab

The MS/MS/MS spectra and proposed fragmentation pattern for compound 63 under positive ion mode (A, B) and negative ion mode (C).

Compound 74 gave accurate mass at m/z 501.18088 in the positive ion mode and m/z 499.16415 in the negative ion mode, and was determined as be C19H32O15 with errors lower than 3 ppm. Its MS2 spectra in the negative ion mode showed ions at m/z 481, 353, 191 and 173 (Fig. 6), indicating [M-H-H2O]-, [M-H-rha]-, [M-H-rha-glu]- and [M-H-rha-glu-H2O]- ions. Typical fragment ions at m/z 191 and 173 were identified as quinic acid according to the literature (Michael et al., 2005, Lin & Harnly, 2007, Deborah et al., 2007). Thus, aglycone was assigned to quinic acid. In the positive ion mode MS2 spectra, the observed losses of 18 and 64 Da were matched the fracture behavior reported before (Cheng et al., 2008, Yuan et al., 2015). In the MS3 spectra, the neutral loss of H2O and CO indicated that the aglycone was polyhydric. Therefore, compound 74 was putatively characterized as quinic acid-Glu-Rha. Compound 75 showed the same fragmentation behavior of 74, and was then characterized as an isomer.

Fig. 6.

Fig. 6

Open in a new tab

The MSn spectra and proposed fragmentation pattern for compound 74 under positive ion mode (A, B) and negative ion mode (C).

Conclusions

In the current study, an online HILIC × HILIC-DAD-ESI/HRMS/MSn system was optimized and applied to analyze the flavonoids and alkaloids of safflower. The mobile phase incompatibility problem was solved by the combination of HILIC and HILIC with high orthogonality. Under optimal conditions, the orthogonality was as high as 88.27%, leading to the detection of 231 peaks on the 2D-TIC contour plot. Among them, 93 compounds were clearly or tentatively identified, including 5 potentially new ones, using the complementary structural information acquired by both negative and positive ion modes. Overall, the established HILIC × HILIC-DAD-ESI/HRMS/MSn system demonstrated its efficiency and powerful to analyze compounds in complex herbal extracts.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by Research Committee of the University of Macau (grant number MYRG2018-00239-ICMS), Macau Science and Technology Development Fund (grant number 147/2019/A3), Guangxi Innovation-driven Development Special Foundation Project (grant number GuiKe AA18118049), and Sichuan Science and Technology Planning Project (grant number 2020YFS0370).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.fochx.2021.100143.

Contributor Information

Hua Yu, Email: bcalecyu@um.edu.mo.

Peng Li, Email: pengli@um.edu.mo.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary data 1
mmc1.doc (406.5KB, doc)

References.

  1. Cao J., Chen Z., Zhu Y., Li Y., Guo C., Gao K.…Wen A. Huangqi-Honghua combination and its main components ameliorate cerebral infarction with Qi deficiency and blood stasis syndrome by antioxidant action in rats. J Ethnopharmacol. 2014;155(2):1053–1060. doi: 10.1016/j.jep.2014.05.061. [DOI] [PubMed] [Google Scholar]
  2. Cao J.L., Wang S.S., Hu H., He C.W., Wan J.B., Su H.X., Li P. Online comprehensive two-dimensional hydrophilic interaction chromatographyxreversed-phase liquid chromatography coupled with hybrid linear ion trap Orbitrap mass spectrometry for the analysis of phenolic acids in Salvia miltiorrhiza. J Chromatogr A. 2017 doi: 10.1016/j.chroma.2017.09.041. [DOI] [PubMed] [Google Scholar]
  3. Cao J.L., Wei J.C., Hu Y.J., He C.W., Chen M.W., Wan J.B., Li P. Qualitative and quantitative characterization of phenolic and diterpenoid constituents in Danshen (Salvia miltiorrhiza) by comprehensive two-dimensional liquid chromatography coupled with hybrid linear ion trap Orbitrap mass. J Chromatogr A. 2016;1427:79–89. doi: 10.1016/j.chroma.2015.11.078. [DOI] [PubMed] [Google Scholar]
  4. Cheng L., Zhang M., Zhang P., Song Z., Ma Z., Qu H. Silver complexation and tandem mass spectrometry for differentiation of triterpenoid saponins from the roots of Pulsatilla chinensis (Bunge) Regel. Rapid Commun Mass Spectrom. 2008;22(23):3783–3790. doi: 10.1002/rcm.v22:2310.1002/rcm.3801. [DOI] [PubMed] [Google Scholar]
  5. Dai Y., Verpoorte R., Choi Y.H. Natural deep eutectic solvents providing enhanced stability of natural colorants from safflower (Carthamus tinctorius) Food Chem. 2014;159:116–121. doi: 10.1016/j.foodchem.2014.02.155. [DOI] [PubMed] [Google Scholar]
  6. Deborah H. Markowicz Bastos , L. A. S., Rodrigo R. Catharino , Alexandra C., & H. F. Sawaya , I. B. S. C., Patrícia O. Carvalho , Marcos N. Eberlin. (2007). Phenolic Antioxidants Identified by ESI-MS from Yerba Maté (Ilex paraguariensis) and Green Tea (Camelia sinensis) Extracts. Molecules, 12, 423-432. https://www.mdpi.com/1420-3049/12/3/423. [DOI] [PMC free article] [PubMed]
  7. Delshad E., Yousefi M., Sasannezhad P., Rakhshandeh H., Ayati Z. Medical uses of Carthamus tinctorius L. (Safflower): A comprehensive review from Traditional Medicine to Modern Medicine. Electron Physician. 2018;10(4):6672–6681. doi: 10.19082/6672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Donato P., Cacciola F., Sommella E., Fanali C., Dugo L., Dacha M., Mondello L. Online comprehensive RPLC x RPLC with mass spectrometry detection for the analysis of proteome samples. Anal Chem. 2011;83(7):2485–2491. doi: 10.1021/ac102656b. [DOI] [PubMed] [Google Scholar]
  9. Fan L., Zhao H.-Y., Xu M., Zhou L., Guo H., Han J.…Guo D.A. Qualitative evaluation and quantitative determination of 10 major active components in Carthamus tinctorius L. by high-performance liquid chromatography coupled with diode array detector. J Chromatogr A. 2009;1216(11):2063–2070. doi: 10.1016/j.chroma.2008.03.046. [DOI] [PubMed] [Google Scholar]
  10. Filgueira M.R., Huang Y., Witt K., Castells C., Carr P.W. Improving peak capacity in fast online comprehensive two-dimensional liquid chromatography with post-first-dimension flow splitting. Anal Chem. 2011;83(24):9531–9539. doi: 10.1021/ac202317m. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. He J., Shen Y., Jiang J.S., Yang Y.N., Feng Z.M., Zhang P.C.…Hou Q. New polyacetylene glucosides from the florets of Carthamus tinctorius and their weak anti-inflammatory activities. Carbohydr Res. 2011;346(13):1903–1908. doi: 10.1016/j.carres.2011.06.015. [DOI] [PubMed] [Google Scholar]
  12. He Y., Wan H., Du Y., Bie X., Zhao T., Fu W., Xing P. Protective effect of Danhong injection on cerebral ischemia-reperfusion injury in rats. J Ethnopharmacol. 2012;144(2):387–394. doi: 10.1016/j.jep.2012.09.025. [DOI] [PubMed] [Google Scholar]
  13. Hong B., Wang Z., Xu T., Li C., Li W. Matrix solid-phase dispersion extraction followed by high performance liquid chromatography-diode array detection and ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometer method for the determination of the main compounds from Carthamus tinctorius L. (Hong-hua) J Pharm Biomed Anal. 2015;107:464–472. doi: 10.1016/j.jpba.2015.01.040. [DOI] [PubMed] [Google Scholar]
  14. Jiang J.S., Chen Z., Yang Y.N., Feng Z.M., Zhang P.C. Two new glycosides from the florets of Carthamus tinctorius. J Asian Nat Prod Res. 2013;15(5):427–432. doi: 10.1080/10286020.2013.780046. [DOI] [PubMed] [Google Scholar]
  15. Kazuma K., Takahashi T., Sato K., Takeuchi H., Matsumoto T., Okuno T. Quinochalcones and flavonoids from fresh florets in different cultivars of Carthamus tinctorius L. Biosci Biotechnol Biochem. 2000;64(8):1588–1599. doi: 10.1271/bbb.64.1588. [DOI] [PubMed] [Google Scholar]
  16. Li D., Duck R., Schmitz O.J. The advantage of mixed-mode separation in the first dimension of comprehensive two-dimensional liquid-chromatography. J Chromatogr A. 2014;1358:128–135. doi: 10.1016/j.chroma.2014.06.086. [DOI] [PubMed] [Google Scholar]
  17. Li D., Jakob C., Schmitz O. Practical considerations in comprehensive two-dimensional liquid chromatography systems (LCxLC) with reversed-phases in both dimensions. Anal Bioanal Chem. 2015;407(1):153–167. doi: 10.1007/s00216-014-8179-8. [DOI] [PubMed] [Google Scholar]
  18. Li D., Schmitz O.J. Comprehensive two-dimensional liquid chromatography tandem diode array detector (DAD) and accurate mass QTOF-MS for the analysis of flavonoids and iridoid glycosides in Hedyotis diffusa. Anal Bioanal Chem. 2015;407(1):231–240. doi: 10.1007/s00216-014-8057-4. [DOI] [PubMed] [Google Scholar]
  19. Li L., Yang Y., Hou X., Gu D., Ba H., Abdulla R.…Aisa H.A. Bioassay-guided separation and purification of water-soluble antioxidants from Carthamus tinctorius L. by combination of chromatographic techniques. Separation and Purification Technology. 2013;104:200–207. doi: 10.1016/j.seppur.2012.11.027. [DOI] [Google Scholar]
  20. Liang Z., Li K., Wang X., Ke Y., Jin Y., Liang X. Combination of off-line two-dimensional hydrophilic interaction liquid chromatography for polar fraction and two-dimensional hydrophilic interaction liquid chromatographyxreversed-phase liquid chromatography for medium-polar fraction in a traditional Chinese medicine. J Chromatogr A. 2012;1224:61–69. doi: 10.1016/j.chroma.2011.12.046. [DOI] [PubMed] [Google Scholar]
  21. Lin L.Z., Harnly J.M. A Screening Method for the Identification of Glycosylated Flavonoids and Other Phenolic Compounds Using a Standard Analytical Approach for All Plant Materials. J. Agric. Food Chem. 2007;55:1084–1096. doi: 10.1021/jf062431s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Liu Y., Guo Z., Feng J., Xue X., Zhang F., Xu Q., Liang X. Development of orthogonal two-dimensional hydrophilic interaction chromatography systems with the introduction of novel stationary phases. J Sep Sci. 2009;32(17):2871–2876. doi: 10.1002/jssc.200900086. [DOI] [PubMed] [Google Scholar]
  23. Clifford Micheal N., Nikolai Kuhnert S.K. Discriminating between the Six Isomers of Dicaffeoylquinic Acid by LC-MSn. J. Agric. Food Chem. 2005;53(10):3821–3832. doi: 10.1021/jf050046h. [DOI] [PubMed] [Google Scholar]
  24. Lu J.X., Zhang C.X., Hu Y., Zhang M.H, Wang Y.N., Qian Y.X.…Guo D.A. Application of multiple chemical and biological approaches for quality assessment of Carthamus tinctorius L. (safflower) by determining both the primary and secondary metabolites. Phytomedicine. 2019;58:152826. doi: 10.1016/j.phymed.2019.152826. [DOI] [PubMed] [Google Scholar]
  25. Paola Dugo O.F., Luppino Rosario, Dugo Giovanni, Mondello Luigi. Comprehensive Two-Dimensional Normal-Phase (Adsorption)-Reversed-Phase Liquid Chromatography. Analytical Chemistry. 2004;76(9):2525–2530. doi: 10.1021/ac0352981. [DOI] [PubMed] [Google Scholar]
  26. Qiao X., Song W., Ji S., Li Y.-jiao., Wang Y., Li R.…Ye M. Separation and detection of minor constituents in herbal medicines using a combination of heart-cutting and comprehensive two-dimensional liquid chromatography. J Chromatogr A. 2014;1362:157–167. doi: 10.1016/j.chroma.2014.08.038. [DOI] [PubMed] [Google Scholar]
  27. Qiao X., Song W., Ji S., Wang Q., Guo D.A., Ye M. Separation and characterization of phenolic compounds and triterpenoid saponins in licorice (Glycyrrhiza uralensis) using mobile phase-dependent reversed-phasexreversed-phase comprehensive two-dimensional liquid chromatography coupled with mass spectrometry. J Chromatogr A. 2015;1402:36–45. doi: 10.1016/j.chroma.2015.05.006. [DOI] [PubMed] [Google Scholar]
  28. Sato H., Kawagishi H., Nishimura T., Yoneyama S., Yoshimoto Y., Sakamura S.…Matsumoto T. Serotobenine, a Novel Phenolic Amide from Safflower Seeds (Carthamus tinctoriusL.) Agricultural and Biological Chemistry. 2014;49(10):2969–2974. doi: 10.1080/00021369.1985.10867182. [DOI] [Google Scholar]
  29. Sato K., Sugimoto N., Ohta M., Yamazaki T., Maitani T., Tanamoto K. Structure determination of minor red pigment in carthamus red colorant isolated by preparative LC/MS. Food Addit Contam. 2003;20(11):1015–1022. doi: 10.1080/02652030310001615177. [DOI] [PubMed] [Google Scholar]
  30. Si W., Yang W., Guo D., Wu J., Zhang J., Qiu S.…Wu W. Selective ion monitoring of quinochalcone C-glycoside markers for the simultaneous identification of Carthamus tinctorius L. in eleven Chinese patent medicines by UHPLC/QTOF MS. J Pharm Biomed Anal. 2016;117:510–521. doi: 10.1016/j.jpba.2015.09.025. [DOI] [PubMed] [Google Scholar]
  31. Sommella E., Ismail O.H., Pagano F., Pepe G., Ostacolo C., Mazzoccanti G., Campiglia P. Development of an improved online comprehensive hydrophilic interaction chromatography x reversed-phase ultra-high-pressure liquid chromatography platform for complex multiclass polyphenolic sample analysis. J Sep Sci. 2017;40(10):2188–2197. doi: 10.1002/jssc.201700134. [DOI] [PubMed] [Google Scholar]
  32. Uliyanchenko E., Cools P.J., van der Wal S., Schoenmakers P.J. Comprehensive two-dimensional ultrahigh-pressure liquid chromatography for separations of polymers. Anal Chem. 2012;84(18):7802–7809. doi: 10.1021/ac3011582. [DOI] [PubMed] [Google Scholar]
  33. Villa C., Costa J., Oliveira M.B., Mafra I. Novel quantitative real-time PCR approach to determine safflower (Carthamus tinctorius) adulteration in saffron (Crocus sativus) Food Chem. 2017;229:680–687. doi: 10.1016/j.foodchem.2017.02.136. [DOI] [PubMed] [Google Scholar]
  34. Wang S.S., Xu H.Y., Ma Y., Wang X.G., Shi Y., Huang B.…Yang H.J. Characterization and rapid identification of chemical constituents of NaoXinTong capsules by UHPLC-linear ion trap/Orbitrap mass spectrometry. J Pharm Biomed Anal. 2015;111:104–118. doi: 10.1016/j.jpba.2015.01.020. [DOI] [PubMed] [Google Scholar]
  35. Wang Y., Lu X., Xu G. Development of a comprehensive two-dimensional hydrophilic interaction chromatography/quadrupole time-of-flight mass spectrometry system and its application in separation and identification of saponins from Quillaja saponaria. J Chromatogr A. 2008;1181(1–2):51–59. doi: 10.1016/j.chroma.2007.12.034. [DOI] [PubMed] [Google Scholar]
  36. Yang W., Si W., Zhang J., Yang M., Pan H., Wu J.…Guo D. Selective and comprehensive characterization of the quinochalcone C-glycoside homologs in Carthamus tinctorius L. by offline comprehensive two-dimensional liquid chromatography/LTQ-Orbitrap MS coupled with versatile data mining strategies†. RSC Advances. 2016;6(1):495–506. doi: 10.1039/C5RA23744K. [DOI] [Google Scholar]
  37. Li X., Stoll D.R., Carr P.W. Equation for Peak Capacity Estimation in Two-Dimensional Liquid Chromatography. Anal. Chem. 2009;81(2):845–850. doi: 10.1021/ac801772u. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yao C.-liang., Yang W.-zhi., Si W., Shen Y., Zhang N.-xia., Chen H.-li.…Guo D.-an. An enhanced targeted identification strategy for the selective identification of flavonoid O-glycosides from Carthamus tinctorius by integrating offline two-dimensional liquid chromatography/linear ion-trap-Orbitrap mass spectrometry, high-resolution diagnostic product ions/neutral loss filtering and liquid chromatography-solid phase extraction-nuclear magnetic resonance. J Chromatogr A. 2017;1491:87–97. doi: 10.1016/j.chroma.2017.02.041. [DOI] [PubMed] [Google Scholar]
  39. Yao D., Wang Z., Miao L., Wang L. Effects of extracts and isolated compounds from safflower on some index of promoting blood circulation and regulating menstruation. J Ethnopharmacol. 2016;191:264–272. doi: 10.1016/j.jep.2016.06.009. [DOI] [PubMed] [Google Scholar]
  40. Yin H.B., He Z.S. A novel semi-quinone chalcone sharing a pyrrole ring C-glycoside from Carthamus tinctorius. Tetrahedron Letters. 2000;41(12):1955–1958. doi: 10.1016/S0040-4039(00)00100-3. [DOI] [Google Scholar]
  41. Yoon H.R. Radical-Scavenging Activities of Four Quinochalcones of Safflower. Journal of the Korean Society for Applied Biological Chemistry. 2008;51(4):346–348. doi: 10.3839/jksabc10.3839/jksabc.2008.061. [DOI] [Google Scholar]
  42. Yuan L., Zhang Z., Hou Z., Yang B., Li A., Guo X.…Li Y. Rapid classification and identification of complex chemical compositions in traditional Chinese medicine based on UPLC-Q-TOF/MS coupled with data processing techniques using the KuDieZi injection as an example. Analytical Methods. 2015;7(12):5210–5217. doi: 10.1039/C4AY03103B. [DOI] [Google Scholar]
  43. Yue S., Tang Y., Li S., Duan J.A. Chemical and biological properties of quinochalcone C-glycosides from the florets of Carthamus tinctorius. Molecules. 2013;18(12):15220–15254. doi: 10.3390/molecules181215220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zhao G., Gai Y., Chu W.J., Qin G.W., Guo L.H. A novel compound N(1), N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine isolated from Carthamus tinctorius L. and acting by serotonin transporter inhibition. Eur Neuropsychopharmacol. 2009;19(10):749–758. doi: 10.1016/j.euroneuro.2009.06.009. [DOI] [PubMed] [Google Scholar]
  45. Zhou X., Tang L., Xu Y., Zhou G., Wang Z. Towards a better understanding of medicinal uses of Carthamus tinctorius L. in traditional Chinese medicine: A phytochemical and pharmacological review. J Ethnopharmacol. 2014;151(1):27–43. doi: 10.1016/j.jep.2013.10.050. [DOI] [PubMed] [Google Scholar]
  46. Zhou Y.Z., Chen H., Qiao L., Xu N., Cao J.Q., Pei Y.H. Two new compounds from Carthamus tinctorius. J Asian Nat Prod Res. 2008;10(5–6):429–433. doi: 10.1080/10286020801892425. [DOI] [PubMed] [Google Scholar]
  47. Xiao J., Capanoglu E., Jassbi A.R., Miron A. Advance on the Flavonoid C-glycosides and Health Benefits. Crit Rev Food Sci Nutr. 2016;56(Suppl 1):S29–S45. doi: 10.1080/10408398.2015.1067595. [DOI] [PubMed] [Google Scholar]
  48. Zhang L.L., Tian K., Tang Z.H., Chen X.J., Bian Z.X., Wang Y.T., Lu J.J. Phytochemistry and Pharmacology of Carthamus tinctorius L. Am J Chin Med. 2016;44(2):197–226. doi: 10.1142/S0192415X16500130. [DOI] [PubMed] [Google Scholar]
  49. Kubica P., Kot-Wasik A., Wasik A., Namiesnik J., Landowski P. Modern approach for determination of lactulose, mannitol and sucrose in human urine using HPLC-MS/MS for the studies of intestinal and upper digestive tract permeability. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;907:34–40. doi: 10.1016/j.jchromb.2012.08.031. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary data 1
mmc1.doc (406.5KB, doc)

Articles from Food Chemistry: X are provided here courtesy of Elsevier

RESOURCES