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ISSN 1615-9306 · JSSCCJ 38 (7) 1073–1262 (2015) · Vol. 38 · No. 7 · April 2015 · D 10609
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J. Sep. Sci. 2015, 38, 1117–1122
Jie Yang1,2 Yuanhong Wang1,2 Ran Zhang1,2 Tifngfu Jiang1,2 Zhihua Lv1,2 1 Key
Laboratory of Marine Drugs, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China 2 Shandong Provincial Key Laboratory of Glycoscience & Glycotechnology, Ocean University of China, Qingdao, China Received November 9, 2014 Revised December 22, 2014 Accepted January 5, 2015
Research Article
Determination of the triterpene glycosides in sea cucumbers by liquid chromatography with evaporative light scattering and mass spectrometry detection Holothurian triterpene glycosides possess various kinds of biological activities, including antifungal, cytotoxic, hemolytic, cytostatic, and immunomodulatory effects. In this study, a rapid extraction method of triterpene glycosides from sea cucumbers using a small column of C18 solid phase was first developed. Furthermore, a novel high-performance liquid chromatography method coupled with evaporative light scattering detection and electrospray ionization mass spectrometry was established for the determination of each triterpene glycosides from different sea cucumbers. Simultaneous separation of all kind of triterpene glycoside were achieved on a C18 column. A gradient of aqueous acetonitrile was applied, and the method was validated. The liquid chromatography method was applied to the online mass detection to identify the triterpene glycosides in the purified extraction of eight kinds of pulverized sea cucumber from the market of Qingdao, China. The negative mode of [M–H]− /[M–Na]− exclusively shown signals corresponding to the triterpene glycosides previously reported and the MS2 product ions of those ions indicate the specific structure of each triterpene glycoside. Keywords: Electrospray ionization mass spectrometry / Evaporative light scattering detection / Sea cucumber / Triterpene glycosides DOI 10.1002/jssc.201401253
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Sea cucumber has been used as a traditional nutritive food for thousands of years. There are many biological materials in the body of sea cucumber, of which the triterpene glycoside is the most important secondary metabolites. Previous reports indicated that the holothurian triterpene glycosides possess various kinds of biological activities, including antifungal, cytotoxic, hemolytic, cytostatic, antitumor, and immunomodulatory effects [1–4]. There have been hundreds of kinds of triterpene glycosides found in the body of different sea cucumbers, and most of them are distributed in the Holothuriidae and Stichopodidae families of Aspidochirotida order and Cucumariidae family of Dendrochirotida order [5]. Due to the high nutritive value and healthcare effects, various kinds of nutriments and tonic products are now made of sea cucumber, including dried products (beche-de-mer), Correspondence: Professor Zhihua Lv, Shandong Provincial Key Laboratory of Glycoscience & Glycotechnology, Ocean University of China, Qingdao 266003, China. E-mail: [email protected] Fax: +86 532 82032064
Abbreviations: ELSD, evaporative light scattering detection; Glc, glucose; MeGlc, methylglucose; Xyl, xylose C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
pickled products, wetter risen products, oral liquid, capsules, etc. [6]. However, the triterpene glycosides have become a very important index for the nutrition evaluation and QC of sea cucumber products. The analytical methods for the quantitative of triterpene glycoside include spectrophotometry, TLC, HPLC–ELSD (where ELSD is evaporative light scattering detection), LC–ESI-MS, LC–UV–NMR, LC–PDA–ESI-MS/MS, and CE [7–12]. Compared with others, the ELSD is a universal, nonspecific detector that can provide a stable baseline even with gradient elution [13]. ESI-MS is an efficient tool for the determination of triterpene glycosides in the crude extracts, due to its high sensitivity, short analysis time, and low consumption of samples [14–16]. ESI-MS can rapidly determine the bioactive compounds in the samples and give their structural information. In recent years, the reports on the combined application of LC and MS to the online analysis of natural products have increased [17–19]. However, the determination of triterpene glycosides from sea cucumber by HPLC–ELSD–MS has not been reported yet. In the present paper, a rapid analytical HPLC method coupled with the ELSD and MS/MS detection has been first established for the QC, which allows simultaneous determination of the total triterpene glycosides in different sea cucumbers. The method includes the pretreatment of sample, www.jss-journal.com
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establishment and validation of the HPLC–ELSD, and determination of the compounds of triterpene glycosides. Using a preparation of Holotoxin A1 as an external standard, the contents of this specific main product were observed, which were isolated from the sea cucumber Apostichopus japonicus (also named after Stichopus japonicus) from different regions. Furthermore, the total triterpene glycosides isolated from eight kinds of pulverized sea cucumber products from the different species were determined. These studies should provide a method for the quantitative determination and structural characterization of the triterpene glycosides in the different sea cucumbers.
2 Materials and methods 2.1 Materials All of the dried sea cucumber samples were purchased from the Nanshan market of Qingdao, China. All other chemical reagents were of analytical grade from Sinopharm Chemical Reagent (Shanghai, China). 2.2 Chromatography conditions The analysis of the total triterpene glycosides were performed on a Waters 1525 liquid chromatograph equipped with Waters 2420 ELSD detector and Hypersil Gold C18 column (Thermo Scientific, USA; 150 mm × 2.1 mm, particle size 3 m). The mobile phases were 5 mM ammonium acetate (solvent A) and acetonitrile (solvent B). A gradient of solvent A was performed from 73 to 58% in 15 min, and kept for 13 min at a flow rate of 0.2 mL/min at 40⬚C. Ten microliters of sample was injected for the analysis. Parameters of the ELSD detector were as follows: the temperature of drift tube was 60⬚C, the atomization efficiency was 50%, and the nebulizer gas nitrogen pressure was 25 psi and the gain was 25. The same LC method was applied to an LC–ESI-MS/MS. The identification of triterpene glycosides was performed in the negative-ion mode using LTQ Orbitrap XL (Thermo Finnegan, San Jose, CA, USA) system equipped with an electrospray source. The source was operated at 3 kV of the ion spray voltage, –43 kV of the capillary voltage, 275⬚C of the capillary temperature, and under the N2 sheath gas set at 8 mbar. For MS/MS, the [M–H]− /[M–Na]– of each triterpene glycoside was selected as a precursor ion and the MS/MS product ions were obtained using the condition of 30% collision energy in the ion trap analyzer.
J. Sep. Sci. 2015, 38, 1117–1122
distributed in 5.0 mL water and washed using petroleum ether, chloroform, and n-butyl alcohol (5.0 mL, three times for each solvent). The crude extracts of the total triterpene glycosides were obtained after the solutions of n-butyl alcohol were pooled and evaporated to dryness. The products were dissolved in 1 mL of water. One hundred microliters of the solution was applied to a small Hypersep C18 column of octadecyl silane SPE (Thermo Scientific, USA). The column was eluted with water (2.0 mL), 40% methanol (2.0 mL), 70% methanol (2.0 mL), and pure methanol. 70% methanol solvent was filtered through a 0.22 m syringe filter and diluted to 2.5 mL. The prepared samples were ready for the HPLC–ELSD analysis. 2.4 Determination of the external standard Holotoxin A1 The triterpene glycoside of Holotoxin A1 , as an external standard, was obtained following the previously described protocol as in Sections 2.2 and 2.3. The peak of Holotoxin A1 was collected after the separation performed on the HPLC column, pooled and lyophilized. The structure and purity of Holotoxin A1 were determined by 1D and 2D NMR spectroscopy and ESI-MS described previously. For the analysis of 1D NMR, 15 mg of sample was dissolved in 400 L of deuterated pyridine (99.9%, Sigma–Aldrich), and determined using JEOL-ECP 600 MHz spectrometer at 25⬚C. The 2D NMR, including the 1 H-1 H COSY, heteronuclear multiple quantum correlation, heteronuclear multiple bond correlation, and nuclear overhauser effect spectroscopy were recorded at 20⬚C. 2.5 Validation of HPLC–ELSD method The standard solutions containing Holotoxin A1 of 2– 200 mg/L were prepared in six concentration levels (excluding the blank sample) and analyzed on HPLC–ELSD to build the calibration curve. The LOQ and LOD values were calculated by determining the S/N values of the lowest measured concentrations and extrapolating to the S/N values of 10 and 3, respectively. The precision and recovery were evaluated by adding accurate amounts of the external standard to 2.0 g of pulverized A. japonicus (from the east coast of Korea), extracted and analyzed as the protocol. The variations were expressed by the RSD%. The average recoveries were determined by the formula: recovery (%) = (observed amount − original amount)/spiked amount × 100%.
2.3 Samples and pretreatments
3 Results and discussion
Two grams of dried sea cucumber samples were powdered and infused in 25 mL of 60% ethanol for a reflux at 75⬚C for 3 h (three times repeated). The extract supernatant was pooled and evaporated to dryness. The products were
3.1 The extraction of the total triterpene glycosides
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extract. When eluted by 70% methanol, a variety of triterpene glycosides were detected by HPLC–ELSD method and no triterpene glycosides were observed in pure methanol elution that followed.
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MS/MS product ions at m/z 423.5 corresponded to the aglycon moiety, the lactone of which was vulnerable to the collision and led to a [M–H–CO2 ]– product ion. The MS data also indicated that the extracted Holotoxin A1 possessed a high purity, which promised its application as an external standard (data not shown).
3.2 The characterization of the extracted external standard Holotoxin A1 3.3 Optimization of the chromatographic conditions The extracted Holotoxin A1 from the HPLC separation was applied to the 1D and 2D NMR spectroscopy and ESI-MS analysis. The 1 H and 13 C NMR spectra showed that Holotoxin A1 is composed of an aglycon moiety and a sugar chain. The 1 H-1 H COSY, heteronuclear multiple quantum correlation, heteronuclear multiple bond correlation, and nuclear Overhauser effect spectroscopy analysis gave information about the adjacent H–H and C–H, the patterns of glycosidic bonds, and the order of the sugar chain. The chemical shifts of C/H of the Holotoxin A1 according to the 1D and 2D NMR data are listed in Supporting Information Table S1, which agreed well with that reported previously [20]. The ESI-MS data revealed the pseudomolecular ion peaks at m/z 1391.6 [M–H]– in the negative-ion mode, and m/z 1410.6 [M+NH4 ]+ in the positive-ion mode, suggesting a molecular weight of 1392.6 Da (Fig. 1). The MS/MS data were obtained by the collision of the deprotonated molecular ion peak of the saponin in the ion trap analyzer. The ions retaining the charge at the reducing terminus are termed Y (the glycosidic cleavage). As described in Fig. 1A, it showed a series of fragments corresponding to the sequential abscission of the monosaccharides, including two O-methylglucose (MeGlc), two xylose (Xyl), a glucose (Glc), and a quinovose (Qui), as follows: m/z 1347.6 [M–H–CO2 ]− , m/z 1171.6 [M–H– CO2 –MeGlc]− , m/z 1153.6 [M–H–H2 O–MeGlc]− , m/z 1039.5 [M–H–CO2 –MeGlc–Xyl]− , m/z 1009.5 [M–H–CO2 –MeGlc– Glc]− , m/z 893.5 [M–H–CO2 –MeGlc–Xyl–Qui]– , and m/z 701.4 [M–H–H2 O–MeGlc–Xyl–MeGlc–Glc]– (Fig. 1B). The
Figure 1. Fragmentation pattern of the [M–H]– ion of Holotoxin A1 (A), and ESI-MS/MS spectrum of [M–H]– ion at m/z 1391.7 of Holotoxin A1 (B).
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The analysis of the total triterpene glycosides by a gradient wash with acetonitrile (from 0 to 100%) indicated that the major compounds of triterpene glycosides could be well separated by 58% acetonitrile. The essential two parameters that can affect the ELSD response are the flow rate of nebulizer gas (pressure) and drift tube temperature. Under the fixed chromatographic conditions, two parameters were evaluated by the injection of the total triterpene glycosides extracted from the pulverized A. japonicus (derived from the east coast of Korea), at the different temperatures from 50 to 90⬚C and the different pressures from 10.0 to 40.0 psi. The optimized conditions in the present work were determined by comparing the peak area values and baseline. The optimized chromatography parameters offer a good separation of compounds and a negligible baseline noise.
3.4 Validation of the HPLC–ELSD method The representative chromatograms of the external standard Holotoxin A1 and the mixture of triterpene glycosides were extracted from 2 g of the pulverized A. japonicus (from the east coast of Korea), as described in Section 2.3 and shown in Fig. 2. The results indicated that the compounds were clearly separated and their quantitative determination in sea cucumbers was possible. The external standard showed a good linearity. The LOD and LOQ were also determined, which were indicated to be lower than previously reported [8, 21]. Five replicates of three different spiked samples containing Holotoxin A1 standard (0.4, 2, and 4 mg) were pretreated as described in Section 2.3, and applied to determine the precision of the method. Moreover, the recovery of the low addition level, medium addition level, and high addition level was tested. The results are shown in Table 1. In 1978, Kitagawa found three kinds of triterpene glycosides from the sea cucumber S. japonicus in coast of Japan, two of which were characterized and assigned to be Holotoxin A and Holotoxin B [22]. Afterwards, Maltsev isolated three triterpene glycosides from the sea cucumber S. japonicus in Posiet Bay of Japan, and two of them possessed the same aglycon moiety as the Holotoxin A and Holotoxin B but a slight difference in the sugar chain, which were named Holotoxin A1 and Holotoxin B1 , respectively [23]. Holotoxin A1 was found to be the main triterpene glycoside in A. japonicus derived from the Bohai sea of China [20]. In the present work, the contents of the main product Holotoxin A1 , in the pulverized www.jss-journal.com
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Figure 2. Chromatograms of external standard, Holotoxin A1 (A) and the extracted triterpene glycosides (B) from pulverized A. japonicus (derived from east coast of Korea). The concentration of Holotoxin A1 is 50 g/mL. Table 1. Validation of the HPLC–ELSD method
Compound
Calibration curvea)
Linear range (g)
LOD (ng)
LOQ (ng)
Spiked samples (mg)
Precision (RSD%)
Recovery (%)
Holotoxin A1
y = 2.73 × 106 x + 327.1, r2 = 0.999
0.0220
10
20
0.4
3.62
94.35
2 4
6.58 4.52
96.64 98.36
RSD% = (SD/mean) × 100%. a) x, concentration (g); y, peak area.
A. japonicus, from the different sea areas are shown in Table 2. The content of Holotoxin A1 in the A. japonicus from the east coast of South Korea was much higher than the others. The results demonstrated that the content of the bioactive compounds extracted from A. japonicus differed from each other, even though they were of the same species. The composition and content of the triterpene glycosides from A. japonicus depended on the regions and environments, which could lead to an obvious distinction. 3.5 HPLC–ESI-MS/MS analysis of the triterpene glycosides extracted from different sea cucumbers The optimized HPLC method was applied to LC–MS for the determination of each compound. All of the triterpene
glycosides were well resolved under the LC–MS conditions used. The mass spectra of the triterpene glycosides from the different sea cucumbers were determined in the negativeion mode to define their molecular weight. The triterpene glycosides with hydroxyl groups were detected with greater sensitivity in the negative-ion mode and showed only peaks corresponding to [M–H]− /[M–Na]− . The chromatographs of LC–ELSD (Fig. 3B) were well agreed with LC–MS (Fig. 3A), where the triterpene glycosides were labeled with the m/z value of [M–H]− /[M–Na]– and identified on the basis of their retention times, m/z values of the intact molecular ions, and MS/MS product ions. The results of each triterpene glycoside extracted from the different sea cucumbers are summarized in Supporting Information Table S2, which are compared to the results described in the previous reports. However, it is difficult to distinguish some triterpene glycosides, which
Table 2. Contents of Holotoxin A1 extracted from A. japonicus derived from five different sea areas
A. japonicus from different sea areas
East coast of South Korea
Qingdao, China
Fujian, China
Kansai, Japan
Kanto, Japan
Content (g/g)
588.20
67.59
21.33
96.72
29.44
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Figure 3. Total ion chromatogram (A) and HPLC– ELSD chromatogram (B) of the extracted triterpene glycosides from pulverized Holothuria mexicana labeled with m/z value of the [M–H]− /[M–Na]– for each peak.
are of the same molecular weight on the MS/MS spectrum, such as 24-dehydroechinoside A, fuscocineroside B/C, and pervicoside C, possessing the same sugar chain and slightly different aglycon moiety. In the present study, eight kinds of pulverized sea cucumbers, which were purchased from the Nanshan market of Qingdao, China, were tested with the method described previously. The representative MS chromatograms of the triterpene glycosides from different sea cucumbers are shown in Fig. 4, where the triterpene glycosides are labeled with the m/z value of [M–H]− /[M–Na]− . Supporting Information Table S3 summarizes the retention times and molecular ratio of the main triterpene glycosides extracted from different sea cucumbers, comparing with that described in previous reports. Besides, the triterpene glycosides in Holothuria mexicana are determined for the first time, and four major kinds of already known triterpene glycosides have been found. Even though there are still many triterpene glycosides that cannot be identified, which could lead to the discovery of a serious new structural triterpene glycosides, the wellestablished methods including the pretreatment of the samples and HPLC–ELSD–MS/MS chromatography could be applied to identify the species of different sea cucumbers and evaluate the commercial production on the fish market.
4 Concluding remarks The HPLC–ELSD–MS method described in the present paper is first reported for the quantitative and identification of the major triterpene glycosides of eight different species of sea cucumbers. The HPLC–ELSD method is effective to analyze the crude extraction of the pulverized production of the sea cucumbers on the market with good accuracy, C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 4. Representative total ion chromatograms of the triterpene glycosides obtained from the different sea cucumbers. (A) The triterpene glycosides from Stichopus horrens and (B) the triterpene glycosides from Bohadschia argus.
precision, and repeatability. Under the ESI-MS/MS conditions, the fragmentation patterns of [M–H]− /[M–Na]– ions exclusively show the signals corresponding to the cleavage of the glycosidic bonds, thus allowing a rapid identification of the triterpene glycosides in the different sea cucumbers. The results demonstrate that the proposed methods could be utilized for the identification and evaluation of the sea cucumber products on the market. www.jss-journal.com
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This research was funded by Natural Science Foundation of China (31171665).
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[11] Song, M., Zhang, S. Y., Xu, X. Y., Hang, T. J., Jia, L., J. Chromatogr. B 2010, 878, 3331–3337. [12] Chen, Q. L., Shi, Zh. Y., Ma, N. N., Wang, W. Q., Wei, Sh. L., Sun, W. J., J. Med. Plants Res. 2012, 6, 1585–1589.
5 References [1] Chludil, H. D., Muniain, C. C., Seldes, A. M., Maier, M. S., J. Nat. Prod. 2002, 65, 860–865. [2] Kalinin, V. I., Prokofieva, N. G., Likhatskaya, G. N., Schentsova, E. B., Agafonova, I. G., Avilov, S. A., Drozdova, O. A., Toxicon 1996, 34, 475–483. [3] Zou, Z. R., Yi, Y. H., Wu, H. M., Wu, J. H., Liaw, C. C., Lee, K. H., J. Nat. Prod. 2003, 66, 1055–1060. ¨ [4] Aminin, D. L., Koy, C., Dmitrenok, P. S., Muller-Hilke, B., Koczan, D., Arbogast, B., Silchenko, A. A., Kalinin, V. I., Avilov, S. A., Stonik, V. A., Collin, P. D., Thiesen, H. J., Deinzer, M. L., Glocker, M. O., J. Proteomics 2009, 72, 886–906. [5] Kalinin, V. I., Silchenko, A. S., Avilov, S. A., Stonik, V. A., Smirnov, A. V., Phytochem. Rev. 2005, 4, 221–236. [6] Dong, P., Xue, C. H., Yu, L. F., Xu, J., Chen, S. G., J. Agric. Food Chem. 2008, 56, 4937–4942. [7] Kurkin, V. A., Akushskaya, A. S., Pharm. Chem. J. 2014, 48, 276–278. [8] Zhu, F. X., Jia, X. B., Cai, Y., Chen, Y., Acta Chromatogr. 2013, 25, 147–157.
[13] Fuzzati, N., J. Chromatogr. B 2004, 812, 119–133. [14] Zhu, J. B., Guo, X. J., Fu, Sh. P., Zhang, X. L., Liang, X. M., J. Pharm. Biomed. 2010, 53, 462–474. [15] Lu, Y. Y., Luo, J. G., Xu, D. R., Huang, X. F., Kong, L. Y., Rapid Commun. Mass Spectrom. 2008, 22, 2447–2452. [16] Liu, Y. Y., Li, J. B., He, J. M., Zeper, A., Qu, J., Yu, Sh. Sh., Ma, Sh. G, Liu, J., Du, D., Rapid Commun. Mass Spectrom. 2009, 23, 667–679. [17] Bankefors, J., Nord, L. I., Kenne, L., Chemom. Intell. Lab. Syst. 2008, 90, 178–187. [18] Han, L. F., Pan, G. X., Wang, Y. F., Song, X. B., Gao, X. M., Ma, B. P., Kang, L. P., J. Pharm. Biomed. 2011, 55, 996–1009. [19] Zhang, P. C., Zheng, C. F., Wu, C. S., Sheng, Y. X., Zhang, J. L., J. Asian Nat. Prod. Res. 2010, 12, 64–69. [20] Wang, Z. L., Zhang, H. W., Yuan, W. H., Gong, W., Tang, H., Liu, B. Sh., Krohn, K., Li, L., Yi, Y. H., Zhang, W., Food Chem. 2012, 132, 295–300. [21] Man, Sh. L., Gao, W. Y., Zhang, Y. J., Wang, J. Y., Zhao, W. Sh., Huang, L. Q., Liu, Ch. X., J. Chromatogr. B 2010, 878, 2943–2948.
[9] Das, B., Paul, T., Apte, K. G., Chauhan, R., Saxena, R. C., J. Pharm. Res. 2013, 6, 93–100.
[22] Kitagawa, I., Yamanaka, H., Kobayashi, M., Nishino, T., Yosioka, I., Sugawara, T., Chem. Pharm. Bull. 1978, 26, 3722–3731.
[10] Zuo, W., Wang, Q., Li, W., Sha, Y., Li, X., Wang, J., Magn. Reson. Chem. 2012, 50, 325–328.
[23] Maltsev, I. I., Stonik, V. A., Kalinovsky, A. I., Elyakov, G. B., Comp. Biochem. Phys. B 1984, 78, 421–426.
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Healthy volunteer BBB protection CSF
Rhubarb Traumatic Brain Injury patient BBB disruption
Rhein
CSF
Rhein
Methods Chromatography · Electroseparation Applications Biomedicine · Foods · Environment
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ISSN 1615-9306 · JSSCCJ 38 (7) 1073–1262 (2015) · Vol. 38 · No. 7 · April 2015 · D 10609
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In vivo
oral
Phellinus igniarius
Methods Chromatography · Electroseparation Applications Biomedicine · Foods · Environment
Wistar rat
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J. Sep. Sci. 2015, 38, 1117–1122
Jie Yang1,2 Yuanhong Wang1,2 Ran Zhang1,2 Tifngfu Jiang1,2 Zhihua Lv1,2 1 Key
Laboratory of Marine Drugs, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China 2 Shandong Provincial Key Laboratory of Glycoscience & Glycotechnology, Ocean University of China, Qingdao, China Received November 9, 2014 Revised December 22, 2014 Accepted January 5, 2015
Research Article
Determination of the triterpene glycosides in sea cucumbers by liquid chromatography with evaporative light scattering and mass spectrometry detection Holothurian triterpene glycosides possess various kinds of biological activities, including antifungal, cytotoxic, hemolytic, cytostatic, and immunomodulatory effects. In this study, a rapid extraction method of triterpene glycosides from sea cucumbers using a small column of C18 solid phase was first developed. Furthermore, a novel high-performance liquid chromatography method coupled with evaporative light scattering detection and electrospray ionization mass spectrometry was established for the determination of each triterpene glycosides from different sea cucumbers. Simultaneous separation of all kind of triterpene glycoside were achieved on a C18 column. A gradient of aqueous acetonitrile was applied, and the method was validated. The liquid chromatography method was applied to the online mass detection to identify the triterpene glycosides in the purified extraction of eight kinds of pulverized sea cucumber from the market of Qingdao, China. The negative mode of [M–H]− /[M–Na]− exclusively shown signals corresponding to the triterpene glycosides previously reported and the MS2 product ions of those ions indicate the specific structure of each triterpene glycoside. Keywords: Electrospray ionization mass spectrometry / Evaporative light scattering detection / Sea cucumber / Triterpene glycosides DOI 10.1002/jssc.201401253
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Sea cucumber has been used as a traditional nutritive food for thousands of years. There are many biological materials in the body of sea cucumber, of which the triterpene glycoside is the most important secondary metabolites. Previous reports indicated that the holothurian triterpene glycosides possess various kinds of biological activities, including antifungal, cytotoxic, hemolytic, cytostatic, antitumor, and immunomodulatory effects [1–4]. There have been hundreds of kinds of triterpene glycosides found in the body of different sea cucumbers, and most of them are distributed in the Holothuriidae and Stichopodidae families of Aspidochirotida order and Cucumariidae family of Dendrochirotida order [5]. Due to the high nutritive value and healthcare effects, various kinds of nutriments and tonic products are now made of sea cucumber, including dried products (beche-de-mer), Correspondence: Professor Zhihua Lv, Shandong Provincial Key Laboratory of Glycoscience & Glycotechnology, Ocean University of China, Qingdao 266003, China. E-mail: [email protected] Fax: +86 532 82032064
Abbreviations: ELSD, evaporative light scattering detection; Glc, glucose; MeGlc, methylglucose; Xyl, xylose C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
pickled products, wetter risen products, oral liquid, capsules, etc. [6]. However, the triterpene glycosides have become a very important index for the nutrition evaluation and QC of sea cucumber products. The analytical methods for the quantitative of triterpene glycoside include spectrophotometry, TLC, HPLC–ELSD (where ELSD is evaporative light scattering detection), LC–ESI-MS, LC–UV–NMR, LC–PDA–ESI-MS/MS, and CE [7–12]. Compared with others, the ELSD is a universal, nonspecific detector that can provide a stable baseline even with gradient elution [13]. ESI-MS is an efficient tool for the determination of triterpene glycosides in the crude extracts, due to its high sensitivity, short analysis time, and low consumption of samples [14–16]. ESI-MS can rapidly determine the bioactive compounds in the samples and give their structural information. In recent years, the reports on the combined application of LC and MS to the online analysis of natural products have increased [17–19]. However, the determination of triterpene glycosides from sea cucumber by HPLC–ELSD–MS has not been reported yet. In the present paper, a rapid analytical HPLC method coupled with the ELSD and MS/MS detection has been first established for the QC, which allows simultaneous determination of the total triterpene glycosides in different sea cucumbers. The method includes the pretreatment of sample, www.jss-journal.com
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establishment and validation of the HPLC–ELSD, and determination of the compounds of triterpene glycosides. Using a preparation of Holotoxin A1 as an external standard, the contents of this specific main product were observed, which were isolated from the sea cucumber Apostichopus japonicus (also named after Stichopus japonicus) from different regions. Furthermore, the total triterpene glycosides isolated from eight kinds of pulverized sea cucumber products from the different species were determined. These studies should provide a method for the quantitative determination and structural characterization of the triterpene glycosides in the different sea cucumbers.
2 Materials and methods 2.1 Materials All of the dried sea cucumber samples were purchased from the Nanshan market of Qingdao, China. All other chemical reagents were of analytical grade from Sinopharm Chemical Reagent (Shanghai, China). 2.2 Chromatography conditions The analysis of the total triterpene glycosides were performed on a Waters 1525 liquid chromatograph equipped with Waters 2420 ELSD detector and Hypersil Gold C18 column (Thermo Scientific, USA; 150 mm × 2.1 mm, particle size 3 m). The mobile phases were 5 mM ammonium acetate (solvent A) and acetonitrile (solvent B). A gradient of solvent A was performed from 73 to 58% in 15 min, and kept for 13 min at a flow rate of 0.2 mL/min at 40⬚C. Ten microliters of sample was injected for the analysis. Parameters of the ELSD detector were as follows: the temperature of drift tube was 60⬚C, the atomization efficiency was 50%, and the nebulizer gas nitrogen pressure was 25 psi and the gain was 25. The same LC method was applied to an LC–ESI-MS/MS. The identification of triterpene glycosides was performed in the negative-ion mode using LTQ Orbitrap XL (Thermo Finnegan, San Jose, CA, USA) system equipped with an electrospray source. The source was operated at 3 kV of the ion spray voltage, –43 kV of the capillary voltage, 275⬚C of the capillary temperature, and under the N2 sheath gas set at 8 mbar. For MS/MS, the [M–H]− /[M–Na]– of each triterpene glycoside was selected as a precursor ion and the MS/MS product ions were obtained using the condition of 30% collision energy in the ion trap analyzer.
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distributed in 5.0 mL water and washed using petroleum ether, chloroform, and n-butyl alcohol (5.0 mL, three times for each solvent). The crude extracts of the total triterpene glycosides were obtained after the solutions of n-butyl alcohol were pooled and evaporated to dryness. The products were dissolved in 1 mL of water. One hundred microliters of the solution was applied to a small Hypersep C18 column of octadecyl silane SPE (Thermo Scientific, USA). The column was eluted with water (2.0 mL), 40% methanol (2.0 mL), 70% methanol (2.0 mL), and pure methanol. 70% methanol solvent was filtered through a 0.22 m syringe filter and diluted to 2.5 mL. The prepared samples were ready for the HPLC–ELSD analysis. 2.4 Determination of the external standard Holotoxin A1 The triterpene glycoside of Holotoxin A1 , as an external standard, was obtained following the previously described protocol as in Sections 2.2 and 2.3. The peak of Holotoxin A1 was collected after the separation performed on the HPLC column, pooled and lyophilized. The structure and purity of Holotoxin A1 were determined by 1D and 2D NMR spectroscopy and ESI-MS described previously. For the analysis of 1D NMR, 15 mg of sample was dissolved in 400 L of deuterated pyridine (99.9%, Sigma–Aldrich), and determined using JEOL-ECP 600 MHz spectrometer at 25⬚C. The 2D NMR, including the 1 H-1 H COSY, heteronuclear multiple quantum correlation, heteronuclear multiple bond correlation, and nuclear overhauser effect spectroscopy were recorded at 20⬚C. 2.5 Validation of HPLC–ELSD method The standard solutions containing Holotoxin A1 of 2– 200 mg/L were prepared in six concentration levels (excluding the blank sample) and analyzed on HPLC–ELSD to build the calibration curve. The LOQ and LOD values were calculated by determining the S/N values of the lowest measured concentrations and extrapolating to the S/N values of 10 and 3, respectively. The precision and recovery were evaluated by adding accurate amounts of the external standard to 2.0 g of pulverized A. japonicus (from the east coast of Korea), extracted and analyzed as the protocol. The variations were expressed by the RSD%. The average recoveries were determined by the formula: recovery (%) = (observed amount − original amount)/spiked amount × 100%.
2.3 Samples and pretreatments
3 Results and discussion
Two grams of dried sea cucumber samples were powdered and infused in 25 mL of 60% ethanol for a reflux at 75⬚C for 3 h (three times repeated). The extract supernatant was pooled and evaporated to dryness. The products were
3.1 The extraction of the total triterpene glycosides
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extract. When eluted by 70% methanol, a variety of triterpene glycosides were detected by HPLC–ELSD method and no triterpene glycosides were observed in pure methanol elution that followed.
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MS/MS product ions at m/z 423.5 corresponded to the aglycon moiety, the lactone of which was vulnerable to the collision and led to a [M–H–CO2 ]– product ion. The MS data also indicated that the extracted Holotoxin A1 possessed a high purity, which promised its application as an external standard (data not shown).
3.2 The characterization of the extracted external standard Holotoxin A1 3.3 Optimization of the chromatographic conditions The extracted Holotoxin A1 from the HPLC separation was applied to the 1D and 2D NMR spectroscopy and ESI-MS analysis. The 1 H and 13 C NMR spectra showed that Holotoxin A1 is composed of an aglycon moiety and a sugar chain. The 1 H-1 H COSY, heteronuclear multiple quantum correlation, heteronuclear multiple bond correlation, and nuclear Overhauser effect spectroscopy analysis gave information about the adjacent H–H and C–H, the patterns of glycosidic bonds, and the order of the sugar chain. The chemical shifts of C/H of the Holotoxin A1 according to the 1D and 2D NMR data are listed in Supporting Information Table S1, which agreed well with that reported previously [20]. The ESI-MS data revealed the pseudomolecular ion peaks at m/z 1391.6 [M–H]– in the negative-ion mode, and m/z 1410.6 [M+NH4 ]+ in the positive-ion mode, suggesting a molecular weight of 1392.6 Da (Fig. 1). The MS/MS data were obtained by the collision of the deprotonated molecular ion peak of the saponin in the ion trap analyzer. The ions retaining the charge at the reducing terminus are termed Y (the glycosidic cleavage). As described in Fig. 1A, it showed a series of fragments corresponding to the sequential abscission of the monosaccharides, including two O-methylglucose (MeGlc), two xylose (Xyl), a glucose (Glc), and a quinovose (Qui), as follows: m/z 1347.6 [M–H–CO2 ]− , m/z 1171.6 [M–H– CO2 –MeGlc]− , m/z 1153.6 [M–H–H2 O–MeGlc]− , m/z 1039.5 [M–H–CO2 –MeGlc–Xyl]− , m/z 1009.5 [M–H–CO2 –MeGlc– Glc]− , m/z 893.5 [M–H–CO2 –MeGlc–Xyl–Qui]– , and m/z 701.4 [M–H–H2 O–MeGlc–Xyl–MeGlc–Glc]– (Fig. 1B). The
Figure 1. Fragmentation pattern of the [M–H]– ion of Holotoxin A1 (A), and ESI-MS/MS spectrum of [M–H]– ion at m/z 1391.7 of Holotoxin A1 (B).
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The analysis of the total triterpene glycosides by a gradient wash with acetonitrile (from 0 to 100%) indicated that the major compounds of triterpene glycosides could be well separated by 58% acetonitrile. The essential two parameters that can affect the ELSD response are the flow rate of nebulizer gas (pressure) and drift tube temperature. Under the fixed chromatographic conditions, two parameters were evaluated by the injection of the total triterpene glycosides extracted from the pulverized A. japonicus (derived from the east coast of Korea), at the different temperatures from 50 to 90⬚C and the different pressures from 10.0 to 40.0 psi. The optimized conditions in the present work were determined by comparing the peak area values and baseline. The optimized chromatography parameters offer a good separation of compounds and a negligible baseline noise.
3.4 Validation of the HPLC–ELSD method The representative chromatograms of the external standard Holotoxin A1 and the mixture of triterpene glycosides were extracted from 2 g of the pulverized A. japonicus (from the east coast of Korea), as described in Section 2.3 and shown in Fig. 2. The results indicated that the compounds were clearly separated and their quantitative determination in sea cucumbers was possible. The external standard showed a good linearity. The LOD and LOQ were also determined, which were indicated to be lower than previously reported [8, 21]. Five replicates of three different spiked samples containing Holotoxin A1 standard (0.4, 2, and 4 mg) were pretreated as described in Section 2.3, and applied to determine the precision of the method. Moreover, the recovery of the low addition level, medium addition level, and high addition level was tested. The results are shown in Table 1. In 1978, Kitagawa found three kinds of triterpene glycosides from the sea cucumber S. japonicus in coast of Japan, two of which were characterized and assigned to be Holotoxin A and Holotoxin B [22]. Afterwards, Maltsev isolated three triterpene glycosides from the sea cucumber S. japonicus in Posiet Bay of Japan, and two of them possessed the same aglycon moiety as the Holotoxin A and Holotoxin B but a slight difference in the sugar chain, which were named Holotoxin A1 and Holotoxin B1 , respectively [23]. Holotoxin A1 was found to be the main triterpene glycoside in A. japonicus derived from the Bohai sea of China [20]. In the present work, the contents of the main product Holotoxin A1 , in the pulverized www.jss-journal.com
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Figure 2. Chromatograms of external standard, Holotoxin A1 (A) and the extracted triterpene glycosides (B) from pulverized A. japonicus (derived from east coast of Korea). The concentration of Holotoxin A1 is 50 g/mL. Table 1. Validation of the HPLC–ELSD method
Compound
Calibration curvea)
Linear range (g)
LOD (ng)
LOQ (ng)
Spiked samples (mg)
Precision (RSD%)
Recovery (%)
Holotoxin A1
y = 2.73 × 106 x + 327.1, r2 = 0.999
0.0220
10
20
0.4
3.62
94.35
2 4
6.58 4.52
96.64 98.36
RSD% = (SD/mean) × 100%. a) x, concentration (g); y, peak area.
A. japonicus, from the different sea areas are shown in Table 2. The content of Holotoxin A1 in the A. japonicus from the east coast of South Korea was much higher than the others. The results demonstrated that the content of the bioactive compounds extracted from A. japonicus differed from each other, even though they were of the same species. The composition and content of the triterpene glycosides from A. japonicus depended on the regions and environments, which could lead to an obvious distinction. 3.5 HPLC–ESI-MS/MS analysis of the triterpene glycosides extracted from different sea cucumbers The optimized HPLC method was applied to LC–MS for the determination of each compound. All of the triterpene
glycosides were well resolved under the LC–MS conditions used. The mass spectra of the triterpene glycosides from the different sea cucumbers were determined in the negativeion mode to define their molecular weight. The triterpene glycosides with hydroxyl groups were detected with greater sensitivity in the negative-ion mode and showed only peaks corresponding to [M–H]− /[M–Na]− . The chromatographs of LC–ELSD (Fig. 3B) were well agreed with LC–MS (Fig. 3A), where the triterpene glycosides were labeled with the m/z value of [M–H]− /[M–Na]– and identified on the basis of their retention times, m/z values of the intact molecular ions, and MS/MS product ions. The results of each triterpene glycoside extracted from the different sea cucumbers are summarized in Supporting Information Table S2, which are compared to the results described in the previous reports. However, it is difficult to distinguish some triterpene glycosides, which
Table 2. Contents of Holotoxin A1 extracted from A. japonicus derived from five different sea areas
A. japonicus from different sea areas
East coast of South Korea
Qingdao, China
Fujian, China
Kansai, Japan
Kanto, Japan
Content (g/g)
588.20
67.59
21.33
96.72
29.44
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Figure 3. Total ion chromatogram (A) and HPLC– ELSD chromatogram (B) of the extracted triterpene glycosides from pulverized Holothuria mexicana labeled with m/z value of the [M–H]− /[M–Na]– for each peak.
are of the same molecular weight on the MS/MS spectrum, such as 24-dehydroechinoside A, fuscocineroside B/C, and pervicoside C, possessing the same sugar chain and slightly different aglycon moiety. In the present study, eight kinds of pulverized sea cucumbers, which were purchased from the Nanshan market of Qingdao, China, were tested with the method described previously. The representative MS chromatograms of the triterpene glycosides from different sea cucumbers are shown in Fig. 4, where the triterpene glycosides are labeled with the m/z value of [M–H]− /[M–Na]− . Supporting Information Table S3 summarizes the retention times and molecular ratio of the main triterpene glycosides extracted from different sea cucumbers, comparing with that described in previous reports. Besides, the triterpene glycosides in Holothuria mexicana are determined for the first time, and four major kinds of already known triterpene glycosides have been found. Even though there are still many triterpene glycosides that cannot be identified, which could lead to the discovery of a serious new structural triterpene glycosides, the wellestablished methods including the pretreatment of the samples and HPLC–ELSD–MS/MS chromatography could be applied to identify the species of different sea cucumbers and evaluate the commercial production on the fish market.
4 Concluding remarks The HPLC–ELSD–MS method described in the present paper is first reported for the quantitative and identification of the major triterpene glycosides of eight different species of sea cucumbers. The HPLC–ELSD method is effective to analyze the crude extraction of the pulverized production of the sea cucumbers on the market with good accuracy, C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 4. Representative total ion chromatograms of the triterpene glycosides obtained from the different sea cucumbers. (A) The triterpene glycosides from Stichopus horrens and (B) the triterpene glycosides from Bohadschia argus.
precision, and repeatability. Under the ESI-MS/MS conditions, the fragmentation patterns of [M–H]− /[M–Na]– ions exclusively show the signals corresponding to the cleavage of the glycosidic bonds, thus allowing a rapid identification of the triterpene glycosides in the different sea cucumbers. The results demonstrate that the proposed methods could be utilized for the identification and evaluation of the sea cucumber products on the market. www.jss-journal.com
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This research was funded by Natural Science Foundation of China (31171665).
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[11] Song, M., Zhang, S. Y., Xu, X. Y., Hang, T. J., Jia, L., J. Chromatogr. B 2010, 878, 3331–3337. [12] Chen, Q. L., Shi, Zh. Y., Ma, N. N., Wang, W. Q., Wei, Sh. L., Sun, W. J., J. Med. Plants Res. 2012, 6, 1585–1589.
5 References [1] Chludil, H. D., Muniain, C. C., Seldes, A. M., Maier, M. S., J. Nat. Prod. 2002, 65, 860–865. [2] Kalinin, V. I., Prokofieva, N. G., Likhatskaya, G. N., Schentsova, E. B., Agafonova, I. G., Avilov, S. A., Drozdova, O. A., Toxicon 1996, 34, 475–483. [3] Zou, Z. R., Yi, Y. H., Wu, H. M., Wu, J. H., Liaw, C. C., Lee, K. H., J. Nat. Prod. 2003, 66, 1055–1060. ¨ [4] Aminin, D. L., Koy, C., Dmitrenok, P. S., Muller-Hilke, B., Koczan, D., Arbogast, B., Silchenko, A. A., Kalinin, V. I., Avilov, S. A., Stonik, V. A., Collin, P. D., Thiesen, H. J., Deinzer, M. L., Glocker, M. O., J. Proteomics 2009, 72, 886–906. [5] Kalinin, V. I., Silchenko, A. S., Avilov, S. A., Stonik, V. A., Smirnov, A. V., Phytochem. Rev. 2005, 4, 221–236. [6] Dong, P., Xue, C. H., Yu, L. F., Xu, J., Chen, S. G., J. Agric. Food Chem. 2008, 56, 4937–4942. [7] Kurkin, V. A., Akushskaya, A. S., Pharm. Chem. J. 2014, 48, 276–278. [8] Zhu, F. X., Jia, X. B., Cai, Y., Chen, Y., Acta Chromatogr. 2013, 25, 147–157.
[13] Fuzzati, N., J. Chromatogr. B 2004, 812, 119–133. [14] Zhu, J. B., Guo, X. J., Fu, Sh. P., Zhang, X. L., Liang, X. M., J. Pharm. Biomed. 2010, 53, 462–474. [15] Lu, Y. Y., Luo, J. G., Xu, D. R., Huang, X. F., Kong, L. Y., Rapid Commun. Mass Spectrom. 2008, 22, 2447–2452. [16] Liu, Y. Y., Li, J. B., He, J. M., Zeper, A., Qu, J., Yu, Sh. Sh., Ma, Sh. G, Liu, J., Du, D., Rapid Commun. Mass Spectrom. 2009, 23, 667–679. [17] Bankefors, J., Nord, L. I., Kenne, L., Chemom. Intell. Lab. Syst. 2008, 90, 178–187. [18] Han, L. F., Pan, G. X., Wang, Y. F., Song, X. B., Gao, X. M., Ma, B. P., Kang, L. P., J. Pharm. Biomed. 2011, 55, 996–1009. [19] Zhang, P. C., Zheng, C. F., Wu, C. S., Sheng, Y. X., Zhang, J. L., J. Asian Nat. Prod. Res. 2010, 12, 64–69. [20] Wang, Z. L., Zhang, H. W., Yuan, W. H., Gong, W., Tang, H., Liu, B. Sh., Krohn, K., Li, L., Yi, Y. H., Zhang, W., Food Chem. 2012, 132, 295–300. [21] Man, Sh. L., Gao, W. Y., Zhang, Y. J., Wang, J. Y., Zhao, W. Sh., Huang, L. Q., Liu, Ch. X., J. Chromatogr. B 2010, 878, 2943–2948.
[9] Das, B., Paul, T., Apte, K. G., Chauhan, R., Saxena, R. C., J. Pharm. Res. 2013, 6, 93–100.
[22] Kitagawa, I., Yamanaka, H., Kobayashi, M., Nishino, T., Yosioka, I., Sugawara, T., Chem. Pharm. Bull. 1978, 26, 3722–3731.
[10] Zuo, W., Wang, Q., Li, W., Sha, Y., Li, X., Wang, J., Magn. Reson. Chem. 2012, 50, 325–328.
[23] Maltsev, I. I., Stonik, V. A., Kalinovsky, A. I., Elyakov, G. B., Comp. Biochem. Phys. B 1984, 78, 421–426.
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