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Dong-Xia Zhao1,2 Bing-Qiang Hu1 Mian Zhang1 Chao-Feng Zhang1 Xiang-Hong Xu1 ∗ 1 Research
Department of Pharmacognosy, China Pharmaceutical University, Nanjing, China 2 Pharmaceutical College, Henan University, Kaifeng, China Received September 15, 2014 Revised November 1, 2014 Accepted November 28, 2014
Short Communication
Simultaneous separation and determination of phenolic acids, pentapeptides, and triterpenoid saponins in the root of Aster tataricus by high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry† We established a qualitative method to analyze the main chemical compositions of the root of Aster tataricus. Most of the peaks were separated on a C18 column packed with 5.0 m particles, and 28 compounds were identified, including 11 chlorogenic acids, ten astins/asterinins, and seven astersaponins, four of which were reported for the first time from A. tataricus. Furthermore, we developed a reliable method for the simultaneous quantification of 3-caffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, astin A, astin B, astin C, astersaponin A, and astersaponin C, and the qualified separations were achieved only on a C18 column packed with 2.7 m particles. The method was used to measure the concentrations of eight components in samples from two major producing areas in China, and the average contents in samples from Bozhou (Anhui) were higher than those in samples from Anguo (Hebei). Keywords: Aster tataricus / Astersaponins / Astins / Chlorogenic acids / Qualitative and quantitative analysis DOI 10.1002/jssc.201401008
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Widely used for thousands of years, the root of Aster tataricus L. f. (RAT, Compositae) is a famous and essential drug in traditional Chinese medicines with the function of relieving cough and dispersing phlegm [1]. Phytochemical studies revealed that RAT mainly contains phenolics, triterpenoids, saponins, pentapeptides, and so on [2–5]. Phenolic acids, such as caffeoylquinic acids (CQAs), have been shown to possess anti-influenza, antioxidative, antiapoptotic, and liver protective activities [6–10]. Triterpenoid saponins and pentapeptides are two characteristic components in RAT. Astersaponins were often considered as the expectorant ingredients [4, 11, 12]. Pentapeptides are composed of Correspondence: Professor Mian Zhang, Research Department of Pharmacognosy, China Pharmaceutical University, #639 Longmian Dadao, Nanjing 211198, P. R. China E-mail: [email protected] Fax: +86 25 86185137
Abbreviations: CQA, caffeoylquinic acid; DCQA, dicaffeoylquinic acid; DAD, diode array detection; RAT, the root of Aster tataricus C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
halogenated cyclic (astins A–P) and linear (asterinins A–F) peptides [13–17]. Astins A–C showed potential antitumor activities, but they are also a kind of hepatotoxic substance [2, 18, 19]. As a commonly used herbal drug, A. tataricus has been cultivated for a long history in China. There are two main producing areas, i.e. Anguo, Hebei and Bozhou, Anhui. However, the chemical compositions and contents of herbal medicines from different areas often vary greatly. So far, the quality of RAT was controlled only by shionone (a triterpenoid) or several flavonoids [1,20,21]. Moreover, the qualities of RAT from different areas still remained unclear. Therefore, it is necessary to develop a comprehensive method for evaluating and comparing the quality of RAT produced from different areas. In the present study, a method of HPLC coupled with Q-TOF-MS has been developed for separation and identification of the major constituents of RAT, and a method of HPLC combined with diode array detection (DAD) was ∗ Additional correspondence: Dr. Xiang-Hong Xu E-mail: [email protected] † This paper is included in the virtual special issue on sample preparation in mass spectrometry available at the Journal of Separation Science website.
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Figure 1. Structures of eight chemical standards (A), representative total ion current chromatograms in the negative/positive mode (B/C) of RAT sample solution, and HPLC chromatograms of standards/RAT sample (D/E) solutions.
established for the determination of eight compounds belonging to CQAs, peptides, and saponins. Both developed methods were applied to evaluate the RAT samples originated from Anguo and Bozhou.
2 Materials and methods
Germany) and Yuwang (Shandong, China), respectively. All other chemicals and solvents were of the highest analytical grade available. RAT samples (Supporting Information Table S1) were collected from Anguo, Hebei and Bozhou, Anhui, All the samples were authenticated by Professor Mian Zhang and deposited in the author’s laboratory.
2.1 Chemicals and materials
2.2 Preparation of sample and standard solutions
The reference standards of 3,4-dicaffeoylquinic acid (3,4DCQA), 3,5-dicaffeoylquinic acids A (3,5-DCQA), astin A, astin B, astin C, astersaponin A, and astersaponin C were isolated from RAT in our laboratory (the structures were confirmed by NMR data), and 3-CQA was purchased from Zelang Pharmaceutical Technology (Nanjing, China). All above standards (Fig. 1A) were of the purity > 98%. HPLC-grade acetonitrile and methanol were purchased from Merck (Darmstadt,
The fine powder (0.2 g) of RAT was accurately weighed and ultrasonicated with 4 mL of 50% v/v methanol for 45 min. After cooled and weighed again, the mixture replenished the loss of weight with the same solvent, mixed well, and centrifuged at 12 000 × g for 10 min. The supernatant was stored as the sample solution. The stock solutions of eight reference standards were prepared in methanol to the final concentrations of 0.5 mg/mL for 3-CQA, 3,5-DCQA, and
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Table 1. Characterization of 28 chemical constituents of RAT by HPLC–ESI-Q-TOF-MS Peak no.
Rt (min)
Formula
Measured mass (Da)
[M-/+H]-/+ (m/z)
Error (ppm)
MS2 fragments [+/-] (m/z)
Assignment
Literature
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
5.951 7.431 7.893 8.378 8.847 10.604 12.870 17.261 19.852 20.037 20.315 20.932 21.355 22.046 22.578 23.185 24.133 25.612 26.461 27.639 32.929 37.539 38.926 41.330 42.255 42.648 45.833 47.527
C22 H28 O14 C16 H18 O9 C16 H18 O9 C16 H18 O9 C17 H20 O9 C16 H18 O9 C17 H20 O9 C25 H33 N5 O9 C25 H24 O12 C28 H26 O15 C25 H24 O12 C25 H33 N5 O8 C28 H26 O15 C25 H33 N5 O8 C25 H33 N5 O8 Cl2 C25 H32 N5 O6 Cl C26 H35 N5 O8 C25 H33 N5 O7 Cl2 C25 H33 N5 O7 Cl2 C25 H33 N5 O7 C25 H33 N5 O6 Cl2 C73 H118 O38 C68 H110 O34 C67 H108 O34 C62 H100 O30 C68 H111 O33 C62 H100 O29 C41 H66 O14
516.1464 354.0954 354.0952 354.0951 368.1109 354.0951 368.1109 547.2292 516.1265 602.1265 516.1268 531.2342 602.1272 531.2335 601.1706 533.2042 545.2489 585.1781 585.1767 515.2385 569.1811 1602.7301 1470.6879 1456.6722 1324.6299 1454.6915 1308.6350 782.4453
[-]a) 515.1393 [−]353.1019 [−]353.1019 [−]353.1069 [−]367.1036 [−]353.1069 [−]367.1231 [+]548.2352 [−]515.1422 [−]601.1444 [−]515.1196 [+]532.2408 [−]601.1444 [+]532.2408 [+]602.1784 [+]534.2109 [+]546.2562 [+]586.1836 [+]586.1836 [+]516.2462 [+]570.1890 [−]1601.7210 [−]1469.6808 [−]1455.6651 [−]1323.6231 [−]1453.6842 [−]1307.6296 [−]781.4711
2.98 −0.85 −0.19 −0.41 −0.45 0.06 −0.39 −2.43 0.56 1.17 −0.09 −1.27 0.05 −1.14 0.06 −0.23 −0.71 −4.12 −1.74 −0.90 −0.55 1.14 −0.15 −0.67 −0.33 −0.61 −1.41 3.84
[−]191.0553, 173.0442, 161.0248, 111.0455 [−]191.0580, 171.0305, 134.0352, 109.2291 [−]191.0551, 171.0240, 161.0220, 134.0370, 109.0288 [−]173.0482, 161.0221, 134.0376, 109.0282 [−]134.0367, 117.0340, 106.0425 [−]191.0556, 171.0312, 134.0367, 109.0296 [−]159.0520, 134.0410, 117.0355, 106.0423 [+]149.0704, 131.0493, 106.0658, 103.0545 [−]191.0561, 161.0204, 135.0441, 111.0434 [−]191.0561, 161.0204, 135.0441, 111.0434 [−]191.0561, 161.0204, 135.0441, 111.0434 [+]131.0490, 106.0655, 105.0706, 103.0547 [−]191.0561, 173.0455, 161.0204, 135.0441, 111.0434 [+]131.0486, 106.0652, 105.0695, 103.0541 [+]137.9862, 131.0487, 106.0651, 103.0544 [+]131.0497, 106.0663, 104.0084, 103.0554 [+]145.0295, 131.0489, 106.0665, 103.0543 [+]223.9380, 137.9871, 131.0490, 106.0653 [+]137.9865, 131.0486, 106.0649, 103.0544 [+]131.0496, 106.0665, 105.0708, 103.0551 [+]137.9872, 131.0493, 106.0656, 103.0551 [−]1469.6802, 781.4581, 687.2275, 541.1732, 409.1315 [−]1305.6038, 781.4364, 687.2324, 541.1793, 409.1326 [−]1323.6199, 781.4581, 673.2297 [−]1191.5604, 781.4381, 541.1779, 337.1174 [−]765.4430, 687.2316, 541.1745, 409.1347 [−]1175.5876, 765.4426, 541.1779, 409.1359, [−]725.4102, 487.3301, 403.5241, 233.0657
Derib) of CQAs 5-CQA 3-CQA 4-CQA Deri of FQAs 1-CQA Deri of FQAs Deri of asterinins 3,4-DCQA Deri of DCQAs 3,5-DCQA Iso-asterinin A Deri of DCQAs Asterinin A Astin K Astin D Asterinin C Astin A Astin B Asterinin D Astin C Astersaponin C Deri of astersaponins Astersaponin A Astersaponin E Deri of astersaponins Astersaponin F Zd)
[22] [23] [23]c) [23] [24, 25] [23] [24, 25] Scifinder [25]c) [26, 27] [25]c) [16] [28] [16] [15] [14] [16] [2]c) [2]c) [17] [2]c) [4]c) [4] [4]c) [11] [11] [11] [12, 15]
a) [−]/[+], in negative/positive ion modes. b) Deri, derivative. c) Compared with authentic compounds. d) Z, 3-O-(␣-D-arabinopyranosyl-(1→6)--D-glucopyranosyl)-2,3,16␣-trihydroxyolean-12-en-28-oic acid.
3,4-DCQA and 1 mg/mL for asins A–C and astersaponins A and C.
2.3 Instrumentation and conditions Qualitative analysis was performed on an Agilent 1260 series system equipped with an autosampler, a vacuum degasser, a quarternary pump, and a DAD, and interfacing to an Agilent 6520 Q-TOF-MS with an ESI source (Agilent Technologies, USA). Samples were separated on a Zorbax C18 column (4.6 × 250 mm, 5 m, Agilent, USA) and the column temperature was kept at 30⬚C with a flow rate of 1 mL/min. The mobile phase consisted of 0.01% formic acid (A) and acetonitrile (B), and the elution program as follows: 10–18% B at 0–13 min, 18–25% B at 13–18 min, 25% B at 18–30 min, 25–28% B at 30–38 min, 28–40% B at 38–45 min, and 40–60% B at 45– 50 min. The injection volume was 10 L. The MS spectrometer was operated in both positive and negative ionization modes. The MS analysis conditions were set as follows: nitrogen at a flow rate of 8.0 L/min at 320⬚C, a nebulizer pressure of 35 psi, a capillary voltage of 4000 V, a skimmer of 65 V, and a fragmentor voltage of 140 V, scanning from m/z 100 to 2000. The sample collision energy was set at 70 V. Data were processed using MassHunter Workstation Data Acquisition Software Ver. B.04.00 (Agilent Technologies). C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Quantitative analysis was performed on the same HPLC equipment. Samples were separated on a Poroshell 120 ECC18 column (4.6 × 150 mm, 2.7 m, Agilent, USA) operated at 30⬚C with a flow rate of 0.5 mL/min. The mobile phase consisted of 0.03% formic acid (A) and acetonitrile (B), and the elution program as follows: 9–25% B at 0–20 min, 25– 28% B at 20–40 min, and 28–44% B at 40–50 min. A detection wavelength was set at 210 nm and the injection volume was 5 L. The calibration curves were prepared by diluting the stock solutions of eight standards to a series of appropriate concentration. The LODs and LOQs were determined at S/N ratio of 3 and 10, respectively. The precision was evaluated with the intra- and interday variations of the mixed standard solution. The stability and repeatability were tested with the same sample, and the accuracy was evaluated by recovery experiments with the same sample.
3 Results and discussion 3.1 Optimization of HPLC conditions To get the most of peaks with good resolution and sensitivity for HPLC–Q-TOF-MS analysis, the chromatographic conditions were optimized. The C18 column packed with 5.0 m particles could obtain much more peaks than that with 2.7 www.jss-journal.com
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Table 2. Contents (mg/g) of eight compounds in 16 batches of RAT (n = 3)
Origin
A1 A2 A3 A4 A5 A6 A7 A8 B1 B2 B3 B4 B5 B6 B7 B8 Average
CQA/DCQA
Astin
Astersaponin
3-
3,4-
3,5-
A
B
C
C
A
1.90 1.78 1.72 2.76 1.82 1.70 2.16 2.28 2.13 1.95 2.07 2.04 1.91 1.89 1.88 2.31 2.02
0.44 0.49 0.45 0.45 0.24 0.28 0.35 0.42 0.72 0.62 0.44 0.67 0.86 0.36 0.31 0.55 0.48
0.46 0.50 0.45 0.76 0.35 0.31 0.40 0.43 0.34 0.37 0.34 0.37 0.45 0.55 0.59 0.74 0.46
0.28 0.36 0.30 0.20 0.15 0.33 0.33 0.25 0.33 0.47 0.33 0.51 0.35 0.47 0.44 0.15 0.33
0.15 0.18 0.13 0.16 0.07 0.16 0.20 0.16 0.22 0.25 0.30 0.34 0.27 0.22 0.25 0.07 0.20
0.44 0.54 0.32 0.49 0.22 0.48 0.54 0.39 0.50 0.64 0.69 0.72 0.60 0.60 0.60 0.30 0.50
0.61 1.20 2.30 1.73 NDb) ND 0.93 1.26 2.01 2.46 2.59 4.39 3.04 4.68 6.75 1.08 2.19
1.73 1.85 3.21 3.23 1.52 2.50 2.65 3.80 2.45 3.81 2.87 2.42 2.87 3.30 4.70 2.43 2.83
Total
6.01 6.90 8.86 9.79 4.36 5.73 7.57 8.99 8.69 10.56 9.62 11.46 10.35 12.07 15.51 7.62 9.01
Relative content (%)a) CQAs
Astins
Astersaponins
46.74 40.22 29.52 40.58 55.18 39.71 38.38 34.86 36.68 27.86 29.65 26.85 31.10 23.17 17.88 47.25 35.35
14.45 15.6 8.33 8.78 9.97 16.79 14.29 8.80 12.09 12.80 13.62 13.80 11.76 10.71 8.28 6.75 11.68
38.81 44.16 62.15 50.64 34.85 43.50 47.33 56.34 51.23 59.34 56.73 59.36 57.14 66.12 73.84 46.01 52.97
a) Relative content (%) = CQAs (astins or astersaponins)/total × 100%. b) ND, not detected.
or 1.8 m particles. Acetonitrile/water could achieve a better separation than methanol/water. The addition of 0.01% formic acid into the mobile phase could greatly improve the separation with sharper peaks and better symmetry. As a result, the solvent of acetonitrile/0.01% formic acid was selected as the mobile phase. For HPLC quantitative analysis, the column with 5.0 m particles could not separate astin A from astin B, 3,4-DCQA from 3,5-DCQA completely. The qualified separations were achieved on the column with 2.7 m particles with a solvent of acetonitrile/0.03% formic acid. An increased concentration of formic acid from 0.01 to 0.03% was necessary for a good separation and peak symmetry.
3.2 HPLC–Q-TOF-MS qualitative analysis Both the positive and negative ion modes were operated for MS/MS analysis. The chromatogram obtained by negative mode was used to identify phenolics and saponins and that obtained by positive mode was used to assign peptides. The total ion current chromatograms of RAT test solution in both negative and positive modes were shown in Fig. 1B and C, and the assignment of 28 peaks were shown in Table 1. By comparing with the authentic standards, the compounds corresponding to peaks 3 (3-CQA), 9 (3,4-DCQA), 11 (3,5-DCQA), 18 (astin A), 19 (astin B), 21 (astin C), 22 (astersaponin C), and 24 (astersaponin A) were unambiguously identified. According to the quasi-molecular ions, the unidentified phenolics could be divided into four groups, i.e. [M-H]− at m/z 353 (CQAs, peaks 2, 4, 6), 367 (feruloylquinic acids, peaks 5, 7), 515 (peak 1), and 601 (peaks 10, 13). The isomers in the C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
first two groups could be assigned by the MS2 ions and the order of peaks [23–25]. Despite with the similar molecular mass, peak 1 has a different molecular formula and a very different polarity from peaks 9 and 11. It was deduced as a CQA glucoside based on its precise molecular mass and MS2 ions (Supporting Information Fig. S1). In the same way, peaks 10 and 13 were speculated to be a type of particular DCQAs derivatives (Supporting Information Fig. S1). Based on the data of the known compounds and the precise molecular mass, unidentified peptides could be assigned by the MS2 ions with high relative abundances at m/z 131.04, 106.06, 103.05 (for both astins and asterinins), 137.98 (for astins), and 105.07 (for asterinins). However, the possible structure of peak 8 (Supporting Information Fig. S1) was greatly different with the known asterinins. The peaks (22–28) corresponding to astersaponins were also easily identified as they varied only in the sorts and numbers of monosacchrides. As a result, 28 identified compounds were composed of 11 phenolics, ten pentapeptides, and seven astersaponins (Table 1).
3.3 HPLC–DAD quantitative analysis The proposed HPLC method for the determination of eight analytes showed good linearity (r > 0.9986), and the LODs and LOQs were no less than 0.29 and 1.08 g/mL, respectively. The precision, stability, and repeatability were all less than 3.0%. The overall recoveries were between 97.0 and 101.5% with RSD less than 3.0% (Supporting Information Tables S2 and S3). The developed method was applied to determine the 16 RAT samples, and the results are shown in Fig. 1D www.jss-journal.com
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and E and Table 2. The average amounts of astersaponins (astersaponins A and C), CQAs (3-CQA, 3,4-DCQA, and 3,5-DCQA), and astins (astins A–C) were 5.02, 2.96, and 1.03 mg/g, respectively. The total levels of the eight compounds were obviously altered from 4.36 (sample A5) to 15.51 mg/g (sample B7). In general, the contents in Bozhou samples (B1–B8) were higher than those in Anguo samples (A1–A8), indicating that the producing area is an important factor for RAT quality.
4 Concluding remarks For the first time, both HPLC–Q-TOF-MS qualitative and HPLC–DAD quantitative methods were developed to analyze the chemical compositions of RAT collected from two major producing areas of China. The present study provides a deeper understanding of the active components of RAT, also provides a reliable HPLC method for the determination of three different components in it. The RAT samples from different areas were similar in chemical components, but different in their contents. This work was financially supported by the National Natural Science Foundation of China (30772702) and the National New Drug Innovation Major Project of China (2011ZX09307–002– 02). The authors have declared no conflict of interest.
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Dong-Xia Zhao1,2 Bing-Qiang Hu1 Mian Zhang1 Chao-Feng Zhang1 Xiang-Hong Xu1 ∗ 1 Research
Department of Pharmacognosy, China Pharmaceutical University, Nanjing, China 2 Pharmaceutical College, Henan University, Kaifeng, China Received September 15, 2014 Revised November 1, 2014 Accepted November 28, 2014
Short Communication
Simultaneous separation and determination of phenolic acids, pentapeptides, and triterpenoid saponins in the root of Aster tataricus by high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry† We established a qualitative method to analyze the main chemical compositions of the root of Aster tataricus. Most of the peaks were separated on a C18 column packed with 5.0 m particles, and 28 compounds were identified, including 11 chlorogenic acids, ten astins/asterinins, and seven astersaponins, four of which were reported for the first time from A. tataricus. Furthermore, we developed a reliable method for the simultaneous quantification of 3-caffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, astin A, astin B, astin C, astersaponin A, and astersaponin C, and the qualified separations were achieved only on a C18 column packed with 2.7 m particles. The method was used to measure the concentrations of eight components in samples from two major producing areas in China, and the average contents in samples from Bozhou (Anhui) were higher than those in samples from Anguo (Hebei). Keywords: Aster tataricus / Astersaponins / Astins / Chlorogenic acids / Qualitative and quantitative analysis DOI 10.1002/jssc.201401008
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Widely used for thousands of years, the root of Aster tataricus L. f. (RAT, Compositae) is a famous and essential drug in traditional Chinese medicines with the function of relieving cough and dispersing phlegm [1]. Phytochemical studies revealed that RAT mainly contains phenolics, triterpenoids, saponins, pentapeptides, and so on [2–5]. Phenolic acids, such as caffeoylquinic acids (CQAs), have been shown to possess anti-influenza, antioxidative, antiapoptotic, and liver protective activities [6–10]. Triterpenoid saponins and pentapeptides are two characteristic components in RAT. Astersaponins were often considered as the expectorant ingredients [4, 11, 12]. Pentapeptides are composed of Correspondence: Professor Mian Zhang, Research Department of Pharmacognosy, China Pharmaceutical University, #639 Longmian Dadao, Nanjing 211198, P. R. China E-mail: [email protected] Fax: +86 25 86185137
Abbreviations: CQA, caffeoylquinic acid; DCQA, dicaffeoylquinic acid; DAD, diode array detection; RAT, the root of Aster tataricus C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
halogenated cyclic (astins A–P) and linear (asterinins A–F) peptides [13–17]. Astins A–C showed potential antitumor activities, but they are also a kind of hepatotoxic substance [2, 18, 19]. As a commonly used herbal drug, A. tataricus has been cultivated for a long history in China. There are two main producing areas, i.e. Anguo, Hebei and Bozhou, Anhui. However, the chemical compositions and contents of herbal medicines from different areas often vary greatly. So far, the quality of RAT was controlled only by shionone (a triterpenoid) or several flavonoids [1,20,21]. Moreover, the qualities of RAT from different areas still remained unclear. Therefore, it is necessary to develop a comprehensive method for evaluating and comparing the quality of RAT produced from different areas. In the present study, a method of HPLC coupled with Q-TOF-MS has been developed for separation and identification of the major constituents of RAT, and a method of HPLC combined with diode array detection (DAD) was ∗ Additional correspondence: Dr. Xiang-Hong Xu E-mail: [email protected] † This paper is included in the virtual special issue on sample preparation in mass spectrometry available at the Journal of Separation Science website.
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Figure 1. Structures of eight chemical standards (A), representative total ion current chromatograms in the negative/positive mode (B/C) of RAT sample solution, and HPLC chromatograms of standards/RAT sample (D/E) solutions.
established for the determination of eight compounds belonging to CQAs, peptides, and saponins. Both developed methods were applied to evaluate the RAT samples originated from Anguo and Bozhou.
2 Materials and methods
Germany) and Yuwang (Shandong, China), respectively. All other chemicals and solvents were of the highest analytical grade available. RAT samples (Supporting Information Table S1) were collected from Anguo, Hebei and Bozhou, Anhui, All the samples were authenticated by Professor Mian Zhang and deposited in the author’s laboratory.
2.1 Chemicals and materials
2.2 Preparation of sample and standard solutions
The reference standards of 3,4-dicaffeoylquinic acid (3,4DCQA), 3,5-dicaffeoylquinic acids A (3,5-DCQA), astin A, astin B, astin C, astersaponin A, and astersaponin C were isolated from RAT in our laboratory (the structures were confirmed by NMR data), and 3-CQA was purchased from Zelang Pharmaceutical Technology (Nanjing, China). All above standards (Fig. 1A) were of the purity > 98%. HPLC-grade acetonitrile and methanol were purchased from Merck (Darmstadt,
The fine powder (0.2 g) of RAT was accurately weighed and ultrasonicated with 4 mL of 50% v/v methanol for 45 min. After cooled and weighed again, the mixture replenished the loss of weight with the same solvent, mixed well, and centrifuged at 12 000 × g for 10 min. The supernatant was stored as the sample solution. The stock solutions of eight reference standards were prepared in methanol to the final concentrations of 0.5 mg/mL for 3-CQA, 3,5-DCQA, and
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Table 1. Characterization of 28 chemical constituents of RAT by HPLC–ESI-Q-TOF-MS Peak no.
Rt (min)
Formula
Measured mass (Da)
[M-/+H]-/+ (m/z)
Error (ppm)
MS2 fragments [+/-] (m/z)
Assignment
Literature
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
5.951 7.431 7.893 8.378 8.847 10.604 12.870 17.261 19.852 20.037 20.315 20.932 21.355 22.046 22.578 23.185 24.133 25.612 26.461 27.639 32.929 37.539 38.926 41.330 42.255 42.648 45.833 47.527
C22 H28 O14 C16 H18 O9 C16 H18 O9 C16 H18 O9 C17 H20 O9 C16 H18 O9 C17 H20 O9 C25 H33 N5 O9 C25 H24 O12 C28 H26 O15 C25 H24 O12 C25 H33 N5 O8 C28 H26 O15 C25 H33 N5 O8 C25 H33 N5 O8 Cl2 C25 H32 N5 O6 Cl C26 H35 N5 O8 C25 H33 N5 O7 Cl2 C25 H33 N5 O7 Cl2 C25 H33 N5 O7 C25 H33 N5 O6 Cl2 C73 H118 O38 C68 H110 O34 C67 H108 O34 C62 H100 O30 C68 H111 O33 C62 H100 O29 C41 H66 O14
516.1464 354.0954 354.0952 354.0951 368.1109 354.0951 368.1109 547.2292 516.1265 602.1265 516.1268 531.2342 602.1272 531.2335 601.1706 533.2042 545.2489 585.1781 585.1767 515.2385 569.1811 1602.7301 1470.6879 1456.6722 1324.6299 1454.6915 1308.6350 782.4453
[-]a) 515.1393 [−]353.1019 [−]353.1019 [−]353.1069 [−]367.1036 [−]353.1069 [−]367.1231 [+]548.2352 [−]515.1422 [−]601.1444 [−]515.1196 [+]532.2408 [−]601.1444 [+]532.2408 [+]602.1784 [+]534.2109 [+]546.2562 [+]586.1836 [+]586.1836 [+]516.2462 [+]570.1890 [−]1601.7210 [−]1469.6808 [−]1455.6651 [−]1323.6231 [−]1453.6842 [−]1307.6296 [−]781.4711
2.98 −0.85 −0.19 −0.41 −0.45 0.06 −0.39 −2.43 0.56 1.17 −0.09 −1.27 0.05 −1.14 0.06 −0.23 −0.71 −4.12 −1.74 −0.90 −0.55 1.14 −0.15 −0.67 −0.33 −0.61 −1.41 3.84
[−]191.0553, 173.0442, 161.0248, 111.0455 [−]191.0580, 171.0305, 134.0352, 109.2291 [−]191.0551, 171.0240, 161.0220, 134.0370, 109.0288 [−]173.0482, 161.0221, 134.0376, 109.0282 [−]134.0367, 117.0340, 106.0425 [−]191.0556, 171.0312, 134.0367, 109.0296 [−]159.0520, 134.0410, 117.0355, 106.0423 [+]149.0704, 131.0493, 106.0658, 103.0545 [−]191.0561, 161.0204, 135.0441, 111.0434 [−]191.0561, 161.0204, 135.0441, 111.0434 [−]191.0561, 161.0204, 135.0441, 111.0434 [+]131.0490, 106.0655, 105.0706, 103.0547 [−]191.0561, 173.0455, 161.0204, 135.0441, 111.0434 [+]131.0486, 106.0652, 105.0695, 103.0541 [+]137.9862, 131.0487, 106.0651, 103.0544 [+]131.0497, 106.0663, 104.0084, 103.0554 [+]145.0295, 131.0489, 106.0665, 103.0543 [+]223.9380, 137.9871, 131.0490, 106.0653 [+]137.9865, 131.0486, 106.0649, 103.0544 [+]131.0496, 106.0665, 105.0708, 103.0551 [+]137.9872, 131.0493, 106.0656, 103.0551 [−]1469.6802, 781.4581, 687.2275, 541.1732, 409.1315 [−]1305.6038, 781.4364, 687.2324, 541.1793, 409.1326 [−]1323.6199, 781.4581, 673.2297 [−]1191.5604, 781.4381, 541.1779, 337.1174 [−]765.4430, 687.2316, 541.1745, 409.1347 [−]1175.5876, 765.4426, 541.1779, 409.1359, [−]725.4102, 487.3301, 403.5241, 233.0657
Derib) of CQAs 5-CQA 3-CQA 4-CQA Deri of FQAs 1-CQA Deri of FQAs Deri of asterinins 3,4-DCQA Deri of DCQAs 3,5-DCQA Iso-asterinin A Deri of DCQAs Asterinin A Astin K Astin D Asterinin C Astin A Astin B Asterinin D Astin C Astersaponin C Deri of astersaponins Astersaponin A Astersaponin E Deri of astersaponins Astersaponin F Zd)
[22] [23] [23]c) [23] [24, 25] [23] [24, 25] Scifinder [25]c) [26, 27] [25]c) [16] [28] [16] [15] [14] [16] [2]c) [2]c) [17] [2]c) [4]c) [4] [4]c) [11] [11] [11] [12, 15]
a) [−]/[+], in negative/positive ion modes. b) Deri, derivative. c) Compared with authentic compounds. d) Z, 3-O-(␣-D-arabinopyranosyl-(1→6)--D-glucopyranosyl)-2,3,16␣-trihydroxyolean-12-en-28-oic acid.
3,4-DCQA and 1 mg/mL for asins A–C and astersaponins A and C.
2.3 Instrumentation and conditions Qualitative analysis was performed on an Agilent 1260 series system equipped with an autosampler, a vacuum degasser, a quarternary pump, and a DAD, and interfacing to an Agilent 6520 Q-TOF-MS with an ESI source (Agilent Technologies, USA). Samples were separated on a Zorbax C18 column (4.6 × 250 mm, 5 m, Agilent, USA) and the column temperature was kept at 30⬚C with a flow rate of 1 mL/min. The mobile phase consisted of 0.01% formic acid (A) and acetonitrile (B), and the elution program as follows: 10–18% B at 0–13 min, 18–25% B at 13–18 min, 25% B at 18–30 min, 25–28% B at 30–38 min, 28–40% B at 38–45 min, and 40–60% B at 45– 50 min. The injection volume was 10 L. The MS spectrometer was operated in both positive and negative ionization modes. The MS analysis conditions were set as follows: nitrogen at a flow rate of 8.0 L/min at 320⬚C, a nebulizer pressure of 35 psi, a capillary voltage of 4000 V, a skimmer of 65 V, and a fragmentor voltage of 140 V, scanning from m/z 100 to 2000. The sample collision energy was set at 70 V. Data were processed using MassHunter Workstation Data Acquisition Software Ver. B.04.00 (Agilent Technologies). C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Quantitative analysis was performed on the same HPLC equipment. Samples were separated on a Poroshell 120 ECC18 column (4.6 × 150 mm, 2.7 m, Agilent, USA) operated at 30⬚C with a flow rate of 0.5 mL/min. The mobile phase consisted of 0.03% formic acid (A) and acetonitrile (B), and the elution program as follows: 9–25% B at 0–20 min, 25– 28% B at 20–40 min, and 28–44% B at 40–50 min. A detection wavelength was set at 210 nm and the injection volume was 5 L. The calibration curves were prepared by diluting the stock solutions of eight standards to a series of appropriate concentration. The LODs and LOQs were determined at S/N ratio of 3 and 10, respectively. The precision was evaluated with the intra- and interday variations of the mixed standard solution. The stability and repeatability were tested with the same sample, and the accuracy was evaluated by recovery experiments with the same sample.
3 Results and discussion 3.1 Optimization of HPLC conditions To get the most of peaks with good resolution and sensitivity for HPLC–Q-TOF-MS analysis, the chromatographic conditions were optimized. The C18 column packed with 5.0 m particles could obtain much more peaks than that with 2.7 www.jss-journal.com
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Table 2. Contents (mg/g) of eight compounds in 16 batches of RAT (n = 3)
Origin
A1 A2 A3 A4 A5 A6 A7 A8 B1 B2 B3 B4 B5 B6 B7 B8 Average
CQA/DCQA
Astin
Astersaponin
3-
3,4-
3,5-
A
B
C
C
A
1.90 1.78 1.72 2.76 1.82 1.70 2.16 2.28 2.13 1.95 2.07 2.04 1.91 1.89 1.88 2.31 2.02
0.44 0.49 0.45 0.45 0.24 0.28 0.35 0.42 0.72 0.62 0.44 0.67 0.86 0.36 0.31 0.55 0.48
0.46 0.50 0.45 0.76 0.35 0.31 0.40 0.43 0.34 0.37 0.34 0.37 0.45 0.55 0.59 0.74 0.46
0.28 0.36 0.30 0.20 0.15 0.33 0.33 0.25 0.33 0.47 0.33 0.51 0.35 0.47 0.44 0.15 0.33
0.15 0.18 0.13 0.16 0.07 0.16 0.20 0.16 0.22 0.25 0.30 0.34 0.27 0.22 0.25 0.07 0.20
0.44 0.54 0.32 0.49 0.22 0.48 0.54 0.39 0.50 0.64 0.69 0.72 0.60 0.60 0.60 0.30 0.50
0.61 1.20 2.30 1.73 NDb) ND 0.93 1.26 2.01 2.46 2.59 4.39 3.04 4.68 6.75 1.08 2.19
1.73 1.85 3.21 3.23 1.52 2.50 2.65 3.80 2.45 3.81 2.87 2.42 2.87 3.30 4.70 2.43 2.83
Total
6.01 6.90 8.86 9.79 4.36 5.73 7.57 8.99 8.69 10.56 9.62 11.46 10.35 12.07 15.51 7.62 9.01
Relative content (%)a) CQAs
Astins
Astersaponins
46.74 40.22 29.52 40.58 55.18 39.71 38.38 34.86 36.68 27.86 29.65 26.85 31.10 23.17 17.88 47.25 35.35
14.45 15.6 8.33 8.78 9.97 16.79 14.29 8.80 12.09 12.80 13.62 13.80 11.76 10.71 8.28 6.75 11.68
38.81 44.16 62.15 50.64 34.85 43.50 47.33 56.34 51.23 59.34 56.73 59.36 57.14 66.12 73.84 46.01 52.97
a) Relative content (%) = CQAs (astins or astersaponins)/total × 100%. b) ND, not detected.
or 1.8 m particles. Acetonitrile/water could achieve a better separation than methanol/water. The addition of 0.01% formic acid into the mobile phase could greatly improve the separation with sharper peaks and better symmetry. As a result, the solvent of acetonitrile/0.01% formic acid was selected as the mobile phase. For HPLC quantitative analysis, the column with 5.0 m particles could not separate astin A from astin B, 3,4-DCQA from 3,5-DCQA completely. The qualified separations were achieved on the column with 2.7 m particles with a solvent of acetonitrile/0.03% formic acid. An increased concentration of formic acid from 0.01 to 0.03% was necessary for a good separation and peak symmetry.
3.2 HPLC–Q-TOF-MS qualitative analysis Both the positive and negative ion modes were operated for MS/MS analysis. The chromatogram obtained by negative mode was used to identify phenolics and saponins and that obtained by positive mode was used to assign peptides. The total ion current chromatograms of RAT test solution in both negative and positive modes were shown in Fig. 1B and C, and the assignment of 28 peaks were shown in Table 1. By comparing with the authentic standards, the compounds corresponding to peaks 3 (3-CQA), 9 (3,4-DCQA), 11 (3,5-DCQA), 18 (astin A), 19 (astin B), 21 (astin C), 22 (astersaponin C), and 24 (astersaponin A) were unambiguously identified. According to the quasi-molecular ions, the unidentified phenolics could be divided into four groups, i.e. [M-H]− at m/z 353 (CQAs, peaks 2, 4, 6), 367 (feruloylquinic acids, peaks 5, 7), 515 (peak 1), and 601 (peaks 10, 13). The isomers in the C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
first two groups could be assigned by the MS2 ions and the order of peaks [23–25]. Despite with the similar molecular mass, peak 1 has a different molecular formula and a very different polarity from peaks 9 and 11. It was deduced as a CQA glucoside based on its precise molecular mass and MS2 ions (Supporting Information Fig. S1). In the same way, peaks 10 and 13 were speculated to be a type of particular DCQAs derivatives (Supporting Information Fig. S1). Based on the data of the known compounds and the precise molecular mass, unidentified peptides could be assigned by the MS2 ions with high relative abundances at m/z 131.04, 106.06, 103.05 (for both astins and asterinins), 137.98 (for astins), and 105.07 (for asterinins). However, the possible structure of peak 8 (Supporting Information Fig. S1) was greatly different with the known asterinins. The peaks (22–28) corresponding to astersaponins were also easily identified as they varied only in the sorts and numbers of monosacchrides. As a result, 28 identified compounds were composed of 11 phenolics, ten pentapeptides, and seven astersaponins (Table 1).
3.3 HPLC–DAD quantitative analysis The proposed HPLC method for the determination of eight analytes showed good linearity (r > 0.9986), and the LODs and LOQs were no less than 0.29 and 1.08 g/mL, respectively. The precision, stability, and repeatability were all less than 3.0%. The overall recoveries were between 97.0 and 101.5% with RSD less than 3.0% (Supporting Information Tables S2 and S3). The developed method was applied to determine the 16 RAT samples, and the results are shown in Fig. 1D www.jss-journal.com
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and E and Table 2. The average amounts of astersaponins (astersaponins A and C), CQAs (3-CQA, 3,4-DCQA, and 3,5-DCQA), and astins (astins A–C) were 5.02, 2.96, and 1.03 mg/g, respectively. The total levels of the eight compounds were obviously altered from 4.36 (sample A5) to 15.51 mg/g (sample B7). In general, the contents in Bozhou samples (B1–B8) were higher than those in Anguo samples (A1–A8), indicating that the producing area is an important factor for RAT quality.
4 Concluding remarks For the first time, both HPLC–Q-TOF-MS qualitative and HPLC–DAD quantitative methods were developed to analyze the chemical compositions of RAT collected from two major producing areas of China. The present study provides a deeper understanding of the active components of RAT, also provides a reliable HPLC method for the determination of three different components in it. The RAT samples from different areas were similar in chemical components, but different in their contents. This work was financially supported by the National Natural Science Foundation of China (30772702) and the National New Drug Innovation Major Project of China (2011ZX09307–002– 02). The authors have declared no conflict of interest.
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