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Page 1
Journal of Separation Science
Characterization of multiple constituents in Kai-Xin-San prescription and rat plasma after oral administration by liquid chromatography with quadrupole time-of-flight tandem mass spectrometry
Xiaowen Zhang b, c
a, c
, Qing Li b, c, Chunxiao Lv b, c, Huarong Xu b, c, Xujia Liu
, Zhenyu Sui b, c, Kaishun Bi b, c, *
a
School of Chinese Material Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
b
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China
c
National and Local United Engineering Laboratory for Key Technology of Chinese Material Medica
Quality Control, Shenyang Pharmaceutical University, Shenyang 110016, China
* Corresponding author at: School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, 110016, China. Tel.: +86 24 23986012; fax: +86 24 23986012. E-mail address: [email protected] (K. Bi)
Received: 02-Feb-2015; Revised: 09-Mar-2015; Accepted: 30-Mar-2015 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jssc.201500123. This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 2
Journal of Separation Science
ABSTRACT A sensitive and reliable ultra high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry method was established to separate and identify the chemical constituents of Kai-Xin-San prescription, a classic traditional Chinese medicine formula that plays an important role in the treatment of Alzheimer’s disease. The detection was performed on an Agilent 6520 Accurate-Mass quadrupole-time-of-flight mass spectrometer equipped with an electrospray ionization source in negative modes. With the optimized conditions, a total of 54 compounds were identified or tentatively characterized. Of the 54 compounds, six compounds were identified by comparing the retention time and MS data with reference standards, the rest were characterized by analyzing MS data and retrieving the reference literatures. Results indicated ginsenosides, polygala saponins, terpenoids and oligosaccharide esters were the major effective constituents in Kai-Xin-San prescription. There were 26 prototype ingredients were assigned for identification in rat plasma. It is also concluded that the developed ultra high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry method with high sensitivity and resolution is suitable for identifying and characterizing the chemical constituents of Kai-Xin-San prescription, and the analysis provides a helpful chemical basis for further research on Kai-Xin-San prescription and the clinical diagnostics of Alzheimer’s disease. Keywords: Alzheimer’s disease; Kai-Xin-San; Traditional Chinese medicine; Ultra high
performance liquid chromatography.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 3
Journal of Separation Science
1. Introduction Alzheimer’s disease (AD) is an irreversible, severe central neurodegenerative epidemic disease, which slowly destroys memory and thinking skills. It eventually damages the ability to implement simplest tasks [1–4]. AD has been officially listed as the fifth-leading cause of death for individuals age 65 and older. Unfortunately, there is no curative medications to treat or retard AD because of its multiple pathogenesis [5]. However, Traditional Chinese Medicine (TCM) is prescribed in combination based on the theory of traditional Chinese herbal medical science to produce synergistic effects at diverse target spots, which could be used for the prevention and treatment of AD. In modern clinical practice, Kai-Xin-San (KXS), a famous traditional Chinese medicinal formula for relieving “sentimental diseases” was reported to possess various pharmacological effects such as enhancing learning and memory, anti-oxidation and anti-depression. It also has been used for the treatment of AD, neurasthenia, and neurosis [4,7–9]. Therefore it is valuable to make the comprehensive investigation on the profile constituents of the formula. KXS, contains four herbs, namely Ginseng Radix et Rhizoma (dried root or rhizome of Panax ginseng C. A. Mey), Poria [dried sclerotia of Poria cocs (Schw.) Wolf], Polygalae Radix (dried root of Polygala tenuifolia Willd or Polygala sibirica L.), and Acori Tatarinowii Rhizoma ( dried root bark of Acorus tatarinowii Schott). It was originally described in Bei Ji Qian Jin Yao Fang and written in ancient China Tang Dynasty by Simiao Sun [4]. Currently, the research of the chemical components in KXS has been mainly based on identification of chemical constituents in individual herb extracts. Meanwhile, there are sparse data for comprehensive investigation on the constituents of KXS [10]. Furthermore, there have been This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 4
Journal of Separation Science
few reports on the absorption and efficacy after oral administration of KXS formula, which is valuable for further studies on the pharmaceutical effect and mechanism of the KXS formula. As the development of sensitive, reliable analytical methods and technology such as MS/MS and quadrupole time-of-fight (Q-TOF) have been proven to be efficient tools for the rapid on-line analysis, the known compounds and elucidation of unknown compounds in complex matrices has become valuable analytical techniques in TCM research [11, 12]. So the main goal of this research was to develop a sensitive, simple and validated LC–Q-TOF-MS method for comprehensive investigation on the profile constituents in KXS formula and the rat plasma after oral administration of this formula. Moreover, the result of this study was expected to be beneficial for improving clinical therapeutic efficacy and further pharmacological studies of KXS formula.
2. Materials and methods 2.1 Animals Six pathogen-free Sprague–Dawley rats of 6–8 weeks of age (weighing 220–250g) purchased from Liaoning Changsheng Biotechnology (Benxi, China) were housed in a SPF grade animal laboratory kept at a temperature of 22 ± 2°C and a relative humidity of 50 ± 10%, with a natural light-dark cycle. The animal study was carried out in accordance with the Guideline for Animal Experimentation of Shenyang Pharmaceutical University, and the protocol was approved by the Animal Ethics Committee of the institution.
This article is protected by copyright. All rights reserved.
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Page 5
Journal of Separation Science
2.2 Chemicals and Materials Ginseng Radixet Rhizoma, Poria, Polygalae Radix, and Acori Tatarinowii Rhizoma were all purchased from Tong-Ren-Tang TCM store (Shenyang, China) and authenticated by professor Ying Jia (Department of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang, China). The reference standards of polygalaxanthone III, ginsenoside Rb1, ginsenoside Rd, ginsenoside Re and ginsenoside Rg1 were obtained from the National Institute for Food and Drug Control (Beijing, China), the purity of these reference standards were all more than 98%. Tumulosic acid (TUM; purity >98%) was isolated from Poria from the author’s laboratory (Department of Pharmaceutical Analysis, Shenyang Pharmaceutical University, Shenyang, China), and the structures were characterized by spectral methods, including MS, 1H and
13
C NMR spectroscopy (data not shown). The data were consistent
with those reported in the literature. Distilled water prepared with demineralized water was employed throughout the experiment. Methanol of HPLC grade was from Fisher Scientific (Fairlawn, NJ, USA). Other HPLC-grade reagents such as formic acid and acetonitrile were provided by Shandong Yuwang Industrial (Yucheng, China). 2.3 Apparatus LC–Q-TOF-MS analytical procedures were performed on an Agilent 6520 Accurate-Mass Q-TOF mass spectrometer with an Agilent 1290 HPLC system (Billerica, USA). The MassHunter Data Aquisation Workstation Software was applied to system operation and data collection. The chromatographic separation was achieved on a ZORBAX Eclipse Plus C18 (100 mm × 2.1 mm, 1.8 μm, USA, Agilent column protected by a high pressure column pre-filter (2 mm; Shimadzu, Japan) at 30°C. Analysis was completed with a gradient elution This article is protected by copyright. All rights reserved.
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Page 6
Journal of Separation Science
of 0.01% formic acid in water (A) and 0.01% formic in methanol (B) within 100.0 min. The 100.0 min chromatographic gradient program was as follows: 5% B→20% B at 0–5.0 min; 20% B at 5–10.0 min; 20% B→35% B at 10.0–15.0 min; 35% B→40% B at 15.0–20.0 min; 40% B→45% B at 20.0–35.0 min; 45% B→50% B at 35.0–38.0 min; 50% B→53% B at 38.0–50.0 min; 53% B→70% B at 50.0–52.0 min; 70% B at 52.0–60.0 min; 70% B→80% B at 60.0–75.0 min; 80% B→90% B at 75.0–85.0 min; 90% B→95% B at 85.0–90.0 min; 95% B→5% B at 90.0–93.0 min; 5% B at 93.0–100.0 min. Efficient and symmetrical peaks were obtained at a flow rate of 0.2 mL min -1 with a sample injection volume of 5 μL. For all LC–Q-TOF-MS experiments, the Q-TOF mass spectrometer was operated in the negative ion mode with an ESI source (ESI–). The capillary voltage of the ion source was set at 3500 V and the fragmentor voltage was set at 130 V. The nebulizer gas pressure was maintained at 40 psi. High purity nitrogen was used as the desolvation and nebulizing gas at a constant temperature of 350°C, and also a flow rate of 9.0 L/min was employed. The full scan range was set at m/z 200–2000, and the MS/MS full scan range was set at m/z 100–2000. The other parameters were adopted for the recommended value of the instrument. 2.4 Preparation of KXS Decoction According to the original composition and preparation method recorded in Bei Ji Qian Jin Yao Fang, KXS decoction was prepared in the following procedures. The four component herbs, Ginseng Radixet Rhizoma (15 g), Poria (15 g), Polygalae Radix (10 g) and Rhizoma Acori Tatarinowii (10 g), were extracted twice by refluxing in boiled water (1:10, w/v) for 2 h. The extracted solutions were combined and concentrated under reduced pressure to a density of 1.5 g crude drug per milliliter. The decoction was stored in the refrigerator at 4°C. This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 7
Journal of Separation Science
2.5 Preparation of rat plasma samples The rats were fasted for 12 h with free access to water before the oral administration of KXS decoction with a dose of 10 g/kg. Blood samples (approx. 1.0 mL) were collected from suborbital vein into heparinized tubes at 0, 0.5, 1, and 2 h after administration and then immediately centrifuged at 12000 rpm for 5 min. Harvested plasma samples were stored at –20°C for later analysis. 3. Results and discussion 3.1 Optimization of UHPLC–Q-TOF-MS conditions Diverse mobile phases including methanol/water, acetonitrile/water, with formic acid in mobile phase and different ratios of formic acid in the mobile phase (from 0.01–0.1%) were all tested to optimize the chromatographic conditions so that the chromatograms with excellent separation and higher ion response could be obtained. Much more effective separation capability for the target ingredients were exhibited by the methanol/water system through the comparison of the acetonitrile/water system. Furthermore, within addition of formic acid in mobile phase, the peak capacities and shapes within the whole chromatogram were enhanced obviously. Consequently, the optimal mobile phase system contained methanol with 0.01% formic acid and 0.01% formic acid aqueous solution on the optimized gradient elution showed an outstanding separation and abundant ion response in negative ion mode. The UHPLC system was adopted to obtain chromatograms with better resolution of adjacent peaks within a short time. The Q-TOF-MS conditions, capillary voltage, fragmentor voltage, nebulizer gas pressure, gas temperature, gas flow and data acquisition, 2 spectra/s, were all optimized to achieve efficient separation and higher response to each one of the This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 8
Journal of Separation Science
ingredients in KXS. Negative ion modes were employed to identify the corresponding signals. Series of experiments were conducted to optimize the LC and MS conditions. 3.2 UHPLC–Q-TOF-MS characterization of ingredients from KXS Agilent MassHunter Qualitative Analysis Workstation Software was used for the MS data information processing. The complete MS data outcomes obtained from the high resolution UHPLC–Q-TOF-MS analysis that was performed using the developed method demonstrated the accurate precursor ion m/z and fragmentation ion m/z data to give the structural identification. According to the mass accuracy (ppm) and retention time, the molecular compositions were also calculated. The precise molecular mass was determined within a reasonable degree of measurement error (
Page 1
Journal of Separation Science
Characterization of multiple constituents in Kai-Xin-San prescription and rat plasma after oral administration by liquid chromatography with quadrupole time-of-flight tandem mass spectrometry
Xiaowen Zhang b, c
a, c
, Qing Li b, c, Chunxiao Lv b, c, Huarong Xu b, c, Xujia Liu
, Zhenyu Sui b, c, Kaishun Bi b, c, *
a
School of Chinese Material Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
b
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China
c
National and Local United Engineering Laboratory for Key Technology of Chinese Material Medica
Quality Control, Shenyang Pharmaceutical University, Shenyang 110016, China
* Corresponding author at: School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, 110016, China. Tel.: +86 24 23986012; fax: +86 24 23986012. E-mail address: [email protected] (K. Bi)
Received: 02-Feb-2015; Revised: 09-Mar-2015; Accepted: 30-Mar-2015 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jssc.201500123. This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 2
Journal of Separation Science
ABSTRACT A sensitive and reliable ultra high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry method was established to separate and identify the chemical constituents of Kai-Xin-San prescription, a classic traditional Chinese medicine formula that plays an important role in the treatment of Alzheimer’s disease. The detection was performed on an Agilent 6520 Accurate-Mass quadrupole-time-of-flight mass spectrometer equipped with an electrospray ionization source in negative modes. With the optimized conditions, a total of 54 compounds were identified or tentatively characterized. Of the 54 compounds, six compounds were identified by comparing the retention time and MS data with reference standards, the rest were characterized by analyzing MS data and retrieving the reference literatures. Results indicated ginsenosides, polygala saponins, terpenoids and oligosaccharide esters were the major effective constituents in Kai-Xin-San prescription. There were 26 prototype ingredients were assigned for identification in rat plasma. It is also concluded that the developed ultra high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry method with high sensitivity and resolution is suitable for identifying and characterizing the chemical constituents of Kai-Xin-San prescription, and the analysis provides a helpful chemical basis for further research on Kai-Xin-San prescription and the clinical diagnostics of Alzheimer’s disease. Keywords: Alzheimer’s disease; Kai-Xin-San; Traditional Chinese medicine; Ultra high
performance liquid chromatography.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 3
Journal of Separation Science
1. Introduction Alzheimer’s disease (AD) is an irreversible, severe central neurodegenerative epidemic disease, which slowly destroys memory and thinking skills. It eventually damages the ability to implement simplest tasks [1–4]. AD has been officially listed as the fifth-leading cause of death for individuals age 65 and older. Unfortunately, there is no curative medications to treat or retard AD because of its multiple pathogenesis [5]. However, Traditional Chinese Medicine (TCM) is prescribed in combination based on the theory of traditional Chinese herbal medical science to produce synergistic effects at diverse target spots, which could be used for the prevention and treatment of AD. In modern clinical practice, Kai-Xin-San (KXS), a famous traditional Chinese medicinal formula for relieving “sentimental diseases” was reported to possess various pharmacological effects such as enhancing learning and memory, anti-oxidation and anti-depression. It also has been used for the treatment of AD, neurasthenia, and neurosis [4,7–9]. Therefore it is valuable to make the comprehensive investigation on the profile constituents of the formula. KXS, contains four herbs, namely Ginseng Radix et Rhizoma (dried root or rhizome of Panax ginseng C. A. Mey), Poria [dried sclerotia of Poria cocs (Schw.) Wolf], Polygalae Radix (dried root of Polygala tenuifolia Willd or Polygala sibirica L.), and Acori Tatarinowii Rhizoma ( dried root bark of Acorus tatarinowii Schott). It was originally described in Bei Ji Qian Jin Yao Fang and written in ancient China Tang Dynasty by Simiao Sun [4]. Currently, the research of the chemical components in KXS has been mainly based on identification of chemical constituents in individual herb extracts. Meanwhile, there are sparse data for comprehensive investigation on the constituents of KXS [10]. Furthermore, there have been This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 4
Journal of Separation Science
few reports on the absorption and efficacy after oral administration of KXS formula, which is valuable for further studies on the pharmaceutical effect and mechanism of the KXS formula. As the development of sensitive, reliable analytical methods and technology such as MS/MS and quadrupole time-of-fight (Q-TOF) have been proven to be efficient tools for the rapid on-line analysis, the known compounds and elucidation of unknown compounds in complex matrices has become valuable analytical techniques in TCM research [11, 12]. So the main goal of this research was to develop a sensitive, simple and validated LC–Q-TOF-MS method for comprehensive investigation on the profile constituents in KXS formula and the rat plasma after oral administration of this formula. Moreover, the result of this study was expected to be beneficial for improving clinical therapeutic efficacy and further pharmacological studies of KXS formula.
2. Materials and methods 2.1 Animals Six pathogen-free Sprague–Dawley rats of 6–8 weeks of age (weighing 220–250g) purchased from Liaoning Changsheng Biotechnology (Benxi, China) were housed in a SPF grade animal laboratory kept at a temperature of 22 ± 2°C and a relative humidity of 50 ± 10%, with a natural light-dark cycle. The animal study was carried out in accordance with the Guideline for Animal Experimentation of Shenyang Pharmaceutical University, and the protocol was approved by the Animal Ethics Committee of the institution.
This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 5
Journal of Separation Science
2.2 Chemicals and Materials Ginseng Radixet Rhizoma, Poria, Polygalae Radix, and Acori Tatarinowii Rhizoma were all purchased from Tong-Ren-Tang TCM store (Shenyang, China) and authenticated by professor Ying Jia (Department of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang, China). The reference standards of polygalaxanthone III, ginsenoside Rb1, ginsenoside Rd, ginsenoside Re and ginsenoside Rg1 were obtained from the National Institute for Food and Drug Control (Beijing, China), the purity of these reference standards were all more than 98%. Tumulosic acid (TUM; purity >98%) was isolated from Poria from the author’s laboratory (Department of Pharmaceutical Analysis, Shenyang Pharmaceutical University, Shenyang, China), and the structures were characterized by spectral methods, including MS, 1H and
13
C NMR spectroscopy (data not shown). The data were consistent
with those reported in the literature. Distilled water prepared with demineralized water was employed throughout the experiment. Methanol of HPLC grade was from Fisher Scientific (Fairlawn, NJ, USA). Other HPLC-grade reagents such as formic acid and acetonitrile were provided by Shandong Yuwang Industrial (Yucheng, China). 2.3 Apparatus LC–Q-TOF-MS analytical procedures were performed on an Agilent 6520 Accurate-Mass Q-TOF mass spectrometer with an Agilent 1290 HPLC system (Billerica, USA). The MassHunter Data Aquisation Workstation Software was applied to system operation and data collection. The chromatographic separation was achieved on a ZORBAX Eclipse Plus C18 (100 mm × 2.1 mm, 1.8 μm, USA, Agilent column protected by a high pressure column pre-filter (2 mm; Shimadzu, Japan) at 30°C. Analysis was completed with a gradient elution This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 6
Journal of Separation Science
of 0.01% formic acid in water (A) and 0.01% formic in methanol (B) within 100.0 min. The 100.0 min chromatographic gradient program was as follows: 5% B→20% B at 0–5.0 min; 20% B at 5–10.0 min; 20% B→35% B at 10.0–15.0 min; 35% B→40% B at 15.0–20.0 min; 40% B→45% B at 20.0–35.0 min; 45% B→50% B at 35.0–38.0 min; 50% B→53% B at 38.0–50.0 min; 53% B→70% B at 50.0–52.0 min; 70% B at 52.0–60.0 min; 70% B→80% B at 60.0–75.0 min; 80% B→90% B at 75.0–85.0 min; 90% B→95% B at 85.0–90.0 min; 95% B→5% B at 90.0–93.0 min; 5% B at 93.0–100.0 min. Efficient and symmetrical peaks were obtained at a flow rate of 0.2 mL min -1 with a sample injection volume of 5 μL. For all LC–Q-TOF-MS experiments, the Q-TOF mass spectrometer was operated in the negative ion mode with an ESI source (ESI–). The capillary voltage of the ion source was set at 3500 V and the fragmentor voltage was set at 130 V. The nebulizer gas pressure was maintained at 40 psi. High purity nitrogen was used as the desolvation and nebulizing gas at a constant temperature of 350°C, and also a flow rate of 9.0 L/min was employed. The full scan range was set at m/z 200–2000, and the MS/MS full scan range was set at m/z 100–2000. The other parameters were adopted for the recommended value of the instrument. 2.4 Preparation of KXS Decoction According to the original composition and preparation method recorded in Bei Ji Qian Jin Yao Fang, KXS decoction was prepared in the following procedures. The four component herbs, Ginseng Radixet Rhizoma (15 g), Poria (15 g), Polygalae Radix (10 g) and Rhizoma Acori Tatarinowii (10 g), were extracted twice by refluxing in boiled water (1:10, w/v) for 2 h. The extracted solutions were combined and concentrated under reduced pressure to a density of 1.5 g crude drug per milliliter. The decoction was stored in the refrigerator at 4°C. This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 7
Journal of Separation Science
2.5 Preparation of rat plasma samples The rats were fasted for 12 h with free access to water before the oral administration of KXS decoction with a dose of 10 g/kg. Blood samples (approx. 1.0 mL) were collected from suborbital vein into heparinized tubes at 0, 0.5, 1, and 2 h after administration and then immediately centrifuged at 12000 rpm for 5 min. Harvested plasma samples were stored at –20°C for later analysis. 3. Results and discussion 3.1 Optimization of UHPLC–Q-TOF-MS conditions Diverse mobile phases including methanol/water, acetonitrile/water, with formic acid in mobile phase and different ratios of formic acid in the mobile phase (from 0.01–0.1%) were all tested to optimize the chromatographic conditions so that the chromatograms with excellent separation and higher ion response could be obtained. Much more effective separation capability for the target ingredients were exhibited by the methanol/water system through the comparison of the acetonitrile/water system. Furthermore, within addition of formic acid in mobile phase, the peak capacities and shapes within the whole chromatogram were enhanced obviously. Consequently, the optimal mobile phase system contained methanol with 0.01% formic acid and 0.01% formic acid aqueous solution on the optimized gradient elution showed an outstanding separation and abundant ion response in negative ion mode. The UHPLC system was adopted to obtain chromatograms with better resolution of adjacent peaks within a short time. The Q-TOF-MS conditions, capillary voltage, fragmentor voltage, nebulizer gas pressure, gas temperature, gas flow and data acquisition, 2 spectra/s, were all optimized to achieve efficient separation and higher response to each one of the This article is protected by copyright. All rights reserved.
www.jss-journal.com
Page 8
Journal of Separation Science
ingredients in KXS. Negative ion modes were employed to identify the corresponding signals. Series of experiments were conducted to optimize the LC and MS conditions. 3.2 UHPLC–Q-TOF-MS characterization of ingredients from KXS Agilent MassHunter Qualitative Analysis Workstation Software was used for the MS data information processing. The complete MS data outcomes obtained from the high resolution UHPLC–Q-TOF-MS analysis that was performed using the developed method demonstrated the accurate precursor ion m/z and fragmentation ion m/z data to give the structural identification. According to the mass accuracy (ppm) and retention time, the molecular compositions were also calculated. The precise molecular mass was determined within a reasonable degree of measurement error (
