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Simultaneous quantification of six major triterpenoid saponins in Schefflera kwangsiensis using highperformance liquid chromatography coupled to orbitrap mass spectrometry a
bc
Leilei Zhang , Yan Wang
& De-Quan Yu
b
a
Click for updates
Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, P.R. China b
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, P.R. China c
Institute of Agro-products Processing Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R. China Published online: 09 Apr 2015.
To cite this article: Leilei Zhang, Yan Wang & De-Quan Yu (2015): Simultaneous quantification of six major triterpenoid saponins in Schefflera kwangsiensis using high-performance liquid chromatography coupled to orbitrap mass spectrometry, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1025397 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1025397
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should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
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Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1025397
Simultaneous quantification of six major triterpenoid saponins in Schefflera kwangsiensis using high-performance liquid chromatography coupled to orbitrap mass spectrometry
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Leilei Zhanga1*, Yan Wangbc1 and De-Quan Yub* a Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, P.R. China; bState Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, P.R. China; cInstitute of Agro-products Processing Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R. China
(Received 25 November 2014; final version received 28 February 2015)
A simple and accurate analytical method was developed for simultaneous and quantitative analysis of six triterpenoid saponins in Schefflera kwangsiensis via highperformance liquid chromatography (HPLC) with mass spectrometry (MS) in this study. Separation was performed on a Thermo hypersil GOLD C18 column (150 mm £ 2.1 mm, 5 mm). A mobile phase consisting of methanol/acetonitrile/8 mM ammonium acetate in water was used with a flow rate of 0.3 mL/min. The analytes were detected by MS with the electrospray ionisation (ESI) source combined with negative monitoring and full scan mode, and were analysed by extracted ion chromatography. This established HPLC-ESI-MS analysis demonstrated good linearity, sensitivity, stability, precision, accuracy and recovery. Therefore, this analytical method has great potential to be a novel tool to qualify S. kwangsiensis. Keywords: Schefflera kwangsiensis; triterpenoid saponins; quantitative analysis; HPLC-ESI-MS
1. Introduction The plant Schefflera kwangsiensis Merr. ex Li, commonly called ‘Han-Tao-Ye’, is a traditional Chinese medicine, which is mainly distributed in Guangdong Province and in the Guangxi Zhuang Autonomous Region in China. It is demonstrated that the aerial parts of this plant have multiple pharmacological activities and are used for the treatment of trigeminal neuralgia, headache,
*Corresponding author. Email: [email protected]; [email protected] q 2015 Taylor & Francis
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sciatica, rheumatalgia, trauma and other ailments. A commercial drug ‘Han-Tao-Ye’ Tablet made from this plant had been recorded in Chinese Pharmacopoeia (Chinese Pharmacopoeia Commission 1977). In previous publications, several organic acids, two triterpenes and three triterpenoid glycosides were reported to be separated from this plant (Pancharoen et al. 1994), probably because the natural products that are rich in this plant are triterpenoid glycosides which are usually difficult and time consuming to extract and mainly due to their high polarity, similar structural features and easy blister. Moreover, owing to few studies on this plant, fumaric acid is the only chemical component usually used to determine the quality of S. kwangsiensis. This quality control criterion was not ideal since fumaric acid exists in most herbal medicines, and there are less convincible evidences to prove that fumaric acid has anti-inflammatory and analgesic effects as S. kwangsiensis has (Cho et al. 2008; Mostafa et al. 2013). Therefore, it is necessary to reasonably determine a natural agent over fumaric acid in S. kwangsiensis to characterise this herb medicine and to accordingly develop a novel analytical method. Recently, systematical investigations have been carried out on the chemical constituents in S. kwangsiensis. More than 60 triterpenes and triterpenoid saponins were isolated and structurally identified from this plant, and several of them were proved to have moderate hepatoprotective activities against D -galactosamine-induced HL-7702 cell damage according to the previous reports (Wang, Wang, et al. 2014; Wang, Zhang, et al. 2014; Wang et al. 2015). Triterpenoid saponins with various of biological and pharmacological activities could be potential therapeutic material basis of S. kwangsiensis (Dinda et al. 2010). Therefore, as major contents of S. kwangsiensis, six abundant triterpenoid saponins, compounds 1– 6 (Figure 1) used as chemical markers, were determined simultaneously. However, due to the inherent characteristics of triterpenoid saponins such as high polarity, similar structures, non-chromophores and low content in plants, it is difficult to quantify them in S. kwangsiensis by regular high-performance liquid chromatography (HPLC)– UV and HPLC – ELSD (evaporative light scattering detector) (de Oliveira et al. 2002; Wang et al. 2007). HPLCcoupled mass spectrometry (MS) detection is a powerful tool to quantitatively analyse triterpenoid
Figure 1. Chemical structures of seven triterpenoid saponins (1 – 7) in S. kwangsiensis.
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saponins because it has a much better detection sensitivity and selectivity than classical analytical methods. In this study, a simple and accurate analytical method was developed and optimised for simultaneous quantification of six major triterpenoid saponins in S. kwangsiensis (Figure 1). Compound 7 was an artificially ethylated product of compound 2 and accordingly was used as an internal standard for quantitation of the rest six triterpenoid saponins. The advantages brought by this analytical method confer its potentials to be used as a tool to qualify S. kwangsiensis.
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2. Results and discussion 2.1. Optimisation of extracting conditions of S. kwangsiensis In order to obtain optimal extracting efficiency, several key factors such as solvent, ultrasonic time and cold soak were optimised (Supporting information Figure S1). According to preliminary exploration, the solvent used in the extracting step is the most important. With regard to high polarity of triterpenoid saponins, water, 45% methanol/water (v/v), 70% methanol/water (v/v) and methanol were chosen as candidates. Among them, 45% methanol/water exhibited the highest efficiency among extracting conditions in parallel. In addition, artificially produced products were commonly found during the extraction and isolation of triterpenoid saponins, such as esterification, methylation, ethylation and butylation. To avoid these artificial modifications to the most extent, instead of conventional heat reflux, a ultrasonic bath was eventually applied to extract major chemical constituents of the plant in 45% (v/v) methanol/water for 30 min. The obtained crude extract was used to qualify S. kwangsiensis in this study. 2.2. Optimisation of HPLC – ESI –MS conditions We optimised parameters of electrospray ionisation MS (ESI-MS) in order to achieve maximum signal for the protonated molecular ions. The ion signal intensities for six triterpenoid saponins were first investigated in both positive ion mode and negative ion mode, in which comparison, negative scan mode offered much greater response regardless of supplementing either formic aid or ammonium acetate in the mobile phase. Therefore, negative ion mode with a capillary voltage of 2.5 kV was set up in this study with 8 mM aqueous ammonium acetate used as an additive in mobile phase to improve analyte ionisation. Next, we manually adjusted other important MS parameters including mass resolution, automatic gain control (AGC) target and maximum injection time. Under the optimised conditions, six isolated triterpenoid saponins were analysed by HPLC coupling ESI-MS (HPLC –ESI-MS). Extracted ion chromatography (EIC) and MS spectra of six triterpenoid saponins and internal standard compound are shown in Figure 2 (total ion chromatogram (TIC) is shown in Supporting information Figure S2). As demonstrated in Figure 2(A, B), ions with high abundance were selected for obtaining EIC of compounds 1– 7 subsequently. EIC of each of these ions will produce a single peak that can be used to accurately quantify each compound. Among them, compounds 3 and 4 are co-eluted (tR ¼ 31.37 min). But these two compounds have different m/z value. Adduct ion [M þ CH3COOZH]22 (m/z 580.3) is observed in the mass spectra of compound 3. Adduct ion [M þ CH3COOZH]22 (m/z 653.3) is observed in the mass spectra of compound 4. Two ions are simultaneously found in one mass spectra. However, m/z 580.3 and 653.3 can be, respectively, chosen, and two single peaks are obtained in the EIC which represents two analytes. 2.3. Validation of HPLC – ESI-MS analysis Under the optimised HPLC–ESI-MS conditions as described above, six calibration curves (peak area ratio of each compound to internal standard vs compound’s concentration) corresponding to six individual triterpenoid saponins were built. All of them exhibited good linear regressions with
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Figure 2. EIC and MS of six triterpenoid saponins (1 – 6) and internal standard (7).
R 2 values between 0.9957 and 0.9989 (Supporting information Table S1). The limit of detection (LOD) and limit of quantification (LOQ) of the six isolated triterpenoid saponins in the analysis were 0.39–0.78 and 1.05–2.80 ng/mL, respectively. In addition, the HPLC – ESI-MS analysis was reproducible with good accuracy for all analytes (Supporting information Table S2). The intra- and inter-day variations were all less than 5%. The recoveries were in the range of 87 – 114% with relative standard deviations (RSDs) less than 12.7% (Supporting information Table S3).
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On the basis of these advantages, we further preliminarily measured six triterpenoid saponins in the crude extract of S. kwangsiensis by using this analytical method once every 5 h in total 25 h. The RSD of concentration variations was even less than 7.9%, which indicates that the six triterpenoid saponins should be stable in the crude extract during the analysis (Supporting information Table S4).
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2.4. Quantitative analysis of six triterpenoid saponins in two batches of S. kwangsiensis HPLC – ESI-MS analysis was eventually applied to quantify the contents of six triterpenoid saponins in two batches of S. kwangsiensis. The TICs of the S. kwangsiensis extract in the negative mode are presented in Figure 3. Although chromatogram peak at 31.88 min is coelution peak of compounds 3 and 4 with similar structures, it does not affect accurate quantitation of these two saponins. All contents were calculated using the internal-standard method, and the mean values from three parallel determinations of each sample are summarised in Table 1. Compounds 1, 2 and 5 were the major constituents in two batches of samples from different regions, their contents were higher than those of the other saponins. However, there is a difference between the two batches. The concentration of compound 1 was highest in batch 1 (328.17 mg/g), the concentration of compound 2 was the highest in batch 2 (271.58 mg/g). 3. Experimental procedures 3.1. Materials and chemicals The aerial parts of plants of S. kwangsiensis were collected in Guangxi Zhuang Autonomous Region in P.R. China and identified by Professor Lin Ma (Institute of Materia Medica, Chinese
Figure 3. TIC of S. kwangsiensis extract in the negative-ionisation mode.
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Table 1. Contents of six triterpenoid saponins in different batches of S. kwangsiensis (n ¼ 3).
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Batch 1 Batch 2
1 (mg/g)
2 (mg/g)
3 (mg/g)
4 (mg/g)
5 (mg/g)
6 (mg/g)
328.17 223.27
217.31 271.58
166.88 134.31
126.26 167.05
248.79 182.45
180.21 65.67
Academy of Medical Sciences and Peking Union Medical College). Voucher specimens were deposited at the herbarium of the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing. The raw plant materials were air-dried and stored at room temperature until use. LC/MS grade acetonitrile (Thermo Fisher Scientific, Fairlawn, NJ, USA) and HPLC grade methanol (Honeywell Burdick & Jackson, Muskegon, MI, USA) were used. Deionised distilled water was Wa Ha-ha purified water. The other reagents were commercially available and of analytical purity. A total of six triterpenoid saponins 1– 6 and one internal standard triterpenoid saponin compound 7 (Figure 1) were isolated from S. kwangsiensis in our laboratory as previously reported (Wang, Zhang, et al. 2014; Wang et al. 2015). All of these samples had a purity of greater than 90% as determined by HPLC analysis and were used as standard compounds for quality analysis (Supporting information Figure S3). 3.2. Sample preparation for LC – MS analysis The dried material of S. kwangsiensis was pulverised into powder. Each sample (1.0 g) was accurately weighed and extracted with 50 mL mixture solution of methanol and water by ultrasonic water bath (power 250 W, 40 kHz) at room temperature for 30 min. Then the extracted solution was adjusted to the original weight, filtered using 0.45 mm membranes and injected into the HPLC system. Each of the seven triterpenoid saponins was accurately weighed into a 1 mL volumetric flask. Compounds 1 and 5– 7 were dissolved in 100 mL DMSO and diluted with 70% methanol/water (v/v) to scales. Compounds 2 –4 were dissolved in 70% methanol – water (v/v). All samples were kept at 48C refrigerator until use. 3.3. HPLC – ESI-MS conditions HPLC – ESI-MS analyses were performed on ThermoFisher Accela liquid chromatography system coupled with Exactive Plus orbitrap MS (Thermo-Fisher, Bremen, Germany) via an ESI interface. The HPLC instrument was equipped with a quaternary solvent delivery system, an autosample, Photo-Diode Array (PDA) detector and a column compartment. The compounds were chromatographically resolved on a Thermo Hypersil GOLD C18 column (150 mm £ 2.1 mm, 5 mm) connected with an Agilent Zorbax SB-C18 guard column (12.5 mm £ 4.6 mm, 5 mm) at room temperature. A three-component mobile phase system was used, which contained the mixture of acetonitrile and methanol (1:1, A), and water containing 8 mM ammonium acetate (B). A gradient programme was used as follows: 0– 48 min, 12 –48% A; 48 –52 min, 48 – 92% A; and 52 –54 min, 92% A. The flow rate was 0.3 mL/min. All the data were processed by using Thermo Xcalibur 2.2 ChemStation software. A 6-min post-run time was set to fully equilibrate the column. The sample injection volume was 10 mL. For MS analysis, high-purity nitrogen (N2) (. 99.99%) was used as the nebulising gas. Ions were generated using electrospray ionisation on the standard Thermo IonMax ESI source. The instrument worked in a full-scan acquisition mode in the range 300– 2000 Da at a mass
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resolution of 17,500 FWHM, allowing a maximum C-Trap fill time of 100 ms. The optimised parameters in the negative ion mode were as follows: capillary temperature and heater temperature were 3508C and 3008C, respectively, spray voltage was 2.5 kV, sheath gas was 40 psi, auxiliary nitrogen pressure was 12 L/min, AGC target valve was set at 1 £ 106 charges.
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3.4. Validation for the HPLC – ESI-MS analysis To qualify the method, the linearity of the method was further investigated. The standard solutions were diluted with 70% methanol to provide a series of standard solutions with the appropriate concentrations. The calibration curves were constructed by plotting peak area ratio of analytes to the internal standard (y) versus the concentrations of each analyte (x, ng/mL). Linearity of each compound was determined with three injections for each concentration and was evaluated by linear regression analysis calculated by the least squares regression method. LOD and LOQ under the present chromatographic conditions were determined on the basis of response at a signal-to-noise ratio of 3 or 10, respectively. The precision of the method was evaluated by analysing the standard solutions containing six standard compounds at three different concentration levels (low, middle and high). The experiment was repeated six times on the same day and additionally on three consecutive days to determine intra- and inter-day variations. Variations were expressed by RSD. Six samples from the same origin were extracted and analysed to measure the method repeatability. The same sample was stored at 258C and analysed at 0, 5, 10, 15, 20 and 25 h for stability test. The recovery test was used to evaluate the accuracy of this quantification method. Accurate amounts of six triterpenoid saponins with three different concentration levels (low, middle and high) were added to known amounts of S. kwangsiensis samples and then extracted and analysed with the HPLC – ESI-MS method. Three replicate extractives at each level were used to calculate the average recoveries which were determined by the following equation: recovery (%) ¼ (detected amount – original amount)/spiked amount £ 100%. 4. Conclusion In this study, a HPLC – ESI-MS method was optimised for quantitation of six major triterpenoid saponins in S. kwangsiensis. To our knowledge, this is the first report about the contents of triterpenoid saponins in S. kwangsiensis. This precise and reproducible analytical method is suitable for the analysis of low content triterpenoid saponins. Therefore, this method facilitates evaluation of S. kwangsiensis in quality. Supplementary material Supplementary material relating to this paper is available online, alongside Tables S1– S4 and Figures S1 –S3. Disclosure statement No potential conflict of interest was reported by the authors.
Funding The project was supported by the National Science and Technology Project of China [grant number 2012ZX09301002-002] and the State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College.
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Note 1. These authors contributed equally to this work.
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References Chinese Pharmacopoeia Commission. 1977. Pharmacopoeia of the People’s Republic of China. Vol. 1. Beijing: China Medical Science Press; p. 193 –195. Cho SM, Jang KY, Park HJ, Park JS. 2008. Analysis of the chemical constituents of Agaricus Brasiliensis. Mycobiology. 36:50–54. doi:10.4489/MYCO.2008.36.1.050. de Oliveira BH, Santos CA, Espı´ndola AP. 2002. Determination of the triterpenoid, betulinic acid, in Doliocarpus schottianus by HPLC. Phytochem Anal. 13(2):95–98. doi:10.1002/pca.628. Dinda B, Debnath S, Mohanta BC, Harigaya Y. 2010. Naturally occurring triterpenoid saponins. Chem Biodivers. 7:2327–2580. doi:10.1002/cbdv.200800070. Mostafa I, Abd El-Aziz E, Hafez S, El-Shazly A. 2013. Chemical constituents and biological activities of Galinsoga parviflora cav. (Asteraceae) from Egypt. Z Naturforsch C. 68:285– 292. Pancharoen O, Tuntiwachwuttikul P, Taylor WC, Picker K. 1994. Triterpenoid glycosides from Schefflera lucantha. Phytochemistry. 35:987–992. doi:10.1016/S0031-9422(00)90653-8. Wang H, Gao J, Zhu D, Yu B. 2007. Quality evaluation of Polygala japonica through simultaneous determination of six bioactive triterpenoid saponins by HPLC-ELSD. J Pharm Biomed Anal. 43:1552–1556. doi:10.1016/j.jpba.2006. 11.012. Wang Y, Wang L, Wang WJ, Zhang XQ, Tian HY, Zhang QW, Li YL, Ye WC. 2014. New triterpenoid saponins from the aerial parts of Schefflera kwangsiensis. Carbohydrate Res. 385:65–71. doi:10.1016/j.carres.2013.07.016. Wang Y, Zhang CL, Liu YF, Liang D, Luo H, Hao ZY, Chen RY, Yu DQ. 2014. Hepatoprotective triterpenoids and saponins of Schefflera kwangsiensis. Planta Med. 80:215–222. Wang Y, Zhang LL, Zhang CL, Liu YF, Liang D, Luo H, Hao ZY, Chen RY, Yu DQ. 2015. Esters of new oleananetype triterpenoid saponins from Schefflera kwangsiensis. Phytochem Lett. 11:95–101. doi:10.1016/j.phytol. 2014.11.019.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Simultaneous quantification of six major triterpenoid saponins in Schefflera kwangsiensis using highperformance liquid chromatography coupled to orbitrap mass spectrometry a
bc
Leilei Zhang , Yan Wang
& De-Quan Yu
b
a
Click for updates
Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, P.R. China b
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, P.R. China c
Institute of Agro-products Processing Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R. China Published online: 09 Apr 2015.
To cite this article: Leilei Zhang, Yan Wang & De-Quan Yu (2015): Simultaneous quantification of six major triterpenoid saponins in Schefflera kwangsiensis using high-performance liquid chromatography coupled to orbitrap mass spectrometry, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1025397 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1025397
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
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This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1025397
Simultaneous quantification of six major triterpenoid saponins in Schefflera kwangsiensis using high-performance liquid chromatography coupled to orbitrap mass spectrometry
Downloaded by [Pennsylvania State University] at 05:33 16 April 2015
Leilei Zhanga1*, Yan Wangbc1 and De-Quan Yub* a Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, P.R. China; bState Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, P.R. China; cInstitute of Agro-products Processing Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R. China
(Received 25 November 2014; final version received 28 February 2015)
A simple and accurate analytical method was developed for simultaneous and quantitative analysis of six triterpenoid saponins in Schefflera kwangsiensis via highperformance liquid chromatography (HPLC) with mass spectrometry (MS) in this study. Separation was performed on a Thermo hypersil GOLD C18 column (150 mm £ 2.1 mm, 5 mm). A mobile phase consisting of methanol/acetonitrile/8 mM ammonium acetate in water was used with a flow rate of 0.3 mL/min. The analytes were detected by MS with the electrospray ionisation (ESI) source combined with negative monitoring and full scan mode, and were analysed by extracted ion chromatography. This established HPLC-ESI-MS analysis demonstrated good linearity, sensitivity, stability, precision, accuracy and recovery. Therefore, this analytical method has great potential to be a novel tool to qualify S. kwangsiensis. Keywords: Schefflera kwangsiensis; triterpenoid saponins; quantitative analysis; HPLC-ESI-MS
1. Introduction The plant Schefflera kwangsiensis Merr. ex Li, commonly called ‘Han-Tao-Ye’, is a traditional Chinese medicine, which is mainly distributed in Guangdong Province and in the Guangxi Zhuang Autonomous Region in China. It is demonstrated that the aerial parts of this plant have multiple pharmacological activities and are used for the treatment of trigeminal neuralgia, headache,
*Corresponding author. Email: [email protected]; [email protected] q 2015 Taylor & Francis
Downloaded by [Pennsylvania State University] at 05:33 16 April 2015
2
L. Zhang et al.
sciatica, rheumatalgia, trauma and other ailments. A commercial drug ‘Han-Tao-Ye’ Tablet made from this plant had been recorded in Chinese Pharmacopoeia (Chinese Pharmacopoeia Commission 1977). In previous publications, several organic acids, two triterpenes and three triterpenoid glycosides were reported to be separated from this plant (Pancharoen et al. 1994), probably because the natural products that are rich in this plant are triterpenoid glycosides which are usually difficult and time consuming to extract and mainly due to their high polarity, similar structural features and easy blister. Moreover, owing to few studies on this plant, fumaric acid is the only chemical component usually used to determine the quality of S. kwangsiensis. This quality control criterion was not ideal since fumaric acid exists in most herbal medicines, and there are less convincible evidences to prove that fumaric acid has anti-inflammatory and analgesic effects as S. kwangsiensis has (Cho et al. 2008; Mostafa et al. 2013). Therefore, it is necessary to reasonably determine a natural agent over fumaric acid in S. kwangsiensis to characterise this herb medicine and to accordingly develop a novel analytical method. Recently, systematical investigations have been carried out on the chemical constituents in S. kwangsiensis. More than 60 triterpenes and triterpenoid saponins were isolated and structurally identified from this plant, and several of them were proved to have moderate hepatoprotective activities against D -galactosamine-induced HL-7702 cell damage according to the previous reports (Wang, Wang, et al. 2014; Wang, Zhang, et al. 2014; Wang et al. 2015). Triterpenoid saponins with various of biological and pharmacological activities could be potential therapeutic material basis of S. kwangsiensis (Dinda et al. 2010). Therefore, as major contents of S. kwangsiensis, six abundant triterpenoid saponins, compounds 1– 6 (Figure 1) used as chemical markers, were determined simultaneously. However, due to the inherent characteristics of triterpenoid saponins such as high polarity, similar structures, non-chromophores and low content in plants, it is difficult to quantify them in S. kwangsiensis by regular high-performance liquid chromatography (HPLC)– UV and HPLC – ELSD (evaporative light scattering detector) (de Oliveira et al. 2002; Wang et al. 2007). HPLCcoupled mass spectrometry (MS) detection is a powerful tool to quantitatively analyse triterpenoid
Figure 1. Chemical structures of seven triterpenoid saponins (1 – 7) in S. kwangsiensis.
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saponins because it has a much better detection sensitivity and selectivity than classical analytical methods. In this study, a simple and accurate analytical method was developed and optimised for simultaneous quantification of six major triterpenoid saponins in S. kwangsiensis (Figure 1). Compound 7 was an artificially ethylated product of compound 2 and accordingly was used as an internal standard for quantitation of the rest six triterpenoid saponins. The advantages brought by this analytical method confer its potentials to be used as a tool to qualify S. kwangsiensis.
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2. Results and discussion 2.1. Optimisation of extracting conditions of S. kwangsiensis In order to obtain optimal extracting efficiency, several key factors such as solvent, ultrasonic time and cold soak were optimised (Supporting information Figure S1). According to preliminary exploration, the solvent used in the extracting step is the most important. With regard to high polarity of triterpenoid saponins, water, 45% methanol/water (v/v), 70% methanol/water (v/v) and methanol were chosen as candidates. Among them, 45% methanol/water exhibited the highest efficiency among extracting conditions in parallel. In addition, artificially produced products were commonly found during the extraction and isolation of triterpenoid saponins, such as esterification, methylation, ethylation and butylation. To avoid these artificial modifications to the most extent, instead of conventional heat reflux, a ultrasonic bath was eventually applied to extract major chemical constituents of the plant in 45% (v/v) methanol/water for 30 min. The obtained crude extract was used to qualify S. kwangsiensis in this study. 2.2. Optimisation of HPLC – ESI –MS conditions We optimised parameters of electrospray ionisation MS (ESI-MS) in order to achieve maximum signal for the protonated molecular ions. The ion signal intensities for six triterpenoid saponins were first investigated in both positive ion mode and negative ion mode, in which comparison, negative scan mode offered much greater response regardless of supplementing either formic aid or ammonium acetate in the mobile phase. Therefore, negative ion mode with a capillary voltage of 2.5 kV was set up in this study with 8 mM aqueous ammonium acetate used as an additive in mobile phase to improve analyte ionisation. Next, we manually adjusted other important MS parameters including mass resolution, automatic gain control (AGC) target and maximum injection time. Under the optimised conditions, six isolated triterpenoid saponins were analysed by HPLC coupling ESI-MS (HPLC –ESI-MS). Extracted ion chromatography (EIC) and MS spectra of six triterpenoid saponins and internal standard compound are shown in Figure 2 (total ion chromatogram (TIC) is shown in Supporting information Figure S2). As demonstrated in Figure 2(A, B), ions with high abundance were selected for obtaining EIC of compounds 1– 7 subsequently. EIC of each of these ions will produce a single peak that can be used to accurately quantify each compound. Among them, compounds 3 and 4 are co-eluted (tR ¼ 31.37 min). But these two compounds have different m/z value. Adduct ion [M þ CH3COOZH]22 (m/z 580.3) is observed in the mass spectra of compound 3. Adduct ion [M þ CH3COOZH]22 (m/z 653.3) is observed in the mass spectra of compound 4. Two ions are simultaneously found in one mass spectra. However, m/z 580.3 and 653.3 can be, respectively, chosen, and two single peaks are obtained in the EIC which represents two analytes. 2.3. Validation of HPLC – ESI-MS analysis Under the optimised HPLC–ESI-MS conditions as described above, six calibration curves (peak area ratio of each compound to internal standard vs compound’s concentration) corresponding to six individual triterpenoid saponins were built. All of them exhibited good linear regressions with
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Figure 2. EIC and MS of six triterpenoid saponins (1 – 6) and internal standard (7).
R 2 values between 0.9957 and 0.9989 (Supporting information Table S1). The limit of detection (LOD) and limit of quantification (LOQ) of the six isolated triterpenoid saponins in the analysis were 0.39–0.78 and 1.05–2.80 ng/mL, respectively. In addition, the HPLC – ESI-MS analysis was reproducible with good accuracy for all analytes (Supporting information Table S2). The intra- and inter-day variations were all less than 5%. The recoveries were in the range of 87 – 114% with relative standard deviations (RSDs) less than 12.7% (Supporting information Table S3).
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On the basis of these advantages, we further preliminarily measured six triterpenoid saponins in the crude extract of S. kwangsiensis by using this analytical method once every 5 h in total 25 h. The RSD of concentration variations was even less than 7.9%, which indicates that the six triterpenoid saponins should be stable in the crude extract during the analysis (Supporting information Table S4).
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2.4. Quantitative analysis of six triterpenoid saponins in two batches of S. kwangsiensis HPLC – ESI-MS analysis was eventually applied to quantify the contents of six triterpenoid saponins in two batches of S. kwangsiensis. The TICs of the S. kwangsiensis extract in the negative mode are presented in Figure 3. Although chromatogram peak at 31.88 min is coelution peak of compounds 3 and 4 with similar structures, it does not affect accurate quantitation of these two saponins. All contents were calculated using the internal-standard method, and the mean values from three parallel determinations of each sample are summarised in Table 1. Compounds 1, 2 and 5 were the major constituents in two batches of samples from different regions, their contents were higher than those of the other saponins. However, there is a difference between the two batches. The concentration of compound 1 was highest in batch 1 (328.17 mg/g), the concentration of compound 2 was the highest in batch 2 (271.58 mg/g). 3. Experimental procedures 3.1. Materials and chemicals The aerial parts of plants of S. kwangsiensis were collected in Guangxi Zhuang Autonomous Region in P.R. China and identified by Professor Lin Ma (Institute of Materia Medica, Chinese
Figure 3. TIC of S. kwangsiensis extract in the negative-ionisation mode.
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Table 1. Contents of six triterpenoid saponins in different batches of S. kwangsiensis (n ¼ 3).
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Batch 1 Batch 2
1 (mg/g)
2 (mg/g)
3 (mg/g)
4 (mg/g)
5 (mg/g)
6 (mg/g)
328.17 223.27
217.31 271.58
166.88 134.31
126.26 167.05
248.79 182.45
180.21 65.67
Academy of Medical Sciences and Peking Union Medical College). Voucher specimens were deposited at the herbarium of the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing. The raw plant materials were air-dried and stored at room temperature until use. LC/MS grade acetonitrile (Thermo Fisher Scientific, Fairlawn, NJ, USA) and HPLC grade methanol (Honeywell Burdick & Jackson, Muskegon, MI, USA) were used. Deionised distilled water was Wa Ha-ha purified water. The other reagents were commercially available and of analytical purity. A total of six triterpenoid saponins 1– 6 and one internal standard triterpenoid saponin compound 7 (Figure 1) were isolated from S. kwangsiensis in our laboratory as previously reported (Wang, Zhang, et al. 2014; Wang et al. 2015). All of these samples had a purity of greater than 90% as determined by HPLC analysis and were used as standard compounds for quality analysis (Supporting information Figure S3). 3.2. Sample preparation for LC – MS analysis The dried material of S. kwangsiensis was pulverised into powder. Each sample (1.0 g) was accurately weighed and extracted with 50 mL mixture solution of methanol and water by ultrasonic water bath (power 250 W, 40 kHz) at room temperature for 30 min. Then the extracted solution was adjusted to the original weight, filtered using 0.45 mm membranes and injected into the HPLC system. Each of the seven triterpenoid saponins was accurately weighed into a 1 mL volumetric flask. Compounds 1 and 5– 7 were dissolved in 100 mL DMSO and diluted with 70% methanol/water (v/v) to scales. Compounds 2 –4 were dissolved in 70% methanol – water (v/v). All samples were kept at 48C refrigerator until use. 3.3. HPLC – ESI-MS conditions HPLC – ESI-MS analyses were performed on ThermoFisher Accela liquid chromatography system coupled with Exactive Plus orbitrap MS (Thermo-Fisher, Bremen, Germany) via an ESI interface. The HPLC instrument was equipped with a quaternary solvent delivery system, an autosample, Photo-Diode Array (PDA) detector and a column compartment. The compounds were chromatographically resolved on a Thermo Hypersil GOLD C18 column (150 mm £ 2.1 mm, 5 mm) connected with an Agilent Zorbax SB-C18 guard column (12.5 mm £ 4.6 mm, 5 mm) at room temperature. A three-component mobile phase system was used, which contained the mixture of acetonitrile and methanol (1:1, A), and water containing 8 mM ammonium acetate (B). A gradient programme was used as follows: 0– 48 min, 12 –48% A; 48 –52 min, 48 – 92% A; and 52 –54 min, 92% A. The flow rate was 0.3 mL/min. All the data were processed by using Thermo Xcalibur 2.2 ChemStation software. A 6-min post-run time was set to fully equilibrate the column. The sample injection volume was 10 mL. For MS analysis, high-purity nitrogen (N2) (. 99.99%) was used as the nebulising gas. Ions were generated using electrospray ionisation on the standard Thermo IonMax ESI source. The instrument worked in a full-scan acquisition mode in the range 300– 2000 Da at a mass
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resolution of 17,500 FWHM, allowing a maximum C-Trap fill time of 100 ms. The optimised parameters in the negative ion mode were as follows: capillary temperature and heater temperature were 3508C and 3008C, respectively, spray voltage was 2.5 kV, sheath gas was 40 psi, auxiliary nitrogen pressure was 12 L/min, AGC target valve was set at 1 £ 106 charges.
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3.4. Validation for the HPLC – ESI-MS analysis To qualify the method, the linearity of the method was further investigated. The standard solutions were diluted with 70% methanol to provide a series of standard solutions with the appropriate concentrations. The calibration curves were constructed by plotting peak area ratio of analytes to the internal standard (y) versus the concentrations of each analyte (x, ng/mL). Linearity of each compound was determined with three injections for each concentration and was evaluated by linear regression analysis calculated by the least squares regression method. LOD and LOQ under the present chromatographic conditions were determined on the basis of response at a signal-to-noise ratio of 3 or 10, respectively. The precision of the method was evaluated by analysing the standard solutions containing six standard compounds at three different concentration levels (low, middle and high). The experiment was repeated six times on the same day and additionally on three consecutive days to determine intra- and inter-day variations. Variations were expressed by RSD. Six samples from the same origin were extracted and analysed to measure the method repeatability. The same sample was stored at 258C and analysed at 0, 5, 10, 15, 20 and 25 h for stability test. The recovery test was used to evaluate the accuracy of this quantification method. Accurate amounts of six triterpenoid saponins with three different concentration levels (low, middle and high) were added to known amounts of S. kwangsiensis samples and then extracted and analysed with the HPLC – ESI-MS method. Three replicate extractives at each level were used to calculate the average recoveries which were determined by the following equation: recovery (%) ¼ (detected amount – original amount)/spiked amount £ 100%. 4. Conclusion In this study, a HPLC – ESI-MS method was optimised for quantitation of six major triterpenoid saponins in S. kwangsiensis. To our knowledge, this is the first report about the contents of triterpenoid saponins in S. kwangsiensis. This precise and reproducible analytical method is suitable for the analysis of low content triterpenoid saponins. Therefore, this method facilitates evaluation of S. kwangsiensis in quality. Supplementary material Supplementary material relating to this paper is available online, alongside Tables S1– S4 and Figures S1 –S3. Disclosure statement No potential conflict of interest was reported by the authors.
Funding The project was supported by the National Science and Technology Project of China [grant number 2012ZX09301002-002] and the State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College.
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Note 1. These authors contributed equally to this work.
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References Chinese Pharmacopoeia Commission. 1977. Pharmacopoeia of the People’s Republic of China. Vol. 1. Beijing: China Medical Science Press; p. 193 –195. Cho SM, Jang KY, Park HJ, Park JS. 2008. Analysis of the chemical constituents of Agaricus Brasiliensis. Mycobiology. 36:50–54. doi:10.4489/MYCO.2008.36.1.050. de Oliveira BH, Santos CA, Espı´ndola AP. 2002. Determination of the triterpenoid, betulinic acid, in Doliocarpus schottianus by HPLC. Phytochem Anal. 13(2):95–98. doi:10.1002/pca.628. Dinda B, Debnath S, Mohanta BC, Harigaya Y. 2010. Naturally occurring triterpenoid saponins. Chem Biodivers. 7:2327–2580. doi:10.1002/cbdv.200800070. Mostafa I, Abd El-Aziz E, Hafez S, El-Shazly A. 2013. Chemical constituents and biological activities of Galinsoga parviflora cav. (Asteraceae) from Egypt. Z Naturforsch C. 68:285– 292. Pancharoen O, Tuntiwachwuttikul P, Taylor WC, Picker K. 1994. Triterpenoid glycosides from Schefflera lucantha. Phytochemistry. 35:987–992. doi:10.1016/S0031-9422(00)90653-8. Wang H, Gao J, Zhu D, Yu B. 2007. Quality evaluation of Polygala japonica through simultaneous determination of six bioactive triterpenoid saponins by HPLC-ELSD. J Pharm Biomed Anal. 43:1552–1556. doi:10.1016/j.jpba.2006. 11.012. Wang Y, Wang L, Wang WJ, Zhang XQ, Tian HY, Zhang QW, Li YL, Ye WC. 2014. New triterpenoid saponins from the aerial parts of Schefflera kwangsiensis. Carbohydrate Res. 385:65–71. doi:10.1016/j.carres.2013.07.016. Wang Y, Zhang CL, Liu YF, Liang D, Luo H, Hao ZY, Chen RY, Yu DQ. 2014. Hepatoprotective triterpenoids and saponins of Schefflera kwangsiensis. Planta Med. 80:215–222. Wang Y, Zhang LL, Zhang CL, Liu YF, Liang D, Luo H, Hao ZY, Chen RY, Yu DQ. 2015. Esters of new oleananetype triterpenoid saponins from Schefflera kwangsiensis. Phytochem Lett. 11:95–101. doi:10.1016/j.phytol. 2014.11.019.
