Research Article Received: 27 October 2014,
Revised: 21 January 2015,
Accepted: 31 January 2015
Published online in Wiley Online Library: 21 March 2015
(wileyonlinelibrary.com) DOI 10.1002/pca.2560
Rapid Identification of Steroidal Saponins in Trillium Tschonoskii Maxim by Ultraperformance Liquid Chromatography Coupled to Electrospray Ionisation Quadrupole Time-of-Flight Tandem Mass Spectrometry Xin Gao,a* Wenjun Sun,b** Qiang Fua and Xiaofeng Niua ABSTRACT: Introduction – Steroidal saponins in Trillium tschonoskii Maxim have many biological activities, including immunological regulation and anti-tumour. Comprehensive ingredient identification is critical for understanding its pharmacological mechanism and establishing quality control protocols. However, it is a challenging problem because of the complexity of steroidal saponins. Objectives – To develop a UPLC–MS method for identifying and characterising steroidal saponins in the root and rhizome of T. tschonoskii. Methods – Methanolic extracts of T. tschonoskii were analysed by using ultraperformance liquid chromatography coupled to electrospray ionisation quadrupole time-of-flight tandem mass spectrometry (UPLC–ESI/QTOF/MS). The UPLC experiments were performed by means of a reversed-phase C18-column and a binary mobile phase system consisting of water and acetonitrile with formic acid under gradient elution conditions. For the UPLC–MS measurements, positive and negative ion modes were used in order to obtain better tandem mass spectra and high-resolution mass spectra. Results – Based on retention times, accurate mass and mass spectrometric fragmentation, a total of 31 saponins distributed over eight steroidal aglycone skeletons were identified or tentatively elucidated from T. tschonoskii. Conclusion – The UPLC–ESI/QTOF/MS method has proven to be a powerful tool for rapid identification of steroidal saponins in T. tschonoskii without tedious and time-consuming isolation of pure constituents. Copyright © 2015 John Wiley & Sons, Ltd. Supporting information can be found in the online version of this article. Keywords: UPLC–MS/MS; steroidal saponins; Trillium tschonoskii Maxim
Introduction
Phytochem. Anal. 2015, 26, 269–278
* Correspondence to: Xin Gao, Department of Pharmaceutical Sciences, School of Pharmacy, Xi’an Jiaotong University, No.76, Yanta West Road, Xi’an, Shaanxi, 710061, China. E-mail: [email protected] ** Correspondence to: Wenjun Sun, Information Department of Science and Technology, Xi’an Xintong Pharmaceutical Research Co., Ltd, No.69, Jinye Road, Xi’an, Shaanxi, 710077, China. E-mail: [email protected] a
Department of Pharmaceutical Sciences, School of Pharmacy, Xi’an Jiaotong University, Xi’an, China
b
Information Department of Science and Technology, Xi’an Xintong Pharmaceutical Research Co., Ltd, Xi’an, China
Copyright © 2015 John Wiley & Sons, Ltd.
269
Trillium tschonoskii Maxim (family Liliaceae) is a herbaceous plant in China, locally known as ‘Yan Ling Cao’, meaning that it is a herb that can prolong human life. Its dried roots and rhizomes are used as a folk medicine for the treatment of hypertension, dizziness, neurasthenia, waist–leg pain, traumatic haemorrhage (Song et al., 2001). Phytochemical examinations revealed that it contains a large number of steroidal saponins, and over 30 saponins including diosgenyl, pennogenyl, protodiosgenyl, kryptgenin and trillenogenin saponins have been isolated (Wang et al., 2007; Yu and Zou, 2008; Zhang et al., 2011; Wei et al., 2012). Steroidal saponins isolated from T. Tschonoskii were found to posses various bioactivities, for example immunological regulation and antiinflammatory, anti-oxidative, especially strong anti-aging and anti-tumour properties (Huang and Xiao, 2006; Yokosuka and Mimaki, 2008; Yu et al., 2008a,2008b; H. Wang et al., 2013). Recently, there has been renewed interest because of their reported beneficial effect in various cancers, as well as senile dementia. Comprehensive ingredient identification including structural characterisation of steroidal saponins from T. tschonoskii is critical for understanding its pharmacological mechanism and
establishing quality control protocols. However, various types of aglycone together with different possibilities of sugar chain composition and attachment cause great natural diversity of saponin structures (Man et al., 2010). Thus, the entire analytical process for characterising saponins could be extremely difficult and labour intensive. Currently, there are only a few analytical research reports on evaluating the quality of T. tschonoskii using HPLC–UV with monitoring hydrolysed sapogenin (diosgenin) or three other saponins (Cui and Liu, 2009; Li and Chen, 2009; Wang et al.,
X. Gao et al. 2013b; Wu et al., 2013). The HPLC–UV method is probably limited in practice as most saponins lack UV absorption. The UPLC– ESI/QTOF/MS method has been established as an ideal tool for the profiling of saponin mixtures, because it offers superior sensitivity, selectivity, considerable structure information and short retention times, and to some extent avoids the time-consuming steps in the isolation of saponins (Ouyang et al., 2014; Qi et al., 2014). In this study, given the complexity of steroidal saponins in T. Tschonoskii and their integrated contribution to therapeutic effects, an approach for rapidly separating, systemically identifying and investigating structural characteristics of steroidal saponins from the extract of T. tschonoskii was established by UPLC– ESI/QTOF/MS.
Experimental Chemicals and materials Acetonitrile (MeCN) and methanol (MeOH) were of HPLC grade from Merck (Darmstadt, Germany); water was produced by a Milli-Q system (Millipore, Bedford, MA, USA); formic acid (HCOOH) was purchased from Tianjin Kermel (Tianjin, China); methanol (analytical grade) was purchased from ChengDu KeLong Chemical Co., Ltd and ChengDu KeLong Chemical Reagent Company (Chendu, China). The roots and rhizomes of T. tschonoskii were collected from Baoji, Shaanxi Province of China in September 2012, and were authenticated by Professor Xiaofeng Niu of the Department of Pharmacognosy, Xi’an Jiaotong University. A voucher specimen (12092801) was deposited in the department of Pharmaceutical Sciences, School of Pharmacy, Xi’an Jiaotong University. Reference standards of trillin, dicosin and diosgenin were purchased from Chendu Must Biotechnology Co., Ltd (Chendu, China), polyphyllin V, polyphyllin VI, polyphyllin VII and pennogenin 3-O-β-chacotrioside were obtained from Chendu Jioute Biotech Co., Ltd (Chendu, China). All the standards used were of the highest purity available (>97%). Pennogenin was isolated and purified from the roots and rhizomes of T. tschonoskii in our laboratory, its purity was > 97% as assayed by HPLC, and its chemical structure was identified by mass and NMR spectra.
duration. Data were collected in centroid mode and mass was corrected TM during acquisition using an external reference (Lock-Spray ) comprising a 100 μL/min solution of leucine–enkephalin via a lockspray interface, + generating a reference ion at 556.2771 Da ([M + H] ) for positive ESI mode and at m/z 554.2615 Da ([M H] ) in negative ion mode to ensure accurate mass measurements over a wide dynamic range. The accurate mass and composition for the precursor ions and for the fragment ions were calculated using the MassLynx 4.1 software (Waters) that was incorporated with the instrument.
Establishment of an in-house molecular formula database By comprehensively searching databases such as Chemspider, PubMed, PubChem Compound and SciFinder we established an in-house molecular formula database of all compounds reported in the literature on T. tschonoskii and related natural products in the same genus and related genera in the Trillaceae family (Paris and Ypsilandra genera) using automated MetaboLynx software. The database includes the compound name, molecular formula, accurate molecular mass, chemical structure and literature citations (Gao et al., 2014).
Results and discussion UPLC–QTOF/MS profiles Optimised chromatographic conditions were achieved through trial and error. A linear gradient elution with acetonitrile and water containing formic acid as the mobile phase gave the best peak resolution. Typical chromatograms with mass spectrometric detection at positive and negative ion modes are presented in Figure 1. Detection at positive ion mode seemed to be more
Preparation of T. tschonoskii samples for UPLC–QTOF/MS analysis The dried root and rhizome of T. tschonoskii were crushed to powder (40 mesh size) and extracted with methanol (1 g/20 mL) twice each for 1 h under reflux. The combined extracts were dissolved in 50 mL of methanol and centrifuged at 13000 rpm for 10 min at 4°C. The supernatant was filtered through a 0.22 μm filter prior to UPLC analysis with triplicate injections.
Chromatography and mass spectrometry TM
270
Chromatographic analysis was conducted on an Acquity Ultra Performance LC system (Waters, Milford, USA) controlled with Masslynx (V4.1). TM Separation was performed using an Acquity UPLC BEH C18-column (2.1 mm × 100 mm, 1.7 μm; Waters), with a mobile phase flow rate of 0.3 mL/min. The column temperature was set at 30°C. The optimal mobile phase consisted of (A) 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. The gradient programme was 0–5 min, 2–20% B; 5–7 min, 20–25% B; 7–10 min, 25–35% B; 10–22 min, 35–99% B. The sample injection volume TM was 5 μL. The Waters Acquity UPLC system equipped with mass spectrometric detection with an electrospray ionisation (ESI) source operated in both positive and negative modes. The optimised mass spectrometric parameters were as follows: nebuliser gas flow rate at 700 L/h at a temperature of 300°C, cone gas at 40 L/h, and source temperature at 110°C, capillary and cone voltages at 3000 V and 40 V, and the extraction cone voltage was 4.0 V in both positive and negative ion mode, respectively. The full-scan MS data were acquired from 100 to 1500 Da with a 0.30 s scan
wileyonlinelibrary.com/journal/pca
Figure 1. The UPLC–MS base peak intensity chromatograms of eight mixed standards in (a) positive ion mode and (b) negative ion mode, and Trillium tschonoskii Maxim in (c) positive ion mode and (d) negative ion mode.
Copyright © 2015 John Wiley & Sons, Ltd.
Phytochem. Anal. 2015, 26, 269–278
Identification of 31 Saponins from T. Tschonoskii by UPLC–MS/MS sensitive and present richer characteristic fragmentation than that at negative ion mode. A complicated chromatographic peak pattern was observed and the major peaks were characterised as steroidal saponins, as described in the following section. Structural identification
Phytochem. Anal. 2015, 26, 269–278
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/pca
271
In negative ion mode, most saponins generated only the solvent adduct ion [M H + HCOOH] and deprotonated ion [M H] of high intensity and without fragmentation, even at higher fragmentation voltage. In positive ion mode, most saponins exhibited quasi-molecular ions of [M + H]+ and/or [M + H H2O]+, plus ions that presented rich structural characteristic fragmentation. Sensitive detection of saponins could be achieved under positive ion mode, while structural characterisation was performed using the data from both positive ion and negative ion mode. The structural identification was based on retention time, precise molecular weight (four decimal places) and MS/MS fragmentation patterns. First, their potential element compositions and precise molecular mass were obtained with specified mass accuracy (error < 5 ppm) and i-FIT (normal, value < 1.0), in addition the degree of non-saturation was obtained. Second, by comprehensively scrutinising the in-house formula database, possible chemical structures were narrowed down to one or a few compounds (Gao et al., 2014). Finally, together with the interpretation of the observed MS/MS spectra using the Masslynx MassFragment function in comparison with those found in the literature (Nohara et al., 1975; Fukuda et al., 1981; Nakano et al., 1983; Ono et al., 1986, 2007a,2007b; Yu and Zou, 2008; Hayes et al., 2009; Xie et al., 2009; Man et al., 2010; Zhang et al., 2011; Kang et al., 2012; Wei et al., 2012; Li et al., 2013; Wang et al., 2013a) or reference standards, the chemical structures were proposed. Thirty-one compounds were tentatively identified and listed in Table 1, which summarises the accurate mass measurements (
Revised: 21 January 2015,
Accepted: 31 January 2015
Published online in Wiley Online Library: 21 March 2015
(wileyonlinelibrary.com) DOI 10.1002/pca.2560
Rapid Identification of Steroidal Saponins in Trillium Tschonoskii Maxim by Ultraperformance Liquid Chromatography Coupled to Electrospray Ionisation Quadrupole Time-of-Flight Tandem Mass Spectrometry Xin Gao,a* Wenjun Sun,b** Qiang Fua and Xiaofeng Niua ABSTRACT: Introduction – Steroidal saponins in Trillium tschonoskii Maxim have many biological activities, including immunological regulation and anti-tumour. Comprehensive ingredient identification is critical for understanding its pharmacological mechanism and establishing quality control protocols. However, it is a challenging problem because of the complexity of steroidal saponins. Objectives – To develop a UPLC–MS method for identifying and characterising steroidal saponins in the root and rhizome of T. tschonoskii. Methods – Methanolic extracts of T. tschonoskii were analysed by using ultraperformance liquid chromatography coupled to electrospray ionisation quadrupole time-of-flight tandem mass spectrometry (UPLC–ESI/QTOF/MS). The UPLC experiments were performed by means of a reversed-phase C18-column and a binary mobile phase system consisting of water and acetonitrile with formic acid under gradient elution conditions. For the UPLC–MS measurements, positive and negative ion modes were used in order to obtain better tandem mass spectra and high-resolution mass spectra. Results – Based on retention times, accurate mass and mass spectrometric fragmentation, a total of 31 saponins distributed over eight steroidal aglycone skeletons were identified or tentatively elucidated from T. tschonoskii. Conclusion – The UPLC–ESI/QTOF/MS method has proven to be a powerful tool for rapid identification of steroidal saponins in T. tschonoskii without tedious and time-consuming isolation of pure constituents. Copyright © 2015 John Wiley & Sons, Ltd. Supporting information can be found in the online version of this article. Keywords: UPLC–MS/MS; steroidal saponins; Trillium tschonoskii Maxim
Introduction
Phytochem. Anal. 2015, 26, 269–278
* Correspondence to: Xin Gao, Department of Pharmaceutical Sciences, School of Pharmacy, Xi’an Jiaotong University, No.76, Yanta West Road, Xi’an, Shaanxi, 710061, China. E-mail: [email protected] ** Correspondence to: Wenjun Sun, Information Department of Science and Technology, Xi’an Xintong Pharmaceutical Research Co., Ltd, No.69, Jinye Road, Xi’an, Shaanxi, 710077, China. E-mail: [email protected] a
Department of Pharmaceutical Sciences, School of Pharmacy, Xi’an Jiaotong University, Xi’an, China
b
Information Department of Science and Technology, Xi’an Xintong Pharmaceutical Research Co., Ltd, Xi’an, China
Copyright © 2015 John Wiley & Sons, Ltd.
269
Trillium tschonoskii Maxim (family Liliaceae) is a herbaceous plant in China, locally known as ‘Yan Ling Cao’, meaning that it is a herb that can prolong human life. Its dried roots and rhizomes are used as a folk medicine for the treatment of hypertension, dizziness, neurasthenia, waist–leg pain, traumatic haemorrhage (Song et al., 2001). Phytochemical examinations revealed that it contains a large number of steroidal saponins, and over 30 saponins including diosgenyl, pennogenyl, protodiosgenyl, kryptgenin and trillenogenin saponins have been isolated (Wang et al., 2007; Yu and Zou, 2008; Zhang et al., 2011; Wei et al., 2012). Steroidal saponins isolated from T. Tschonoskii were found to posses various bioactivities, for example immunological regulation and antiinflammatory, anti-oxidative, especially strong anti-aging and anti-tumour properties (Huang and Xiao, 2006; Yokosuka and Mimaki, 2008; Yu et al., 2008a,2008b; H. Wang et al., 2013). Recently, there has been renewed interest because of their reported beneficial effect in various cancers, as well as senile dementia. Comprehensive ingredient identification including structural characterisation of steroidal saponins from T. tschonoskii is critical for understanding its pharmacological mechanism and
establishing quality control protocols. However, various types of aglycone together with different possibilities of sugar chain composition and attachment cause great natural diversity of saponin structures (Man et al., 2010). Thus, the entire analytical process for characterising saponins could be extremely difficult and labour intensive. Currently, there are only a few analytical research reports on evaluating the quality of T. tschonoskii using HPLC–UV with monitoring hydrolysed sapogenin (diosgenin) or three other saponins (Cui and Liu, 2009; Li and Chen, 2009; Wang et al.,
X. Gao et al. 2013b; Wu et al., 2013). The HPLC–UV method is probably limited in practice as most saponins lack UV absorption. The UPLC– ESI/QTOF/MS method has been established as an ideal tool for the profiling of saponin mixtures, because it offers superior sensitivity, selectivity, considerable structure information and short retention times, and to some extent avoids the time-consuming steps in the isolation of saponins (Ouyang et al., 2014; Qi et al., 2014). In this study, given the complexity of steroidal saponins in T. Tschonoskii and their integrated contribution to therapeutic effects, an approach for rapidly separating, systemically identifying and investigating structural characteristics of steroidal saponins from the extract of T. tschonoskii was established by UPLC– ESI/QTOF/MS.
Experimental Chemicals and materials Acetonitrile (MeCN) and methanol (MeOH) were of HPLC grade from Merck (Darmstadt, Germany); water was produced by a Milli-Q system (Millipore, Bedford, MA, USA); formic acid (HCOOH) was purchased from Tianjin Kermel (Tianjin, China); methanol (analytical grade) was purchased from ChengDu KeLong Chemical Co., Ltd and ChengDu KeLong Chemical Reagent Company (Chendu, China). The roots and rhizomes of T. tschonoskii were collected from Baoji, Shaanxi Province of China in September 2012, and were authenticated by Professor Xiaofeng Niu of the Department of Pharmacognosy, Xi’an Jiaotong University. A voucher specimen (12092801) was deposited in the department of Pharmaceutical Sciences, School of Pharmacy, Xi’an Jiaotong University. Reference standards of trillin, dicosin and diosgenin were purchased from Chendu Must Biotechnology Co., Ltd (Chendu, China), polyphyllin V, polyphyllin VI, polyphyllin VII and pennogenin 3-O-β-chacotrioside were obtained from Chendu Jioute Biotech Co., Ltd (Chendu, China). All the standards used were of the highest purity available (>97%). Pennogenin was isolated and purified from the roots and rhizomes of T. tschonoskii in our laboratory, its purity was > 97% as assayed by HPLC, and its chemical structure was identified by mass and NMR spectra.
duration. Data were collected in centroid mode and mass was corrected TM during acquisition using an external reference (Lock-Spray ) comprising a 100 μL/min solution of leucine–enkephalin via a lockspray interface, + generating a reference ion at 556.2771 Da ([M + H] ) for positive ESI mode and at m/z 554.2615 Da ([M H] ) in negative ion mode to ensure accurate mass measurements over a wide dynamic range. The accurate mass and composition for the precursor ions and for the fragment ions were calculated using the MassLynx 4.1 software (Waters) that was incorporated with the instrument.
Establishment of an in-house molecular formula database By comprehensively searching databases such as Chemspider, PubMed, PubChem Compound and SciFinder we established an in-house molecular formula database of all compounds reported in the literature on T. tschonoskii and related natural products in the same genus and related genera in the Trillaceae family (Paris and Ypsilandra genera) using automated MetaboLynx software. The database includes the compound name, molecular formula, accurate molecular mass, chemical structure and literature citations (Gao et al., 2014).
Results and discussion UPLC–QTOF/MS profiles Optimised chromatographic conditions were achieved through trial and error. A linear gradient elution with acetonitrile and water containing formic acid as the mobile phase gave the best peak resolution. Typical chromatograms with mass spectrometric detection at positive and negative ion modes are presented in Figure 1. Detection at positive ion mode seemed to be more
Preparation of T. tschonoskii samples for UPLC–QTOF/MS analysis The dried root and rhizome of T. tschonoskii were crushed to powder (40 mesh size) and extracted with methanol (1 g/20 mL) twice each for 1 h under reflux. The combined extracts were dissolved in 50 mL of methanol and centrifuged at 13000 rpm for 10 min at 4°C. The supernatant was filtered through a 0.22 μm filter prior to UPLC analysis with triplicate injections.
Chromatography and mass spectrometry TM
270
Chromatographic analysis was conducted on an Acquity Ultra Performance LC system (Waters, Milford, USA) controlled with Masslynx (V4.1). TM Separation was performed using an Acquity UPLC BEH C18-column (2.1 mm × 100 mm, 1.7 μm; Waters), with a mobile phase flow rate of 0.3 mL/min. The column temperature was set at 30°C. The optimal mobile phase consisted of (A) 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. The gradient programme was 0–5 min, 2–20% B; 5–7 min, 20–25% B; 7–10 min, 25–35% B; 10–22 min, 35–99% B. The sample injection volume TM was 5 μL. The Waters Acquity UPLC system equipped with mass spectrometric detection with an electrospray ionisation (ESI) source operated in both positive and negative modes. The optimised mass spectrometric parameters were as follows: nebuliser gas flow rate at 700 L/h at a temperature of 300°C, cone gas at 40 L/h, and source temperature at 110°C, capillary and cone voltages at 3000 V and 40 V, and the extraction cone voltage was 4.0 V in both positive and negative ion mode, respectively. The full-scan MS data were acquired from 100 to 1500 Da with a 0.30 s scan
wileyonlinelibrary.com/journal/pca
Figure 1. The UPLC–MS base peak intensity chromatograms of eight mixed standards in (a) positive ion mode and (b) negative ion mode, and Trillium tschonoskii Maxim in (c) positive ion mode and (d) negative ion mode.
Copyright © 2015 John Wiley & Sons, Ltd.
Phytochem. Anal. 2015, 26, 269–278
Identification of 31 Saponins from T. Tschonoskii by UPLC–MS/MS sensitive and present richer characteristic fragmentation than that at negative ion mode. A complicated chromatographic peak pattern was observed and the major peaks were characterised as steroidal saponins, as described in the following section. Structural identification
Phytochem. Anal. 2015, 26, 269–278
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/pca
271
In negative ion mode, most saponins generated only the solvent adduct ion [M H + HCOOH] and deprotonated ion [M H] of high intensity and without fragmentation, even at higher fragmentation voltage. In positive ion mode, most saponins exhibited quasi-molecular ions of [M + H]+ and/or [M + H H2O]+, plus ions that presented rich structural characteristic fragmentation. Sensitive detection of saponins could be achieved under positive ion mode, while structural characterisation was performed using the data from both positive ion and negative ion mode. The structural identification was based on retention time, precise molecular weight (four decimal places) and MS/MS fragmentation patterns. First, their potential element compositions and precise molecular mass were obtained with specified mass accuracy (error < 5 ppm) and i-FIT (normal, value < 1.0), in addition the degree of non-saturation was obtained. Second, by comprehensively scrutinising the in-house formula database, possible chemical structures were narrowed down to one or a few compounds (Gao et al., 2014). Finally, together with the interpretation of the observed MS/MS spectra using the Masslynx MassFragment function in comparison with those found in the literature (Nohara et al., 1975; Fukuda et al., 1981; Nakano et al., 1983; Ono et al., 1986, 2007a,2007b; Yu and Zou, 2008; Hayes et al., 2009; Xie et al., 2009; Man et al., 2010; Zhang et al., 2011; Kang et al., 2012; Wei et al., 2012; Li et al., 2013; Wang et al., 2013a) or reference standards, the chemical structures were proposed. Thirty-one compounds were tentatively identified and listed in Table 1, which summarises the accurate mass measurements (
