1742 Aihua Zhang Di Zou Guangli Yan Yunlong Tan Hui Sun Xijun Wang ∗ Department of Pharmaceutical Analysis, National TCM Key Laboratory of Serum Pharmacochemistry, Heilongjiang University of Chinese Medicine, Harbin, China Received January 31, 2014 Revised April 11, 2014 Accepted April 15, 2014
J. Sep. Sci. 2014, 37, 1742–1747
Research Article
Identification and characterization of the chemical constituents of Simiao Wan by ultra high performance liquid chromatography with mass spectrometry coupled to an automated multiple data processing method The chemical constituents of Simiao Wan (SW), a traditional Chinese medicine preparation, are difficult to determine and remain unclear. To more efficiently detect ions, a multiple data processing approach has been used in the characterization of the compounds. In this study, a rapid and sensitive method based on ultra high performance liquid chromatography with mass spectrometry and the multiple data processing approach was established to characterize the chemical constituents of SW. Ultra high performance liquid chromatography with mass spectrometry coupled with the multiple data processing approach could efficiently remove nonrelated ion signals from accurate mass data. We report the application of the multiple data processing approach for comprehensive detection and rapid identification of chemical constituents of SW. Of note, the total analysis time for separation was less than 20 min without losing any resolution. In the variable, importance in projection plot of orthogonal projection to latent structure-discriminant analysis, a total of 72 ions of interest (37 ions in positive mode, 38 ions in negative mode and three ions in both mode) were extracted or tentatively characterized based on their retention times, exact mass measurement for each molecular ion and subsequent fragment ions. In summary, the methodology proposed in this study could be valuable for the structural characterization and identification of the multiple constituents in the traditional Chinese medicine formula SW. Keywords: Multiple data processing approach / Principal component analysis / Simiao Wan identification / Ultra high performance liquid chromatography DOI 10.1002/jssc.201400105
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Over the past decades, traditional Chinese medicine (TCM) has been an important resource for providing a large number of potential lead compounds for drug discovery, and represents a cornucopia of plant-derived remedies to discover novel lead molecules for the development of new drugs [1, 2]. As TCM plays an important role in drug discovery and human health, investigation on phytochemical constituents of TCM has become a hotspot, with more and more publications conCorrespondence: Dr. Aihua Zhang, Department of Pharmaceutical Analysis, National TCM Key Laboratory of Serum Pharmacochemistry, Heilongjiang University of Chinese Medicine, Heping Road 24, Harbin 150040, China E-mail: [email protected] Fax: +86-451-82110818
Abbreviations: Mdpa, multiple data processing approach; PCA, principal component analysis; SW, Simiao Wan; TCM, traditional Chinese medicine C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
cerning TCM published per year [3–5]. It is well known that TCM is commonly believed to operate due to the synergistic effects of their multicomponents. Therefore, the detection and identification of components may be equally important for understanding the pharmacological basis. In spite of this, the actual value of TCM has not been fully recognized worldwide, due to its complex components. Since almost every TCM is a multicomponent system, it is important to establish selective, sensitive, and feasible analytical methods for recognition, separation, and identification of multicomponents present in TCM [6]. Recent improvements in analytical instrumentation with improved sensitivity and precision to allow greater resolution of multicomponents are driving the investigation of TCM [7]. In order to discern the chemical profiles of TCM formulas, nowadays, UHPLC–Q-TOF-MS combined with the rapid ∗ Additional Corresponding Author: Professor Xijun Wang, Email: [email protected] Colour Online: See the article online to view Figs. 1–4 in colour.
www.jss-journal.com
J. Sep. Sci. 2014, 37, 1742–1747
Liquid Chromatography
1743
separation capability of LC and exact mass measurement for both MS and MS/MS has been developed as an efficient method to analyze complex TCMs [8, 9]. It provided improved chromatographic parameters resulting in significantly increased sample throughput including lower solvent consumption and lower limits of quantitation for most of target analytes compared to common method employing conventional HPLC separation [10]. Recently, we established UHPLC–MS technology with an automated multiple data processing approach (Mdpa) (Fig. S1) for the screening and identification of phytochemical constituents in Simiao Wan (SW) [11]. SW, a well-known and widely used TCM formula, has been extensively used as an adjuvant to chemotherapy for rheumatoid arthritis disease treatment in clinic [12,13]. However, its chemical constituents have not been investigated so far. UHPLC coupled with mass spectrometry provides a great deal of data, which is best used for structural elucidation and plays an important role in the analysis of the complex constituents of the herb medicine. In this study, a UHPLC–ESIQ-TOF-MS system using a C18 column (1.7 um particle size) was established for the rapid separation and structural identification of the constituents in SW. Of note, it was combined with Mdpa to provide unique high-throughput capabilities for a phytochemical study with excellent MS mass accuracy and enhanced MS data acquisition. The results obtained in this research will provide a basis for quality control and further in vivo study of SW.
2 Materials and methods 2.1 Chemical and materials HPLC-grade acetonitrile, formic acid, and methanol were purchased from Merck (Merck, Darmstadt, Germany); Distilled water was purchased from Watson’s Food & Beverage (Guangzhou, China). The distilled water was used for the extraction and preparation of samples. Leucine enkephalin was purchased from Sigma–Aldrich (MO, USA). Cortex Phellodendri Chinensis, Rhizoma Atractylodis, Radix Achyranthis Bidentatae, and Semen Coicis were purchased from Harbin Tongrentang Drug Store (Harbin, China), and authenticated by Professor Xijun Wang, Department of Pharmacognosy of Heilongjiang University of Chinese Medicine. The voucher specimens were deposited in the authors’ laboratory.
2.2 Preparation of SW samples for LC–MS analysis According to the original composition and preparation method of SW recorded in “Chinese Pharmacopeia,” SW was prepared by the following procedure. The methanol extract from SW was made by a common method. Briefly, Cortex Phellodendri Chinensis (100 g), Rhizoma Atractylodis (50 g), Radix Achyranthis Bidentatae (50 g), and Semen Coicis (100 g) were mixed and extracted twice with ten times the volume v/w C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Multiple data processing approach for accurate mass UHPLC–MS data of SW in positive mode. PCA of UHPLC–MS spectra of SW group vs. control group in positive mode (A); 3D PCA of UHPLC–MS spectra in positive mode (B); VIP-plot for accurate mass UHPLC–MS data.
of 75% v/v methanol in distilled water under reflux for 1.5 h. After filtration and reclaiming the methanol, the extract was lyophilized to obtain a powder. 25 mg of SW powder was accurately weighed into a volumetric flask and subjected to ultrasonic treatment at room temperature with 50% methanol for 15 min. The supernatant was collected and filtered through a 0.22 m membrane before use.
2.3 UHPLC condition Analysis was performed on a Waters AcquityTM ultra performance LC system coupled with accurate-mass Q-TOF www.jss-journal.com
1744
J. Sep. Sci. 2014, 37, 1742–1747
A. Zhang et al.
range was set at m/z 100–1000 Da with a 0.3 s scan time. The optimal conditions of MS/MS detector were as follows: ESI+ mode, capillary voltage of 3.0 kV, sampling cone voltage was 35.0 V, extraction cone voltage was 4.0 V. The temperature was set at 110⬚C, desolvation gas temperature was 300⬚C, desolvation gas flow was 800 L/h. Nitrogen was used as nebulizer and auxiliary gas. A mass spectrometry (Elevated Energy) (MS(E)) data collection technique was performed on ESI-QTOF-MS setup with a collision energy ramp of 10–30 eV (MSE parameters: low energy, 10 eV; and high energy, 20–30 eV). Data were collected in centroid mode and mass was corrected during acquisition using an external reference (Lock-Spray) comprising a 200 pg/mL solution of leucine-enkephalin via a lockspray interface, generating a reference ion at 556.2771 Da ([M+H]+ ) for positive ESI mode and, while at m/z 554.2615 Da ([M-H]− ) in negative ion mode. All the acquisition and analysis of data were controlled by the Ezinfo Software 2.0 (Waters, Manchester, UK). The data were processed using MassLynxTM 4.1 software with the MSE program (Waters, Milford, MA, USA).
2.5 Data processing The centroid LC–MS data files were further processed with multiple data processing approach using the Waters EZinfo 2.0 software (Waters, Milford, MA, USA). For further confirmation the structure and the source of the chemical constituents of a Chinese herbal formula SW, all data matrices were introduced to MetaboLynxTM software. The ions which were present in the SW group and absent in the control group were extracted with the help of the corresponding loading plot, and further these ions were identified with a combination of elemental composition tool and MS/MS fragment mass spectra. Figure 2. Multiple data processing approach for accurate mass UHPLC–MS data of SW in negative mode. PCA of UHPLC–MS spectra of SW group vs. control group in negative mode (A); 3-D PCA of UHPLC–MS spectra in negative mode (B); VIP-plot for accurate mass UHPLC–MS data.
mass spectrometry (Waters, Milford, USA) equipped with an ESI source. The separation of all samples was performed on a UPLCTM BEH C18 column (2.1×100 mm, 1.7 m) at 35⬚C. The mobile phases were composed of acetonitrile with 0.1% v/v formic acid (A) and water with 0.1% formic acid (B) using a linear gradient program of 1–16% A (0–1.5 min), 16–20% A (1.5–5 min), 20–25% A (5–7 min), 25–35% A (7–10 min), and 35–99% A (10–20 min) at a flow rate kept at 0.3 mL/min. The injection volume was 5 L.
2.4 High-resolution accurate MS The MS instrument consisted of a Waters AcquityTM Synapt mass spectrometer equipped with ESI mode, and the mass C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3 Results and discussion 3.1 Optimization of UHPLC–ESI-Q-TOF-MS conditions In order to obtain chromatograms with good separation and strong total ion current, several mobile phase systems including methanol/water, acetonitrile/water, methanol with 0.1% formic acid and acetonitrile with 0.1% formic acid were tested to optimize the chromatographic conditions. When formic acid was added to the mobile phase, the peak capacities and shapes of all the chromatographic peaks were enhanced remarkably. The acetonitrile/water system showed more powerful separation ability and elution power for investigated compounds through the comparison of the methanol/water system. As a result, the optimal solvent systems consisting of acetonitrile and 0.1% formic acid aqueous solution on the optimized gradient mode showed a good separation and abundant signal response both in positive and negative www.jss-journal.com
J. Sep. Sci. 2014, 37, 1742–1747
Liquid Chromatography
1745
Figure 3. UHPLC–MS BPI chromatograms of EW in positive mode (A) and in negative mode (B). Each peak number is consistent with Tables S1 and S2, respectively.
ion scan mode, which increased the efficiency of ionization and gave satisfactory sensitivity, were ultimately tested as mobile phase. The selection of the fast UHPLC conditions was guided by the requirement for obtaining chromatograms with better resolution of adjacent peaks within a short time. The MS conditions, capillary voltage, sampling cone voltage, extraction cone voltage, desolvation gas temperature, and desolvation gas flow were optimized in order to achieve efficient separation and good responses to all chemical components in SW. And both positive and negative ion modes were employed to identify the corresponding signals. Series of experiments were conducted to optimize the LC chromatographic and MS conditions as described in Sections 2.3 and 2.4. And both positive and negative ion modes were employed to identify the corresponding signals.
3.2 UHPLC–ESI-Q-TOF-MS analysis of SW The experimental setup for UHPLC–ESI-Q-TOF-MS coupled with multiple data processing approach analysis is shown in Fig. S1. In order to gain the details of differences, principal component analysis (PCA), is the most widely used exploratory techniques in multivariate analysis, could convert multidimensional data space into a low dimensional model plane. In our study, PCA method was employed to phenotype the differences between the SW and control group. PCA of LC–MS spectra of SW group vs. control group in positive and negative mode was shown in Figs. 1A and 2A. Figures 1B and 2B showed the 3-D PCA of UHPLC–ESI-Q-TOF-MS spectra in positive and negative mode, respectively. The ions of interest that were present only in the SW group and absent in the control group were extracted easily by utilizing VIP-plot that could clearly display leading contributing markers. As shown in Figs. 2C and 3C, the points in the red frame were at higher level in SW group. As demonstrated above, 72 ions of interest (37 ions in positive mode, 38 ions in negative mode and three ions in both modes) were extracted, identified or tentatively C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
characterized based on their retention times, exact mass measurement for each molecular ion and subsequent fragment ions, and their information is shown in Tables S1 and S2, respectively. The mass error for molecular ions of all identified compounds was within ±6 ppm. 72 major constituents (Fig. 3A and B) including flavonoids, phenylpropanoids, alkaloids, isoflavonoids, organic acids, and saponins were tentatively characterized (Table S1).
3.3 MS/MS characterization of chemical constituents from SW All information of MS data obtained using the aforementioned protocol indicated the retention time and precise molecular mass and provided the MS/MS data which was necessary for the structural identification. The precise molecular mass was determined within a reasonable degree of measurement error (
J. Sep. Sci. 2014, 37, 1742–1747
Research Article
Identification and characterization of the chemical constituents of Simiao Wan by ultra high performance liquid chromatography with mass spectrometry coupled to an automated multiple data processing method The chemical constituents of Simiao Wan (SW), a traditional Chinese medicine preparation, are difficult to determine and remain unclear. To more efficiently detect ions, a multiple data processing approach has been used in the characterization of the compounds. In this study, a rapid and sensitive method based on ultra high performance liquid chromatography with mass spectrometry and the multiple data processing approach was established to characterize the chemical constituents of SW. Ultra high performance liquid chromatography with mass spectrometry coupled with the multiple data processing approach could efficiently remove nonrelated ion signals from accurate mass data. We report the application of the multiple data processing approach for comprehensive detection and rapid identification of chemical constituents of SW. Of note, the total analysis time for separation was less than 20 min without losing any resolution. In the variable, importance in projection plot of orthogonal projection to latent structure-discriminant analysis, a total of 72 ions of interest (37 ions in positive mode, 38 ions in negative mode and three ions in both mode) were extracted or tentatively characterized based on their retention times, exact mass measurement for each molecular ion and subsequent fragment ions. In summary, the methodology proposed in this study could be valuable for the structural characterization and identification of the multiple constituents in the traditional Chinese medicine formula SW. Keywords: Multiple data processing approach / Principal component analysis / Simiao Wan identification / Ultra high performance liquid chromatography DOI 10.1002/jssc.201400105
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Over the past decades, traditional Chinese medicine (TCM) has been an important resource for providing a large number of potential lead compounds for drug discovery, and represents a cornucopia of plant-derived remedies to discover novel lead molecules for the development of new drugs [1, 2]. As TCM plays an important role in drug discovery and human health, investigation on phytochemical constituents of TCM has become a hotspot, with more and more publications conCorrespondence: Dr. Aihua Zhang, Department of Pharmaceutical Analysis, National TCM Key Laboratory of Serum Pharmacochemistry, Heilongjiang University of Chinese Medicine, Heping Road 24, Harbin 150040, China E-mail: [email protected] Fax: +86-451-82110818
Abbreviations: Mdpa, multiple data processing approach; PCA, principal component analysis; SW, Simiao Wan; TCM, traditional Chinese medicine C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
cerning TCM published per year [3–5]. It is well known that TCM is commonly believed to operate due to the synergistic effects of their multicomponents. Therefore, the detection and identification of components may be equally important for understanding the pharmacological basis. In spite of this, the actual value of TCM has not been fully recognized worldwide, due to its complex components. Since almost every TCM is a multicomponent system, it is important to establish selective, sensitive, and feasible analytical methods for recognition, separation, and identification of multicomponents present in TCM [6]. Recent improvements in analytical instrumentation with improved sensitivity and precision to allow greater resolution of multicomponents are driving the investigation of TCM [7]. In order to discern the chemical profiles of TCM formulas, nowadays, UHPLC–Q-TOF-MS combined with the rapid ∗ Additional Corresponding Author: Professor Xijun Wang, Email: [email protected] Colour Online: See the article online to view Figs. 1–4 in colour.
www.jss-journal.com
J. Sep. Sci. 2014, 37, 1742–1747
Liquid Chromatography
1743
separation capability of LC and exact mass measurement for both MS and MS/MS has been developed as an efficient method to analyze complex TCMs [8, 9]. It provided improved chromatographic parameters resulting in significantly increased sample throughput including lower solvent consumption and lower limits of quantitation for most of target analytes compared to common method employing conventional HPLC separation [10]. Recently, we established UHPLC–MS technology with an automated multiple data processing approach (Mdpa) (Fig. S1) for the screening and identification of phytochemical constituents in Simiao Wan (SW) [11]. SW, a well-known and widely used TCM formula, has been extensively used as an adjuvant to chemotherapy for rheumatoid arthritis disease treatment in clinic [12,13]. However, its chemical constituents have not been investigated so far. UHPLC coupled with mass spectrometry provides a great deal of data, which is best used for structural elucidation and plays an important role in the analysis of the complex constituents of the herb medicine. In this study, a UHPLC–ESIQ-TOF-MS system using a C18 column (1.7 um particle size) was established for the rapid separation and structural identification of the constituents in SW. Of note, it was combined with Mdpa to provide unique high-throughput capabilities for a phytochemical study with excellent MS mass accuracy and enhanced MS data acquisition. The results obtained in this research will provide a basis for quality control and further in vivo study of SW.
2 Materials and methods 2.1 Chemical and materials HPLC-grade acetonitrile, formic acid, and methanol were purchased from Merck (Merck, Darmstadt, Germany); Distilled water was purchased from Watson’s Food & Beverage (Guangzhou, China). The distilled water was used for the extraction and preparation of samples. Leucine enkephalin was purchased from Sigma–Aldrich (MO, USA). Cortex Phellodendri Chinensis, Rhizoma Atractylodis, Radix Achyranthis Bidentatae, and Semen Coicis were purchased from Harbin Tongrentang Drug Store (Harbin, China), and authenticated by Professor Xijun Wang, Department of Pharmacognosy of Heilongjiang University of Chinese Medicine. The voucher specimens were deposited in the authors’ laboratory.
2.2 Preparation of SW samples for LC–MS analysis According to the original composition and preparation method of SW recorded in “Chinese Pharmacopeia,” SW was prepared by the following procedure. The methanol extract from SW was made by a common method. Briefly, Cortex Phellodendri Chinensis (100 g), Rhizoma Atractylodis (50 g), Radix Achyranthis Bidentatae (50 g), and Semen Coicis (100 g) were mixed and extracted twice with ten times the volume v/w C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Multiple data processing approach for accurate mass UHPLC–MS data of SW in positive mode. PCA of UHPLC–MS spectra of SW group vs. control group in positive mode (A); 3D PCA of UHPLC–MS spectra in positive mode (B); VIP-plot for accurate mass UHPLC–MS data.
of 75% v/v methanol in distilled water under reflux for 1.5 h. After filtration and reclaiming the methanol, the extract was lyophilized to obtain a powder. 25 mg of SW powder was accurately weighed into a volumetric flask and subjected to ultrasonic treatment at room temperature with 50% methanol for 15 min. The supernatant was collected and filtered through a 0.22 m membrane before use.
2.3 UHPLC condition Analysis was performed on a Waters AcquityTM ultra performance LC system coupled with accurate-mass Q-TOF www.jss-journal.com
1744
J. Sep. Sci. 2014, 37, 1742–1747
A. Zhang et al.
range was set at m/z 100–1000 Da with a 0.3 s scan time. The optimal conditions of MS/MS detector were as follows: ESI+ mode, capillary voltage of 3.0 kV, sampling cone voltage was 35.0 V, extraction cone voltage was 4.0 V. The temperature was set at 110⬚C, desolvation gas temperature was 300⬚C, desolvation gas flow was 800 L/h. Nitrogen was used as nebulizer and auxiliary gas. A mass spectrometry (Elevated Energy) (MS(E)) data collection technique was performed on ESI-QTOF-MS setup with a collision energy ramp of 10–30 eV (MSE parameters: low energy, 10 eV; and high energy, 20–30 eV). Data were collected in centroid mode and mass was corrected during acquisition using an external reference (Lock-Spray) comprising a 200 pg/mL solution of leucine-enkephalin via a lockspray interface, generating a reference ion at 556.2771 Da ([M+H]+ ) for positive ESI mode and, while at m/z 554.2615 Da ([M-H]− ) in negative ion mode. All the acquisition and analysis of data were controlled by the Ezinfo Software 2.0 (Waters, Manchester, UK). The data were processed using MassLynxTM 4.1 software with the MSE program (Waters, Milford, MA, USA).
2.5 Data processing The centroid LC–MS data files were further processed with multiple data processing approach using the Waters EZinfo 2.0 software (Waters, Milford, MA, USA). For further confirmation the structure and the source of the chemical constituents of a Chinese herbal formula SW, all data matrices were introduced to MetaboLynxTM software. The ions which were present in the SW group and absent in the control group were extracted with the help of the corresponding loading plot, and further these ions were identified with a combination of elemental composition tool and MS/MS fragment mass spectra. Figure 2. Multiple data processing approach for accurate mass UHPLC–MS data of SW in negative mode. PCA of UHPLC–MS spectra of SW group vs. control group in negative mode (A); 3-D PCA of UHPLC–MS spectra in negative mode (B); VIP-plot for accurate mass UHPLC–MS data.
mass spectrometry (Waters, Milford, USA) equipped with an ESI source. The separation of all samples was performed on a UPLCTM BEH C18 column (2.1×100 mm, 1.7 m) at 35⬚C. The mobile phases were composed of acetonitrile with 0.1% v/v formic acid (A) and water with 0.1% formic acid (B) using a linear gradient program of 1–16% A (0–1.5 min), 16–20% A (1.5–5 min), 20–25% A (5–7 min), 25–35% A (7–10 min), and 35–99% A (10–20 min) at a flow rate kept at 0.3 mL/min. The injection volume was 5 L.
2.4 High-resolution accurate MS The MS instrument consisted of a Waters AcquityTM Synapt mass spectrometer equipped with ESI mode, and the mass C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3 Results and discussion 3.1 Optimization of UHPLC–ESI-Q-TOF-MS conditions In order to obtain chromatograms with good separation and strong total ion current, several mobile phase systems including methanol/water, acetonitrile/water, methanol with 0.1% formic acid and acetonitrile with 0.1% formic acid were tested to optimize the chromatographic conditions. When formic acid was added to the mobile phase, the peak capacities and shapes of all the chromatographic peaks were enhanced remarkably. The acetonitrile/water system showed more powerful separation ability and elution power for investigated compounds through the comparison of the methanol/water system. As a result, the optimal solvent systems consisting of acetonitrile and 0.1% formic acid aqueous solution on the optimized gradient mode showed a good separation and abundant signal response both in positive and negative www.jss-journal.com
J. Sep. Sci. 2014, 37, 1742–1747
Liquid Chromatography
1745
Figure 3. UHPLC–MS BPI chromatograms of EW in positive mode (A) and in negative mode (B). Each peak number is consistent with Tables S1 and S2, respectively.
ion scan mode, which increased the efficiency of ionization and gave satisfactory sensitivity, were ultimately tested as mobile phase. The selection of the fast UHPLC conditions was guided by the requirement for obtaining chromatograms with better resolution of adjacent peaks within a short time. The MS conditions, capillary voltage, sampling cone voltage, extraction cone voltage, desolvation gas temperature, and desolvation gas flow were optimized in order to achieve efficient separation and good responses to all chemical components in SW. And both positive and negative ion modes were employed to identify the corresponding signals. Series of experiments were conducted to optimize the LC chromatographic and MS conditions as described in Sections 2.3 and 2.4. And both positive and negative ion modes were employed to identify the corresponding signals.
3.2 UHPLC–ESI-Q-TOF-MS analysis of SW The experimental setup for UHPLC–ESI-Q-TOF-MS coupled with multiple data processing approach analysis is shown in Fig. S1. In order to gain the details of differences, principal component analysis (PCA), is the most widely used exploratory techniques in multivariate analysis, could convert multidimensional data space into a low dimensional model plane. In our study, PCA method was employed to phenotype the differences between the SW and control group. PCA of LC–MS spectra of SW group vs. control group in positive and negative mode was shown in Figs. 1A and 2A. Figures 1B and 2B showed the 3-D PCA of UHPLC–ESI-Q-TOF-MS spectra in positive and negative mode, respectively. The ions of interest that were present only in the SW group and absent in the control group were extracted easily by utilizing VIP-plot that could clearly display leading contributing markers. As shown in Figs. 2C and 3C, the points in the red frame were at higher level in SW group. As demonstrated above, 72 ions of interest (37 ions in positive mode, 38 ions in negative mode and three ions in both modes) were extracted, identified or tentatively C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
characterized based on their retention times, exact mass measurement for each molecular ion and subsequent fragment ions, and their information is shown in Tables S1 and S2, respectively. The mass error for molecular ions of all identified compounds was within ±6 ppm. 72 major constituents (Fig. 3A and B) including flavonoids, phenylpropanoids, alkaloids, isoflavonoids, organic acids, and saponins were tentatively characterized (Table S1).
3.3 MS/MS characterization of chemical constituents from SW All information of MS data obtained using the aforementioned protocol indicated the retention time and precise molecular mass and provided the MS/MS data which was necessary for the structural identification. The precise molecular mass was determined within a reasonable degree of measurement error (
