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| MODERN RESEARCH ON CHINESE MATERIA MEDICA |
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| Year : 2015 | Volume
: 1
| Issue : 3 | Page : 27-39 |
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Double off-line two-dimensional liquid chromatography for separation and identification of compounds in Salvia Miltiorrhiza (Danshen)
Ji-xia Wang1, Xiu-li Zhang1, Fan Yang1, Hong-li Jin1, Li-ying Shi2, Wei-jia Zhou1, Yan-fang Liu1, Xin-miao Liang1
1 Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China
2 Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences; Institute of Materia Medica, Dalian University, Dalian, China
| Date of Web Publication | 11-Sep-2020 |
Correspondence Address:
Xiu-li Zhang
Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian
China
 Source of Support: None, Conflict of Interest: None  | 5 |
DOI: 10.15806/j.issn.2311-8571.2015.0020
Background: Danshen is an important traditional Chinese medicine (TCM) used for the treatment of cardiovascular and cerebrovascular diseases. Separation and analysis of its components have been widely investigated. However, the systematical two dimensional liquid chromatography (2D-LC) methods have not been developed to comprehensively separate and characterize its components.
Objective: In this work, double off-line 2D-LC methods were aimed to develop for the systematical separation of compounds from Danshen.
Methods: Using solid phase extraction (SPE), the Danshen extract was divided into a medium-polar fraction (Sample I) and a weak-polar fraction (Sample II) according to their polarities. Based on reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) modes, a 2D-HILIC × RPLC system and a 2D-RPLC × RPLC system were designed for the separation of Sample I and Sample II, respectively. According to reversed-phase and HILIC columns selectivities characterized in our previous reports, ZIC-HILIC and XTerra C18 were employed to build the 2D-HILIC × RPLC system and Click TE-CD and XTerra C18 for the 2D-RPLC × RPLC system, respectively.
Results: The 2D-HILIC × RPLC and 2D-RPLC × RPLC systems exhibited excellent orthogonality for the separation of Sample I and Sample II, respectively. Their orthogonalities were 88.42% and 63.24%. Based on these double 2D-LC systems combined with mass spectrometry, at least 200 compounds were found and 33 compounds of them were identified, including 16 phenolic acids and 17 diterpenoid quinines.
Conclusion: These results suggest that these two off-line 2D-LC methods are effective for the separation and characterization of components in Danshen.
Keywords: two-dimensional liquid chromatography, separation, identification, Danshen
How to cite this article:
Wang Jx, Zhang Xl, Yang F, Jin Hl, Shi Ly, Zhou Wj, Liu Yf, Liang Xm. Double off-line two-dimensional liquid chromatography for separation and identification of compounds in Salvia Miltiorrhiza (Danshen). World J Tradit Chin Med 2015;1:27-39 |
How to cite this URL:
Wang Jx, Zhang Xl, Yang F, Jin Hl, Shi Ly, Zhou Wj, Liu Yf, Liang Xm. Double off-line two-dimensional liquid chromatography for separation and identification of compounds in Salvia Miltiorrhiza (Danshen). World J Tradit Chin Med [serial online] 2015 [cited 2023 Dec 1];1:27-39. Available from: https://www.wjtcm.net/text.asp?2015/1/3/27/294835 |
| Introduction | |  |
Traditional Chinese medicines (TCMs) are now receiving considerable attention for drug discovery given their wide variety of biological activities. Analysis and purification of compounds from TCMs is a critical step for biochemical, pharmaceutical and clinical research[1],[2],[3]. High-performance liquid chromatography (HPLC) is the most widely used separation technique for analysis and purification of compounds from TCMs[4],[5],[6]. However, one-dimensional liquid chromatography fails to provide sufficient resolving power for the separation of targeted compounds from TCMs, which usually contain hundreds of thousands of compounds with great differences in category, polarity and concentration. Therefore, two-dimensional liquid chromatography (2D-LC), introduced by Frei and Erni in 1978, was developed to improve peak capacity and reduce sample complexity to an acceptable level[7]. A 2D-LC method can be developed based on the same or different chromatography modes, including reversed-phase, ion exchange, size exclusion or hydrophilic interaction chromatography. Among these modes, reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) are the most widely used in sample separation.
2D-RPLC × RPLC is one of the most prospective separation systems due to its robustness, the outstanding peak capacity in each dimension and the compatibility of mobile phase in each dimension with mass spectrometry (MS)[8],[9],[10]. The resolving power of 2D-RPLC × RPLC systems depends on the different selectivity of the two stationary phases in both dimensions. Due to traditional C18 columns with high similarities, some new stationary phases have been synthesized and used in constructing 2D-RPLC × RPLC systems with good orthogonality. Chen et al. designed a comprehensive 2D-RPLC × RPLC system using CN and ODS columns for the separation of components in Rhizoma chuanxiong[11] Based on these two columns, a 2D-RPLC × RPLC system was established for analysis of components in Swertia franchetiana Smith[12] In light of differences in selectivity between C18 and phenyl columns, they were used to design a 2D-RPLC × RPLC system for the evaluation of retention behaviors of polycyclic aromatic hydrocarbons[13]. A novel click oligo(ethylene glycol) (Click OEG) stationary phase and a C18 column were employed to develop a excellent orthogonal system to separate LignumDalbergiae Odoriferae[141. Furthermore, this 2D-RPLC × RPLC system was used to purify compounds from this herb[15]. Although 2D-RPLC × RPLC systems have been widely used in the separation of TCMs, their orthogonality has a certain limitation for the similar retention mechanisms.
HILIC is an effective technique for the separation of polar compounds[16]. For its retention mechanisms different from RPLC, HILIC provides complementary selectivity to RPLC, which is useful for constructing highly orthogonal 2D-LC systems. Based on XTerra C18 and Click β-CD, a 2D-RPLC × HILIC system was developed for the separation of polar and medium-polar components in TCMs[8]. This 2D-RPLC × HILIC system was also used for the isolation of flavonoids from licorice extract[17]. Guo et al. used a C18 column and an XAmide column to develop a 2D-RPLC × HILIC system for the separation of saponins from leaves of Panax notoginseng[181. These 2D-LC methods provide a powerful means for the analysis of TCMs.
Danshen, the dried root of Salvia miltiorrhiza, is a traditional Chinese medicine (TCM) widely used in China for the treatment of cardiovascular and cerebrovascular diseases[19],[20]. The Danshen Dripping Pill is currently in phase III clinical trial with a great hope to be the first Food and Drug Administration (FDA) approved TCM. Danshen extract contains two main types of ingredients, including water-soluble phenolic acids and lipophilic diterpenoid quinines. Until now, more than 100 compounds have been isolated[21]. High-speed counter-current chromatography[22],[23],[24], capillary zone electrophoresis[25],[26] and HPLC[27],[28],[29],[30],[31] with UV detector or mass spectrometry have been used to separate and identify compounds from Danshen. Zhu et al. has identified forty constituents from Radix Salvia miltiorrhizae using RPLC combined with diode-array detection, electrospray ionization time-of-flight mass spectrometry and electrospray ionization quadrupole ion trap mass spectrometry[27]. Using normal phase HPLC, tanshinone I, tanshinone IIA and cryptotanshinone were successfully separated from Salvia miltiorrhiza Bunge[28]. Although the analysis of components from Danshen has been widely investigated[20], the proper 2D-LC methods have not been developed for its systematical separation.
In this work, the Danshen extract was divided into the medium-polar fraction and the weak-polar fraction using solid phase extraction (SPE). Subsequently, a 2D-RPLC × HILIC system and a 2D-RPLC × RPLC system were developed for the separation and identification of components in the medium-polar fraction and the weak-polar fraction, respectively. And by combination with MS, compounds in these fractions were identified or tentatively characterized.
| Experimental | | |
1. Reagents and chemicals
Acetonitrile (ACN) and methanol (MeOH) of HPLC grade were purchased from Merck (Darmstadt, Germany). Formic acid (FA) was from Acros (Fair Lawn, NJ, USA). Water (H2O) was prepared by a Milli-Q water purification system (Billerica, MA, USA). XTerra C18, Atlantis T3 and Atlantis HILIC Silica were supplied by Waters (Milford, MA, USA). XAqua C18, XAqua CN, XAmide and Unitary NH2 were from Acchrom (Beijing, China). Click OEG, Click TE-CD and Click TE-Cys were homemade[32],[33],[34]. TSKgel Amide- 80 and ZIC-HILIC were purchased from Tosoh Biosciences (Shanghai, China) and Merck SeQuant (Darmstadt, Germany), respectively. Except as noted, all of these columns were 150 mm × 4.6 mm with 5 μm particle size.
2. Sample preparation
Danshen, the dried root of Salvia miltiorrhiza, was collected in Henan province (China) and authenticated by Institute of Medication, Xiyuan hospital of China Academy of Traditional Chinese Medicine. The procedures of extraction were as follows: the dried root was ground into powder and sieved through a No. 40 mesh. 1 g of powder sample was extracted for 30 min by sonication using 25 mL of MeOH-H2O (70/30, v/v). After centrifugation for 15 min at 5000 rpm, 5 mL of the supernatant was filtered through a 0.45 μm cellulose membrane and evaporated to 3 mL under a gentle stream of nitrogen. The obtained solution contained about MeOH-H2O (50/50, v/v).
An XAqua C18 cartridge (200 mg, Acchrom, Beijing, China) was first activated with 1 mL of MeOH and equilibrated with 1 mL of MeOH-H2O (50/50, v/v). 600 μL of the obtained solution was loaded onto this cartridge. It was eluted with 1 mL of ACN-H2O containing 0.5% FA (50/50, v/v) for Sample I and eluted with 1 mL of ACN-H2O containing 0.5% FA (90/10, v/v) for Sample II. This step was repeated thrice and the respective fractions were combined. These fractions were evaporated to dryness under a gentle stream of nitrogen. The Sample I and Sample II were dissolved in 800 μL of MeOH-H2O (50/50, v/v) and 700 μL of MeOH-H2O (65/35, v/v), respectively. These solutions were stored at approximate 4 oC before use.
3. Chromatographic conditions
The samples were analyzed by HPLC system (Waters, MA, USA) equipped with an Alliance 2695 quaternary pump and an ultraviolet detector (UV) at 280 nm. The mobile phases consisted of 0.5% FA in H2O (A) and ACN (B) at a constant flow rate of 1.0 mL/min. The column temperature was set at 30 oC. The total sample was separated on the XAqua C18 using the following gradients: (1) 0-15 min, 10%-30% B; (2) 15-20 min, 30%–60% B; (3) 20–30 min, 60%–95% B; (4) 3035 min, 95%–95% B.
3.1. Chromatographic conditions for Sample I
The Sample I was separated on different columns. After optimization, the suitable gradient conditions were obtained. The gradients performed on the XTerra C18 were from 5% B to 25% B within 20 min and then increased linearly to 45% B within another 10 min. The gradients on TSKgel Amide-80 were as follows: (1) 0–20 min, 95%–90% B; (2) 20–30 min, 90%–60% B. The gradients on XAmide started from 90% B to 60% B within 30 min. The eluted conditions on ZIC-HILIC were as follows: (1) 0–20 min, 95%–83% B; (2) 20–30 min, 83%–50% B. The gradients on Click TE-Cys, Atlantis HILIC Silica and Unitary NH2 were: 0 ~ 15 ~ 30 min, 95% ~ 85% ~ 10% B; 0 ~ 20 ~ 30 min, 95% ~ 95% ~ 60% B; 0 ~ 30 min, 95% ~ 10% B; respectively.
3.2. Chromatographic conditions for Sample II
To effectively separate Sample II, different columns were tested. The gradients performed on XAqua C18 were as follows: (1) 0–20 min, 50%–75% B; (2) 20–30 min, 75%–90% B. The gradients on Atlantis T3 were the same as those on XAqua C18. The gradients on XTerra C18 were from 40% B to 65% B within 20 min and then linearly increased to 80% B within another 10 min. The gradients on Click TE-CD were: 0 ~ 25 ~ 30 min, 25% ~ 30% ~ 45% B. The gradients on Click OEG started from 25% B to 55% B within 30 min. The gradients performed on XAqua CN were as follows: (1) 0–20 min, 20–35% B; (2) 20–30 min, 35–70% B.
3.3. Two-dimensional liquid chromatography for separation of Sample I and II
The separation conditions of Sample I were as follows: ZIC-HILIC was used as the first dimensional column. A volume of 50 μL of Sample I was injected into the first dimension. The linear gradient of the mobile phase was as follows: 0 ~ 20 ~ 30 min, 95% ~ 83% ~ 50% B. Fractions were collected manually from 3 min to 18 min at 1-min interval and denoted orderly as Fraction SI-F1 to Fraction SI-F16. Each fraction was concentrated to dryness and redissolved in 100 μL of MeOH-H2O (50/50, v/v). 10 μL of each fraction was injected directly into the second dimension. XTerra C18 (150 mm × 2.1 mm, I.D. 5 μm) was used as the second dimensional column with the flow rate at 0.2 mL/min. The linear gradient elution on this column was: 0 ~ 20 ~ 30 min, 8% ~ 28% ~ 48% B.
The separation conditions of Sample II were as follows: Click TE-CD was used as the first dimensional column. A volume of 50 μL of Sample II was injected into the first dimension. The linear gradient of the mobile phase was as follows: 0 ~ 20 ~ 25 min, 25% ~ 29% ~ 55% B. Fractions were collected manually from 6 min to 21 min at 1-min interval and denoted orderly as Fraction SII-F1 to Fraction SII-F16. Each fraction was concentrated to dryness and redissolved in 100 μL of MeOH-H2O (50/50, v/v). 10 μL of each fraction was injected directly into the second dimension. XTerra C18 (150 mm ×2.1 mm, I.D. 5 μm) was used as the second dimensional column with the flow rate at 0.2 mL/min. The linear gradient elution on this column was: 0 ~ 20 ~ 30 min, 45% ~ 70% ~ 85% B.
4. LC-MS analysis
Compounds of fractions in Section 3.3 (EXPERIMENTAL) were characterized using an Agilent 1290 Infinity LC instrument coupled to Agilent 6540 series Q-TOF-MS (Agilent Technologies Inc., USA), which equipped with electrospray ionization (ESI) source. Fractions of Sample I and Sample II were separated using the same chromatographic conditions as in Section 3.3 (EXPERIMENTAL). Compounds in fractions of Sample I were detected in the negative ion mode. While compounds in fractions of Sample II were detected in the positive ion mode. The mass spectrometer conditions were as follows: nebulizer gas pressure (35 psi), drying gas flow rate (8 L/min), gas temperature (350 °C), capillary voltage (3500 V) and collision energy (25 eV). The MS scan ranged from 100 to 1000 m/z and the MS/MS scan ranged from 50 to 1000 m/z.
5. Data analysis
The orthogonality of any two chromatographic systems was calculated according to the literature[8],[35]. Retention times in the second dimension are normalized according to Eq. (1),
where tRmax and tRmin represent the retention times of the most and least retained solute in the data set, respectively. The retention times are converted to the normalized tRi(norm) ranging from 0 to 1. For the x-axis, the grid number is the number of fractions in the first dimension and the grid number of the y-axis is calculated according to Eq. (2),
where Nump is the number of peaks detected by the two dimensional chromatography and Fracx is the grid number of the x-axis. The coverage of data points can describe the orthogonality calculated by Eq. (3),
where Σbins is the number of bins containing data points in the two dimensional plot and Pmax is the total peak capacity obtained as a sum of all bins. Σbins(blank) is the number of bins containing data points in the two dimensional plot when the fractions are analyzed using identical conditions for the first and second dimensions, namely a non-orthogonal system, in which the data points would be lined up along the diagonal and the surface coverage should be 10%. The data was calculated using Microsoft excel 2010 and Origin 8.0.
| Results and Discussion | | |
1. Design of 2D-LC separation systems
1.1. Pretreatment of Danshen extracts
Given that the chemical compositions of Chinese herbs are very complex, it is necessary to reduce sample complexity by pretreatment procedures. The Danshen extracts was first separated on an XCharge C18 column [Figure 1]A. According to the retention of compounds on this column under this gradient conditions, the extracts mainly contained mediumpolar components and weak-polar components. After SPE pretreatment, the extracts can be divided into two sections: the medium-polar components (Sample I) eluted with ACN-H2O containing 0.5% FA (50/50, v/v) and the weak-polar components (Sample II) eluted with ACN-H2O containing 0.5% FA (90/10, v/v) [Figure 1]B and [Figure 1]C. Because the medium-polar components have good retention under RPLC and HILIC modes and these two modes have different separation selectivity for their different retention mechanisms. Herein, a 2D-HILIC × RPLC system was designed to separate Sample I. While the weak-polar components could not be retained by HILIC columns but had good retention on RPLC columns. Therefore, a 2D-RPLC × RPLC system was designed to separate Sample II. Thus, double off-line 2D-LC systems were designed for the systematical separation of Danshen. | Figure 1: The chromatograms of samples on XCharge C18 column. (A) the total extracts of Danshen; (B) Sample I (the medium-polar components); (C) Sample II (the weak-polar components).
Click here to view |
1.2. Optimization of separation conditions for Sample II
12 kinds of reversed-phase columns have been characterized using linear solvation energy relationships (LSERs) combined with fundamental retention equations in our previous work[36]. According to these column selectivities, representative columns were selected, including Xterra C18, Atlantis T3, XAqua C18, XAqua CN, Click OEG and Click TE-CD. The separation conditions of Sample II on these columns were optimized. Sample II was separated on these columns under optimal chromatographic conditions and their chro-matographs are shown in [Figure 2]. As expected, the elution orders of the components of Sample II on XTerra C18 [Figure 2]A, Atlantis T3 [Figure 2]B and XAqua C18 [Figure 2]C are similar, meaning that these columns are near-equivalent. Among these three columns, XTerra C18 could provide sharpest peaks and was selected. Based on the elution orders of these components on XAqua CN, Click OEG and Click TE-CD [Figure 2]D, [Figure 2]E, [Figure 2]F, Click TE-CD can provided highest difference in pattern from XTerra C18. Additionally, samples are well separated in the second dimension, which is useful for the improvement of the resolving power of 2D-LC[37]. Therefore, Click TE-CD and XTerra C18 were used in the first and second dimension respectively to develop a 2D-RPLC × RPLC system for the separation of Sample II. | Figure 2: The chromatograms of Sample II on XTerra C18 (A), Atlantis T3 (B), XAqua C18 (C), XAqua CN (D), Click OEG (E) and Click TE-CD (F).
Click here to view |
1.3. Optimization of separation conditions for Sample I
In the process of optimization of separation conditions for Sample II, XTerra C18 could provide better separation power and was tested for the separation of Sample I [Figure 3]A. It can be seen that Sample I is well separated on XTerra C18. Therefore, this column was selected and used for constructing a orthogonal system for the separation of Sample I. According to selectivities of HILIC columns evaluated using our hydrophilic-subtraction model[38], representative columns were selected, including TSKgel Amide-80, XAmide, ZIC-HILIC, Click TE-Cys, Atlantis HILIC Silica and Unitary NH2. Separation of Sample I on these HILIC columns under the optimal experimental conditions is showed in [Figure 3]. The elution orders of components of Sample I on TSKgel Amide- 80, XAmide, ZIC-HILIC and Click TE-Cys are similar [Figure 3]B, [Figure 3]C, [Figure 3]D, [Figure 3]E. Among these columns, the sharp peaks of components are obtained on ZIC-HILIC [Figure 3]D. It can be explained as follows: Click TE-Cys is synthesized by immobilizing cysteine onto the silica surface and it possesses positive charges on the stationary phase surface under the acid conditions[34], which will interact with the partially ionized phenolic acids, resulting in peak tailing [Figure 3]E. The neutral TSKgel Amide-80 and XAmide cannot provide symmetry peaks of the main components for molecular interactions between them [Figure 3]B and [Figure 3]C. While ZIC-HILIC is prepared by grafting sulfobetaine to the silica surface with negatively charged sulfonate groups as a distal moiety and positively charged quaternary ammonium in the proximal location to the silica surface[39]. The negative charges of this stationary phase surface lead to a certain ionic repulsion to phenolic acids, which is useful for obtaining good peaks [Figure 3]D. This similar phenomenon has also been reported in the separation of basic compounds[40]. Due to the relatively weak hydrophilicity of Atlantis HILIC Silica, Sample I was eluted at dead time even under the condition of 95% ACN [Figure 3]F. When Sample I was separated on Unitary NH2, the peak high decreased greatly for death absorption of phenolic acids [Figure 3]G. In summary, ZIC-HILIC was the optimal one for the separation of Sample I. Therefore, ZIC-HILIC and XTerra C18 were used in the first and second dimension respectively to develop a 2D-HILIC × RPLC system for the separation of Sample I. | Figure 3: The chromatograms of Sample I on XTerra C18 (A), TSKgel Amide-80 (B), XAmide (C), ZIC-HILIC (D), Click TE-Cys (E), Atlantis HILIC Silica (F) and Unitary NH2 (G).
Click here to view |
2. 2D-HILIC × RPLC system for separation of sample I
Using the 2D-HILIC × RPLC system developed in Section 1.3 (RESULTS AND DISCUSSION), ZIC-HILIC and XTerra C18 were employed as the first and second dimension columns respectively to separate Sample I. Of note, XTerra C18 (150 mm × 2.1 mm, I.D. 5 μm) was used in the second dimension to enhance the detected concentration of components. During the separation of Sample I, 366 peaks were detected in SI-F1 ~ SI-F16 fractions. According to Eq. (2), the normalized data points were distributed in space of 16 × 23 bins (368, which was approximate to 366) and bins containing data points were 257 [Figure 4]A. Σbins(blank) was calculated by the non-orthogonal system that ZIC-HILIC was used in both dimensions to analyze these 16 fractions. As presented in [Figure 4]B, Σbins(blank) was 52. According to Eq. (3), the degree of orthogonality of ZIC-HILIC and XTerra C18 in the separation of Sample I was 88.42%. The corresponding three dimensional chromatogram is displayed in [Figure 5]. The results demonstrated that this 2D-HILIC × RPLC system was highly orthogonal in the separation of medium-polar components in Danshen. | Figure 4: Normalized plots of this 2D-HILIC × RPLC system for the separation of Sample I: (A) ZIC-HILIC × XTerra C18 system; (B) ZIC-HILIC × ZIC-HILIC system.
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 | Figure 5: Three dimensional chromatogram of SI-F1 ~ SI-F16 fractions analyzed on XTerra C18.
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3. 2D-RPLC × RPLC system for separation of sample II
Using the 2D-RPLC × RPLC system developed in Section 1.2 (RESULTS AND DISCUSSION), Click TE-CD and XTerra C18 (150 mm ×2.1 mm, I.D. 5 μm) were employed as the first and second dimension columns respectively to separate Sample II. During the separation of Sample II, 129 peaks were detected in SII-F1 ~ SII-F16 fractions. According to Eq. (2), the normalized data points were distributed in space of 16 × 8 bins (128, which was approximate to 129) and bins containing data points were 71 [Figure 6]A. Σbins(blank) was calculated by the non-orthogonal system that Click TE-CD was used in both dimensions to analyze these 16 fractions. As presented in [Figure 6]B, Σbins(blank) was 20. According to Eq. (3), the degree of orthogonality of Click TE-CD and XTerra C18 in the separation of Sample II was 63.24%. The corresponding three dimensional chromatogram is displayed in [Figure 7]. The results demonstrated that this 2D-RPLC × RPLC system was highly orthogonal in the separation of weak-polar components in Danshen. | Figure 6: Normalized plots of this 2D-HILIC × RPLC system for the separation of Sample II: (A) Click TE-CD × XTerra C18 system; (B) Click TE-CD × Click TE-CD system.
Click here to view |
 | Figure 7: Three dimensional chromatogram of SII-F1 ~ SII-F16 fractions analyzed on XTerra C18.
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4. Characterization of components in sample I and sample II
Fractions of Sample I and Sample II were analyzed using high resolution ESI-Q-TOF-MS/MS. At least 200 compounds were found. Generally, water-soluble phenolic acids and lipophilic diterpenoid quinines in Danshen extract are believed to be active ingredients. In these fractions, a total of 33 compounds including 16 phenolic acids and 17 diterpenoid quinines were identified in [Table 1]. Among them, 11 compounds were identified unambiguously by comparing their retention times and mass spectra with standard compounds, including protocatechuic aldehyde (1), caffeic acid (2), danshensu (4), rosmarinic acid (5), salvianolic acid C (6), salvianolic acid A (11), lithospermic acid (13), salvianolic acid B (16), dihydrotanshinone (23), cryptotanshinone (32) and tanshinone IIA (33). And other compounds were identified tentatively based on MS/MS fragmentation rules reported in reference[27],[41],[42]. As shown in [Table 1], retention times of compounds 8 and 10 were 21.86 min and 21.93 min in the second dimension, but they were fractioned into Fraction SI-F8 and Fraction SI-F10 in the first dimension, respectively. The same phenomenon can be found in compounds 24 and 30. Therefore, compounds with very close retention times in one-dimensional LC system were identified obviously by such off-line 2D-LC separation systems. Compared to the published work using a one-dimensional LC system[27],[41],[42], these off-line 2D-LC separation systems provided great potential in separating more compounds, especially in isomer compounds or compounds with close retention times in one-dimensional LC system.
Additionally, the unidentified components were listed in [Table S1], which were difficult to be identified based on the current information and needed to be characterized via further work.
| Conclusion | | |
Double off-line 2D-LC methods were successfully developed for the systematical separation of compounds from Danshen. The Danshen extract was first divided into the medium-polar fraction (Sample I) and the weak-polar fraction (Sample II) using solid phase extraction (SPE). Subsequently, ZIC-HILIC and XTerra C18 were used to construct the 2D-HILIC×RPLC system for the separation of Sample I and Click TE-CD and XTerra C18 were used to establish the 2D-RPLC×RPLC system for the separation of Sample II. These two off-line 2D-LC systems exhibited excellent orthogonality, reaching 88.42% and 63.24%, respectively. By identification using MS, 33 compounds were identified, including 16 phenolic acids and 17 diterpenoid quinines. These double off-line 2D-LC methods exhibited excellent performance in the separation and characterization of components in Danshen.
| Acknowledgements | | |
This work was funded by Project of National Science Foundation of China (81473436, 81274077 and 81403100).
Summary
This supporting information file includes additional information and results as described in the article.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1]
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