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. 2015 Nov 18;20(11):20518–20537. doi: 10.3390/molecules201119712

Analysis of the Constituents in “Zhu She Yong Xue Shuan Tong” by Ultra High Performance Liquid Chromatography with Quadrupole Time-of-Flight Mass Spectrometry Combined with Preparative High Performance Liquid Chromatography

Lin-Lin Wang 1,2, Li-Feng Han 1,2, He-Shui Yu 1, Mang-Mang Sang 1,2, Er-Wei Liu 1,2, Yi Zhang 1,2, Shi-Ming Fang 1,2, Tao Wang 1,2, Xiu-Mei Gao 1,*
Editor: Derek J McPhee
PMCID: PMC6332010  PMID: 26593895

Abstract

“Zhu She Yong Xue Shuan Tong” lyophilized powder (ZSYXST), consists of a series of saponins extracted from Panax notoginseng, which has been widely used in China for the treatment of strokes. In this study, an ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) combined with preparative high performance liquid chromatography (PHPLC) method was developed to rapidly identify both major and minor saponins in ZSYXST. Some high content components were removed through PHPLC in order to increase the sensitivity of the trace saponins. Then, specific characteristic fragment ions in both positive and negative mode were utilized to determine the types of aglycone, saccharide, as well as the saccharide chain linkages. As a result, 94 saponins, including 20 pairs of isomers and ten new compounds, which could represent higher than 98% components in ZSYXST, were identified or tentatively identified in commercial ZSYXST samples.

Keywords: ZSYXST, saponins, UHPLC-Q-TOF/MS

1. Introduction

“Zhu She Yong Xue Shuan Tong” lyophilized powder, containing saponins of Panax notoginseng, is commonly used for treating strokes in the clinic. It can dilate blood vessels, promote blood circulation [1], and prevent thrombosis [2,3,4]. It is also reported to have a therapeutic effect on diabetes [5,6]. Its efficacy has been confirmed and is widely accepted in clinical application. The average annual sales of ZSYXST in China are about a hundred million dollars. Although it is widely used in China, its chemical constituents, especially the minor compounds, are not understood very well. Recently, there was a chemical analysis of ZSYXST by an LC/MS method, however, only 30 compounds were identified because the researchers only used a normal LC/MS method [7]. Thus, the aim of this study is to establish a comprehensive analytical method to profile the constituents of ZSYXST as much as possible.

Ultra-high performance liquid chromatography (UHPLC) is characterized by the advantages of high resolution, good sensitivity, high speed of analysis and high peak capacity [8]. Quadrupole time of flight mass spectrometry (Q-TOF/MS) has already been widely used for structural characterization of unknown saponins [9,10,11]. Thus, combining UHPLC with Q-TOF/MS (UHPLC-Q-TOF/MS) could be an effective method to identify the chemical constituents in ZSYXST. There are four major saponins (notoginsenoside R1, ginsenosides Rg1, Re, and Rb1) which together represent more than 85% in ZSYXST, and these four major saponins could significantly decrease the sensitivity of minor saponins in the LC-MS fingerprint. Preparative high performance liquid chromatography (PHPLC) is a separation and purification technology with the advantages of high separation efficiency, sensitive detection and automated collection of fractions. This study utilized PHPLC to remove the major ingredients from ZSYXST samples in order to decrease the influence of the major saponins on the MS detection of the minor saponins.

In this paper, the structural characteristics of saponins from ZSYXST were investigated and illuminated using UHPLC-Q-TOF/MS and a target MS/MS data acquisition strategy. The full MS scan provided protonated or deprotonated molecules in their intact form, while the target MS/MS scan provided fragment information. The fragmentation patterns of the reference saponins were investigated first, and the types of aglycone, sequences and linkage positions of saccharide chains could be deduced accurately according to some diagnostic fragments pathways in both positive and negative modes. Finally, with the help of fragmentation pathway rules and finding compounds by the molecule feature in the Agilent Mass Hunter Workstation Software (Version B.02.00), 94 saponins, including 20 pairs of isomers, which could represent more than 98% of the components were identified or tentatively identified in commercial ZSYXST samples. Based on the literature and the SciFinder database, compounds 1, 2, 18, 20, 21, 22, 50, 51, 59, 90 were speculated to be new saponins in ZSYXST.

2. Results and Discussion

2.1. Optimization of MS Conditions

In order to obtain better MS response, cone voltage and CE were optimized. According to our research and literature data [12,13,14,15], the cone voltage was set to 120 V and 175 V in positive and negative mode, respectively. CE was dynamically adjusted from 45 to 70 V according to the m/z of precursor ions in the negative MS/MS mode, such as 45 V for m/z 650–750, 50 V for m/z 750–850, 55 V for m/z 850–950, 60 V for m/z 950–1050, 65 or 70 V for m/z > 1050.

2.2. MS Cleavage Rules of Saponins in ZSYXST

According to cleavage pathway of reference compounds and literatures, some diagnostic rules for the identification of compounds in ZSYXST can be summarized.

2.2.1. Differentiation and Classification of Diverse Saponins in ZSYXST

There were three main types of saponins in ZSYXST (Figure 1), namely protopanaxadiol (PPD), protopanaxatriol (PPT), and ocotillol (OCO) types [16,17]. According to the literature [12,15], there are some fragmentation rules for the sugar chains of saponins in P. notoginseng. The sugar moieties of PPD type are usually attached at the C-3 or C-3, 20 positions, while PPT sugars are attached at C-6 or C-6, C-20. OCO type usually form glycosides at the C-6 position when there as a five membered epoxy ring at C-20 [18,19,20]. According to [12,21] and our results, some characteristic ions could be used for deducing aglycone types, for example, PPD-type could be identified by diagnostic fragment ions at m/z 443, 425 and 407, PPT-type by m/z 441, 423 and 405, and OCO-type by m/z 457, 439 and 421 (Figure 1).

Figure 1.

Figure 1

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A diagram for rapid classification and identification of saponins by characteristic ions in MS and MS/MS mode.

2.2.2. Differentiation of Sub-Types of Aglycone

With the help of positive MS spectra data, the aglycone types could be deduced. However, there were several aglycone sub-types. Fortunately, those sub-types could be distinguished by characteristic fragment ions between m/z 390 to 500 in the negative MS/MS spectra. Specifically, PPD-A type could be observed at m/z 459, PPD-B type at m/z 477, PPD-C type at m/z 475, and PPD-D type at m/z 457, while the ions at m/z 391–475 could be attributed to PPT-A type, m/z 457 to PPT-B type, m/z 493 to PPT-C type, m/z 473 to PPT-D type; in OCO types, the ions at m/z 415–491 belonged to OCO-A or -C type, however, a neutral loss of 180 Da from precursor ions, it could be attributed to OCO-A type, as for OCO-B, -D, -E types, diagnostic ions at m/z 391–491, 473, and 489 could be observed, respectively (Figure 1 and Figure 2).

Figure 2.

Figure 2

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The structures of different types of saponin aglycone.

2.2.3. Differentiation of Sugar Moieties

In negative MS/MS spectra, it could be found that the terminal sugar moiety exposed to the outside in the spatial structure would cleave first. For example, in PPD type, the end sugar moiety at C-3 was cleaved first, and then the terminal sugar moiety at C-20 would cleave afterwards. However, in the PPT type, the terminal sugar moiety at C-20 was eliminated first, and then the end sugar moiety at C-6 would cleave later. OCO type saponins also conformed to this rule.

Sugar moieties linked to an aglycone through glycoside ether bonds at C-3, C-6 and C-20 or other positions, cleaved successively from the saponins. The most common sugar neutral losses were 132 Da, 146 Da, 162 Da, 278 Da, 294 Da and 324 Da, which correspond to arabinose/xylose (Ara/Xyl), rhamnose (Rha), glucose (Glc), Rha-Ara/Xyl, Glc-Ara/Xyl and Glc-Glc, respectively. In the lower mass region of negative MS/MS spectra, characteristic fragment ions could be observed to further identify sugar moieties, such as m/z 161, 179, 119, 113, 101 for Glc, m/z 145, 163 for Rha, m/z 131, 149 for Xyl or Ara, m/z 323 or 221 for Glc-Glc, m/z 307, 205 for Glc-Rha, and m/z 293,191 for Glc-Xyl/Ara (Figure 1).

In addition, in the lower mass region of positive MS spectra, characteristic fragment ions also could be observed to further authenticate sugar moieties, such as m/z 325 for Glc-Glc, m/z 309 for Glc-Rha, m/z 295 for Glc-Xyl/Ara (Table 1).

Table 1.

The characteristic ions of saponins in ZSYXST.

Comp. [M − H]− [M + COOH]− [M − H]−/[M + COOH]− Diagnostic Ions of Sugar Moieties in Negative Mode Diagnostic Ions of Aglycone Types in Positive Mode
1 965.5198 1011.5278 0.1 131.0312, 191.0668 439.3453, 457.3560
2 965.5237 1011.5291 0.15 131.0310, 191.1745 439.3459, 457.3559
3 979.5387 1025.5451 0.2 205.0648 421,1231, 439.1502
4 979.5407 1025.5464 0.2 145.0321, 205.0667 439.3480, 457.3572
5 801.4635 847.4662 0.1 221.0687 407.3257, 425.3360
6 815.4807 861.4881 0.14 221.0117 421.3436, 439.3547
7 947.5170 993.5230 0.18 131.0287, 191.0432, 221.0223 421.3586, 457.3660
8 815.4807 861.4881 0.14 221.0654 421.3446, 439.3581
9 815.4669 861.4817 0.02 323.0378 421.3324, 439.3512, 457.2314
10 815.4814 861.4820 0.09 323.0665 421.3477, 439.3594, 457.3701
11 815.4701 861.4836 0.1 323.0656 421.3577, 439.3659, 457.3730
12 817.4872 863.4982 0.8 323.2112 423.3736, 441.3848
13 799.4312 845.4598 20 221.0599 405.3408, 423.3536, 441.3938
14 961.5332 1007.5382 0.1 145.0595, 205.0690 421.3660, 439.3758, 457.3886
15 961.5328 1007.5333 0.15 205.4532 421.3748, 439.3861, 457.3971
16 815.4673 861.4815 0.1 323.0454 439.3907, 457.4193
17 815.4668 861.4817 0.15 323.0452 421.3840, 439.3967, 457.4080
18 813.4677 859.4739 0.1 421.3640, 439.3767, 457.4070
19 961.5323 1007.5366 0.2 205.9385 421.3975, 439.4145, 457.4258
20 959.5194 1005.5220 0.25 221.0431 421.3841, 439.3967, 457.4081
21 813.4591 859.4652 0.1 421.4140, 439.4235, 457.4046
22 959.5158 1005.5205 0.3 221.0446 421.3880, 439.4228
23 1093.5734 1139.5771 40 221.0995, 323.0662 405.4047, 423.4172, 441.4303, 325.1562, 295.1434
24 1093.5719 1139.5767 14 221.0676 405.3985, 423.4120, 441.4245, 325.1519, 295.1366
25 961.5409 1007.5460 1.5 221.0670 405.3973, 423.4204, 441.4223, 325.1495
26 1093.5705 1139.5761 10 191.0538, 221.0668 405.3913, 423.4036, 441.3970
27 961.5305 1007.5341 0. 1 221.0597 405.3926, 423.4051, 441.4172
28 1107.5870 1153.5909 0.5 205.0700, 221.0662 405.3926, 423.4051, 441.4132
29 961.5303 1007.5361 0.8 221.0663 405.3910, 423.4035, 441.4157, 325.1456
30 1107.5789 1153.5908 0.5 205.0684, 221.0661 405.3886, 423.4016, 441.4128, 309.1526
31 961.5294 1007.5278 0.3 221.0662 405.3874, 423.4002, 441.4121
32 961.5289 1007.5335 0.3 221.0650 405.3848, 423.3977, 441.4089
33 931.5210 977.5270 0.2 131.0349, 191.0570 405.3806, 423.3936, 441.4050
34 961.5297 1007.5350 0.4 221.0661 405.3794, 423.3909, 441.4028
35 961.5295 1007.5324 0.4 221.0663 405.3898, 423.3911, 441.4001
36 931.5179 977.5240 0.1 191.0561, 221.0598 405.3765, 423.3878, 441.3995
37 961.5277 1007.5328 0.75 221.0660 405.3738, 423.3857, 441.3973
38 1121.5639 1167.5655 10 221.0599 407.3630, 425.3740, 443.3849
39 959.1231 1005.1253 0.35 221.0664 407.3597, 425.3721, 443.3820
40 799.4673 845.4847 0.1 405.3730, 423.3859, 441.3968
41 945.5324 991.5386 0.2 145.0346, 205.1696 405.3655, 423.3810, 441.3920, 309.1347
42 1255.6201 1301.6254 10 131.0295, 191.0599, 221.0661 405.3766, 423.3890, 441.3995, 295.1264, 325.1313
43 1141.5875 1187.5921 3 221.0661 423.3764, 441.3898
44 979.5352 1025.5410 100 221.0665 423.3764, 441.3898
45 1123.5721 1169.5763 5 221.0660, 323.0961 405.3629, 423.3588
46 901.5177 947.5063 0.88 191.0660 405.3629, 423.3588, 441.3587
47 769.3977 815.4032 0.15 131.0231, 191.0623 405.3629, 423.3588
48 901.5008 947.5050 0.8 191.0601 405.3629, 423.3588, 441.3887
49 769.4713 815.4661 0.2 191.0600 405.3587, 423.3712, 441.3816
50 959.5032 1005.5082 0.4 221.0672 421.3554, 439.3660, 457.3768
51 1109.5496 1155.5567 2.5 221.0670 421.3541, 439.3654
52 769.4673 815.4610 0.15 191.0643 405.3584, 423.3694
53 961.5108 1007.5159 0.4 221.0667 405.3558 , 423.3683, 441.3798
54 1123.5610 1169.5643 2.5 221.0661 405.3558 , 423.3683, 441.3798
55 769.4412 815.4537 0.1 131.0329 405.3565, 423.3679, 441.3781
56 915.5043 961.5123 60 131.331, 205.0690 405.3565, 423.3679, 441.3781
57 1123.5456 1169.5495 4 221.0653 423.3781, 441.3878, 325.1177
58 1123.5427 1169.5475 4 221.0644, 323.0924 405.3552, 423.3661, 441.3768
59 1121.5226 1167.5242 5 221.0655, 323.0970 421.3487, 439.3613, 457.3717, 325.1176
60 1125.5476 1171.5509 4 221.0657, 323.0960 407.3671, 425.3739
61 1123.5306 1169.5326 4 221.0651 405.3552, 423.3649, 441.3748
62 797.4781 843.4276 0.1 421.3488, 439.3595, 457.3695
63 1123.5154 1169.5164 3 221.0645 405.3554, 423.3641, 441.3708
64 947.4713 993.4633 0.25 191.0598, 221.0661 421.3447, 439.3612, 457.3701
65 961.4723 1007.4753 0.3 221.0644 407.3661, 425.3773
66 781.4321 827.42225 0.1 405.3531, 423.3637, 441.3738
67 961.4687 1007.4704 0.2 221.0660 405.3523, 423.3623, 441.3754
68 961.4623 1007.4634 6 221.0664 405.3534, 423.3665, 441.3771
69 799.4215 845.4232 0.3 221.0612 405.3503, 423.3621, 441.3719
70 961.4620 1007.4617 0.2 221.0618 405.1917, 423.3524, 441.3701
71 961.4630 1007.4635 0.7 221.0660 405.3501, 423.3633, 441.3721
72 769.4185 815.4207 0.35 131.0304, 191.0624 405.3499, 423.3609, 441.3706
73 799.4331 845.4304 0.8 221.0588, 323.0889 405.3489, 423.3597, 441.3700, 325.1119
74 959.4537 1005.4554 0.22 221.0580 439.3546, 457.3632, 325.1057
75 1371.5895 1417.5705 100 131.0298, 221.0645, 293.0659, 323.0926 407.3640, 425.3740, 443.3839, 325.1109, 295.1006
76 901.4601 947.4621 0.8 191.0599 407.3629, 4253724, 443.3830
77 901.4614 947.4655 0.6 131.0334, 191.0621 407.3621, 4253733, 443.3843
78 783.4448 829.4493 0.1 145.0247, 205.0579 405.3472, 423.3587, 441.3686
79 1239.5732 1285.5715 15 221.0642, 293.0883, 323.1087 407.3631, 425.3733, 443.3838, 325.1097, 295.0995
80 637.3796 683.4054 0.1 405.3461, 423.3562, 441.3665
81 1269.5894 1315.5809 50 221.0468, 323.0734 407.3614, 425.3722, 443.3799
82 1239.5892 1315.5879 85 131.0342, 191.0660, 221.0661, 323.0979 407.3624, 425.3726, 443.3827
83 1105.5357 1151.5401 0.1 221.0664 405.3459, 423.3565, 441.3661
84 1105.5425 1151.5449 0.1 221.0596 405.3421, 423.3554, 441.3667
85 1239.2331 1285.2377 40 131.0121, 191.0366, 221.0663 407.3665, 425.3779, 443.3760
86 1107.5643 1153.5662 10 221.0488, 323.0729 407.3613, 425.3719, 443.3813
87 1107.5632 1153.5643 6.6 221.0599 407.3601, 425.3677, 443.3805
88 1077.5696 1123.5746 3.3 131.0257, 191.0466, 221.0540 407.3583, 425.3682, 443.3781
89 1077.5705 1123.5761 3.3 131.0265, 191.0453, 221.0536 407.3583, 425.3690, 443.3800
90 943.5121 989.5183 8 221.0662 405.3433, 423.3545, 441.3794
91 945.5351 991.5398 0.3 221.5863 407.3622, 425.3730, 443.3826, 325.1087
92 945.5458 991.5516 0.35 221.0664, 323.0721 407.3829, 425.3941, 443.4048, 325.1278
93 619.4279 665.4239 0.1 405.4064, 423.4219, 441.4347
94 619.4255 665.4245 0.1 405.4064, 423.4202, 441.4332
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2.2.4. Identification of Sugar Chains at C-20 by the [M − H]− to [M + COOH]− Peak Ratio

There was an interesting phenomenon in the negative MS spectra. The ratio of quasi−molecular ions {[M − H]− to [M + COOH]−} were related to the sugar chains at C-20. When there were more than one sugar located at C-20, the peak ratio of [M − H]− to [M + COOH]− was higher than 0.5, however, when there was only one sugar or none linked at C-20, the peak ratio would be lower than 0.5 (Figure 3A, Figure 4A and Figure 5A). These characteristics could be explained by the existence of the space effect.

Figure 3.

Figure 3

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The typical TOF-MS spectra and fragmentation pathways of compound 91. (A) MS spectrum in negative mode; (B) MS spectrum in positive mode; (C) MS/MS spectrum in negative mode.

Figure 4.

Figure 4

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The typical TOF-MS spectra and fragmentation pathways of compound 72. (A) MS spectrum in negative mode; (B) MS spectrum in positive mode; (C) MS/MS spectrum in negative mode.

Figure 5.

Figure 5

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The typical TOF-MS spectra and fragmentation pathways of compound 9. (A) MS spectrum in negative mode; (B) MS spectrum in positive mode; (C) MS/MS spectrum in negative mode.

2.3. Identification of Compounds in ZSYXST

2.3.1. Identification of PPD Type Saponins

In Figure 3A,B, the molecular weight of compound 91 could be deduced as 946 through the quasi−molecular ions at m/z 945.5351 [M − H]−, 991.5398 [M + COOH]− and 969.5266 [M + Na]+. The peak ratio of [M − H]− to [M + COOH]− was about 0.3, which indicated that the number of sugar moities at C-20 was less than one (Figure 3A). Furthermore, diagnostic ions at m/z 407, 443, 425 could be observed which indicated that the saponin should be PPD Type (Figure 1 and Figure 3B). In the MS/MS spectrum of [M − H]− (Figure 3C), fragment ions at m/z 783.4954, 621.4422 and 459.3887 could be deduced as three successive neutral losses of glucose.

From the diagnostic ions at m/z 459, the aglycone could be deduced to be PPD-A type (Figure 1 and Figure 2). Fragment ions at m/z 221 indicated there was a Glc-Glc chain linked to the aglycone. According to the above diagnostic ions and comparison of the retention time with a reference, 91 could unambiguously be identified as ginsenoside Rd. Similar fragment ions were observed in 92.

Compound 75 produced [M − H]− ions at m/z 1371.5895 and the peak ratio of [M − H]− to [M + COOH]− was 100 in negative mode. In MS/MS mode, diagnostic fragment ions of sugar neutral loss at m/z 1107.5948 [M − H − 132 − 132]−, 945.5704 [M − H − 132 − 132 − 162]−, 783.4893 [M − H − 132 − 132 − 162 − 162]−, 459.5118 [M − H − 132 − 132 − 162 − 162 − 162 − 162]− and Glc-Glc diagnostic fragment ions at m/z 221.0645, 293.0659, 323.0926 were observed. In positive mode, m/z 407.3640, 425.3740, 443.3839 indicate 75 belongs to the PPD type. According to the above MS cleavage rules and the literature [17], compound 75 was easily identified as notoginsenoside D.

Compound 88 generated [M − H]− and [M + COOH]− ions at m/z 1077.5696 and 1123.5746, the peak ratio of [M − H]− to [M + COOH]− was 3.3 in negative mode. PPD Type diagnostic ions at m/z 407.3583, 425.3682, 443.3781 were obtained in positive mode. According to negative MS/MS diagnostic fragment ions, such as m/z 945.4812 [M − H − 132]−, 783.4405 [M − H − 132 − 162]−, 621.4014 [M − H − 132 − 162 − 162]−, 459.3421 [M − H − 132 − 162 − 162 − 162]−, as well as literature data [12], 88 was identified as notoginsenoside L. Similar fragment ions were observed in compound 89.

In the positive MS spectrum, compound 60 produced ions at m/z 407.3671, 425.3739 which could be used to identify it as PPD type. In the negative MS spectrum, the peak ratio of [M − H]− (m/z 1125.5476) and [M + COOH]− (m/z 1171.5509) was about 4, which indicated that there was more than one sugar linked at C-20. In the MS/MS spectrum, characteristic fragment ions at m/z 963.5411 [M − H − 162]−, 801.4900 [M − H − 162 − 162]−, 635.4389 [M − H − 162 − 162 − 162]−, 477.3889 [M − H − 162 − 162 − 162 − 162]−, Glc-Glc diagnostic fragment ions at m/z 221.0657, 323.0960, and PPD-B type characteristic fragment ions at m/z 477.3889 were all observed. According to the MS fragment rules, 60 was tentatively identified as PPD-B-S1(Glc-Glc)-S2(Glc-Glc).

Similarly, with the help of MS cleavage rules, reference compounds and literature data, another eight PPD type saponins were identified. Thus compounds 5, 38, 39, 79, 81, 82, 85, 86, 87 were identified or tentatively identified as ginsenoside C-Y1, quinquenoside IV, notoginsenoside G, chikusetsusaponin VI, ginsenoside Ra0/quinquenoside V, notoginsenoside Fa, ginsenoside Ra3, ginsenoside Rb1, isomer of ginsenoside Rb1, respectively (Table 1 and Table 2).

Table 2.

Identification of compounds in ZSYXST.

Comp. Rt (min) MS/MS m/z Area% Identification
1 7.54 965.5198 785.9465 [M − H − 180]−, 653.3969 [M − H − 180 − 132]−, 491.3843 [M − H − 180 − 132 − 162]−, 415.3350 0.019 OCO-A-S1(Glc)-S2(Glc-Xyl/Ara) *
2 8.611 965.5237 785.9489 [M − H − 180]−, 653.3794 [M − H − 180 − 132]−, 491.3654 [M − H − 180 − 132 − 162]−, 415.3229 0.006 isomer of OCO-A-S1(Glc)-S2(Glc-Xyl/Ara) *
3 8.811 979.5387 799.4633 [M − H − 180]−, 635.3669 [M − H − 180 − 146]−, 491.3370 [M − H − 180 − 146 − 162]−, 415.3301 0.016 OCO-A-S1(Glc)-S2(Glc-Rha) [14]
4 10.189 979.5407 799.4532 [M − H − 180]−, 635.3868 [M − H − 180 − 146]−, 491.3569 [M − H − 180 − 146 − 162]−, 415.3277 0.006 isomer of OCO-A-S1(Glc)-S2(Glc-Rha) [14]
5 12.992 801.4635 639.4097 [M − H − 162]−, 477.3605 [M − H − 162 − 162]− 0.006 ginsenoside C-Y1 [22]
6 13.699 815.4807 653.4212 [M − H − 162]−, 491.3770 [M − H − 162 − 162]−, 391.2872 0.032 OCO-B-S1(Glc-Glc)-S2(OH) [23]
7 13.852 947.5170 785.4339 [M − H − 162]−, 623.4062 [M − H − 162 − 162]−, 491.4131 [M − H − 162 − 162 − 132]−, 415.3310 0.032 vinaginsenoside R5 or yesanchinoside C [24,25]
8 14.151 815.4801 653.4276 [M − H − 162]−, 491.3788 [M − H − 162 − 162]−, 391.2878 0.032 isomer of OCO-B-S1(Glc-Glc)-S2(OH)
9 14.252 861.4892 653.4263 [M − H − 162]−, 491.3777 [M − H − 162 − 162]−, 415.3253 0.010 majonoside R1 [12,19]
10 14.582 815.4814 653.4162 [M − H − 162]−, 491.3716 [M − H − 162 − 162]−, 415.3533 0.019 isomer of majonoside R1 [12,19]
11 15.03 815.4701 653.4162 [M − H − 162]−, 491.3716 [M − H − 162 − 162]−, 415.3519 0.151 isomer of majonoside R1 [12,19]
12 15.948 817.4872 655.4462 [M − H − 162]−,493.3934 [M − H − 162 − 162]− 0.048 PPT-C-S1(Glc-Glc)-S2(H) [26]
13 16.431 799.4312 799.4678 [M − H − 162]−, 637.4793 [M − H − 162 − 162]−, 475.3866 [M − H − 162 − 162 − 162]−, 391.2904 0.013 ginsenoside Rf [27]
14 16.714 961.5332 799.4679 [M − H − 162]−, 653.4002 [M − H − 162 − 146]−, 491.3304 [M − H − 162 − 146 − 162]−, 391.2772 0.032 OCO-B-S1(Glc-Rha-Glc)-S2(CH3) [26]
15 17.597 961.5328 799.5297 [M − H − 162]−, 653.4002 [M − H − 162 − 146]−, 491.3693 [M − H − 162 − 146 − 162]−, 391.8943 0.032 isomer of OCO-B-S1(Glc-Rha-Glc)-S2(CH3)
16 18.398 815.4868 653.4263 [M − H − 162]−, 491.3777 [M − H − 162 − 162]−, 415.3361 0.515 isomer of majonoside R1 [12,19]
17 18.092 815.4673 653.4373 [M − H − 162]−, 491.3788 [M − H − 162 − 162]−, 415.3415 0.032 isomer of majonoside R1 [12,19]
18 19.481 859.4723 651.4111 [M − H − 162]−, 489.3579 [M − H − 162 − 162]− 0.019 OCO-E-S1(Glc-Glc) *
19 20.247 961.5323 799.4824 [M − H − 162]−, 653.4002 [M − H − 162 − 146]−, 491.3558 [M − H − 162 − 146 − 162]−, 391.3441 0.016 isomer of OCO-B-S1(Glc-Rha-Glc)-S2(CH3) [21]
20 21.142 959.5194 797.4207 [M − H − 162]−, 635.3567 [M − H − 162 − 162]−, 473.3460 [M − H − 162 − 162 − 162]− 0.016 OCO-D-S1(Glc-Glc-Glc) *
21 22.39 859.4721 651.4113 [M − H − 162]−, 489.3635 [M − H − 162 − 162]− 0.023 isomer of OCO-E-S1(Glc-Glc) *
22 23.909 959.5158 797.4575 [M − H − 162]−, 635.4076 [M − H − 162 − 162]−, 473.3611 [M − H − 162 − 162 − 162]− 0.016 isomer of OCO-D-S1(Glc-Glc-Glc) *
23 26.936 1093.5734 961.5389 [M − H − 132]−, 637.4333 [M − H − 132 − 162 − 162]−, 475.3832 [M − H − 132 − 162 − 162 − 162]−, 391.2872 0.093 sanchirhinoside A6
24 28.314 1093.5719 961.5374 [M − H − 132]−, 637. 4382 [M − H − 132 − 162 − 162]−, 475.3753 [M − H − 132 − 162 − 162 − 162]−, 391.2883 0.064 isomer of sanchirhinoside A6 [19,28]
25 28.962 961.5374 799.4883 [M − H − 162]−, 637.4389 [M − H − 162 − 162]−, 475.3826 [M − H − 162 − 162 − 162]−, 391.2872 0.328 notoginsenoside R3
26 29.751 1093.5705 961.5272 [M − H − 132]−, 637.4367 [M − H − 132 − 162 − 162]−, 475.3753 [M − H − 132 − 162 − 162 − 162]−, 391.2879 0.028 isomer of sanchirhinoside A6 [19,28]
27 30.175 961.5305 799.4817 [M − H − 162]−, 637.4185 [M − H − 162 − 162]−, 475.3757 [M − H − 162 − 162 − 162]−, 391.2901 0.187 notoginsenoside N
28 30.175 1107.5870 961.5311 [M − H − 146]−, 799.4749 [M − H − 146 − 162]−, 637.4310 [M − H − 146 − 162 − 162]−, 475.3794 [M − H − 146 − 162 − 162 − 162]−, 391.2892 − yesanchinoside E [17]
29 30.599 961.5303 637.4328 [M − H − 162 − 162]−, 475.3781 [M − H − 162 − 162 − 162]−, 391.2912 0.170 notoginsenoside R6
30 30.999 1107.5789 961.5299 [M − H − 146]−, 799.4747 [M − H − 146 − 162]−, 637.4224 [M − H − 146 − 162 − 162]−, 475.3760 [M − H − 146 − 162 − 162 − 162]−, 391.2902 0.026 isomer of yesanchinoside E [17]
31 31.788 961.5294 799.4784 [M − H − 162]−, 637.4286 [M − H − 162 − 162]−, 475.3782 [M − H − 162 − 162 − 162]−, 391.2899 0.715 20-O-glucoginsenoside Rf
32 32.259 961.5289 799.4824 [M − H − 162]−, 637.4403 [M − H − 162 − 162]−, 475.3832 [M − H − 162 − 162 − 162]−, 391.2876 0.052 isomer of 20-O-glucoginsenoside Rf
33 33.661 931.5210 637.4369 [M − H − 132 − 162]−, 475.3824 [M − H − 132 − 162 − 162]−,391.3270 13.39 notoginsenoside R1
34 34.296 961.5297 799.4803 [M − H − 162]−, 637.4268 [M − H − 162 − 162]−, 475.3785 [M − H − 162 − 162 − 162]−, 391.2913 0.151 notoginsenoside M or N [19,21]
35 34.991 961.5295 799.4811 [M − H − 162]−, 637.4194 [M − H − 162 − 162]−, 475.3634 [M − H − 162 − 162 − 162]−, 391.2920 0.067 isomer of notoginsenoside M or N [19,21]
36 35.392 931.5179 637.4281 [M − H − 132 − 162]−, 475.3762 [M − H − 132 − 162 − 162]−, 391.2901 0.138 gypenoside LXIV [29]
37 35.957 961.5277 799.4883 [M − H − 162]−, 637.4255 [M − H − 162 − 162]−, 475.3787 [M − H − 162 − 162 − 162]−, 391.2877 0.041 isomer of notoginsenoside R3 or isomer of notoginsenoside R6 [17,19]
38 36.31 1121.5639 959.3973 [M − H − 162]−, 797.4040 [M − H − 162 − 162]−, 473.3085 [M − H − 162 − 162 − 162 − 162]− 0.229 quinquenoside IV [27]
39 36.31 959.1231 797.3990 [M − H − 162]−, 635.4110 [M − H − 162 − 162]−, 473.3675 [M − H − 162 − 162 − 162]− − notoginsenoside G
40 36.781 799.4673 637.4352 [M − H − 162]−, 475.3815 [M − H − 162 − 162]−, 391.2930 27.59 ginsenoside Rg1 [27]
41 37.3 945.5324 783.4775 [M − H − 162]−, 637.4309 [M − H − 162 − 146]−, 475.3791 [M − H − 162 − 146 − 162]−, 391.3001 7.670 ginsenoside Re
42 37.676 1255.6201 1123.1027 [M − H − 132]−, 961.2952 [M − H − 132 − 162]−, 799.3770 [M − H − 132 − 162 − 162]−, 637.4266 [M − H − 132 − 162 − 162 − 162]−, 475.3701 [M − H − 132 − 162 − 162 − 162 − 162]−, 391.2900 0.032 PPT-A-S1(Glc-Glc-Xyl/Ara)-S2(Glc-Glc) [21]
43 39.349 1141.5875 817.4870 [M − H − 162 − 162]−, 655.4351 [M − H − 162 − 162 − 162]−, 493.3991 [M − H − 162 − 162 − 162 − 162]− 0.222 quinquenoside L16 [18]
44 39.349 979.5352 817.4912 [M − H − 162]−, 655.4429 [M − H − 162 − 162]−, 493.4046 [M − H − 162 − 162 − 162]− − PPT-C-S1(Glc)-S2(Glc-Glc) [30]
45 41.41 1123.5721 961.5210 [M − H − 162]−, 799.4757 [M − H − 162 − 162]−, 637.4238 [M − H − 162 − 162 − 162]−, 475.3770 [M − H − 162 − 162 − 162 − 162]−, 391.2870 0.248 PPT-A-S1(Glc-Glc)-S2(Glc-Glc) [31]
46 41.41 901.5177 637.4241 [M − H − 132 − 132]−, 475.3827 [M − H − 132 − 132 − 162]−, 391.2881 − chikusetsusaponin L5 [27]
47 41.41 769.3977 637.3997 [M − H − 162]−, 475.3876 [M − H − 162 − 132]−, 391.2851 − pseudoginsenoside Rt3
48 42.717 901.5008 637.4230 [M − H − 132 − 132]−, 475.3744 [M − H − 132 − 132 − 162]−, 391.2901 0.077 isomer of chikusetsusaponin L5 [27]
49 43.223 769.4713 637.4342 [M − H − 132]−, 475.3828 [M − H − 132 − 162]−, 391.2873 0.090 notoginsenoside R2 [19]
50 43.8 959.5032 797.4575 [M − H − 162]−, 635.4076 [M − H − 162 − 162]−, 473.3611 [M − H − 162 − 162 − 162]− 0.145 isomer of OCO-D-S1(Glc-Glc-Glc)*
51 44.377 1109.5496 785.4931 [M − H − 162 − 162]−, 623.4047 [M − H − 162 − 162 − 162]−, 491.3606 [M − H − 162 − 162 − 162 − 132]−, 391.8654 0.032 OCO-B-S1(Xyl-Glc-Glc-Glc)-S2(OH)*
52 44.79 769.4673 637.4342 [M − H − 132]−, 475.3828 [M − H − 132 − 162]−, 391.2890 0.045 isomer of notoginsenoside R2 [19]
53 45.108 961.5108 799.4836 [M − H − 162]−, 637.4239 [M − H − 162 − 162]−, 475.3783 [M − H − 162 − 162 − 162]−, 391.2903 0.174 isomer of 20-O-glucoginsenoside Rf [32,33]
54 45.108 1123.5610 961.5215 [M − H − 162]−, 799.4657 [M − H − 162 − 162]−, 637.4338 [M − H − 162 − 162 − 162]−, 475.3770 [M − H − 162 − 162 − 162 − 162]−, 391.2891 − isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) [31]
55 46.344 769.4412 637.4341 [M − H − 132]−,475.3807 [M − H − 132 − 162]−,391.2891 0.196 sanchirhinoside A3 [19]
56 46.344 915.5043 783.4801 [M − H − 132]−, 637.4801 [M − H − 132 − 146]−, 475.3756 [M − H − 132 − 146 − 162]−, 391.3001 − PPT-A-S1(Rha-Xyl)-S2(Glc) [31]
57 46.909 1123.5456 961.5244 [M − H − 162]−, 799.4729 [M − H − 162 − 162]−, 637.4283 [M − H − 162 − 162 − 162]−, 475.3721 [M − H − 162 − 162 − 162 − 162]−, 391.2911 0.051 isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) [31]
58 47.169 1123.5427 961.5227 [M − H − 162]−, 799.4758 [M − H − 162 − 162]−, 637.4274 [M − H − 162 − 162 − 162]−, 475.3728 [M − H − 162 − 162 − 162 − 162]−, 391.2893 0.012 isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) [31]
59 47.84 1121.5226 959.5057 [M − H − 162]−, 797.4549 [M − H − 162 − 162]−, 635.4088 [M − H − 162 − 162 − 162]−, 473.3567 [M − H − 162 − 162 − 162 − 162]− 0.077 OCO-D-S1(Glc-Glc-Glc-Glc) *
60 48.17 1125.5476 963.5411 [M − H − 162]−, 801.4900 [M − H − 162 − 162]−, 635.4389 [M − H − 162 − 162 − 162]−, 477.3889 [M − H − 162 − 162 − 162 − 162]− 0.066 PPD-B-S1(Glc-Glc)-S2(Glc-Glc) [34]
61 48.594 1123.5306 961.5171 [M − H − 162]−, 799.4745 [M − H − 162 − 162]−, 637.4232 [M − H − 162 − 162 − 162]−, 475.3754 [M − H − 162 − 162 − 162 − 162]−, 391.2891 0.035 isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) [31]
62 48.864 797.4781 635.4190 [M − H − 162]−, 473.3695 [M − H − 162 − 162]− 0.039 PPT-d-S1(Glc-Glc) [21]
63 49.63 1123.5254 961.5203 [M − H − 162]−, 799.4698 [M − H − 162 − 162]−, 637.4431 [M − H − 162 − 162 − 162]−, 475.3701 [M − H − 162 − 162 − 162 − 162]−, 391.2973 0.097 isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) [31]
64 49.63 947.4713 815.3567 [M − H − 132]−, 653.3778 [M − H − 132 − 162]−, 491.3608 [M − H − 132 − 162 − 162]−, 391.8554 − OCO-B-S1(Glc-Glc-Xyl/Ara)-S2(OH) [35]
65 50.101 961.4723 799.4722 [M − H − 162]−, 637.4220 [M − H − 162 − 162]−, 475.3720 [M − H − 162 − 162 − 162]− 0.1160 vina−ginsenoside R4
66 50.254 781.4321 619.3533 [M − H − 162]−, 457.3661 [M − H − 162 − 162]− 0.032 sanchirhinoside B
67 50.619 961.4687 799.4598 [M − H − 162]−, 637.4037 [M − H − 162 − 162]−, 475.3770 [M − H − 162 − 162 − 162]−, 391.2866 0.058 isomer of 20-O-glucoginsenoside Rf [32,33]
68 51.762 961.4623 781.4714 [M − H − 180]−, 637.4232 [M − H − 180 − 144]−, 475.3801 [M − H − 180 − 144 − 162]−, 391.2909 0.019 isomer of notoginsenoside R3 or isomer of notoginsenoside R6 [17,19]
69 52.221 799.4215 637.4209 [M − H − 162]−, 475.3722 [M − H − 162 − 162]−, 391.2877 0.068 isomer of ginsenoside Rg1 [36,37]
70 53.552 961.4620 799.4434 [M − H − 162]−, 637.4263 [M − H − 162 − 162]−, 475.4024 [M − H − 162 − 162 − 162]−, 391.2907 0.039 isomer of 20-O-glucoginsenoside Rf [32,33]
71 54.376 961.4630 799.4695 [M − H − 162]−, 637.4074 [M − H − 162 − 162]−, 475.3638 [M − H − 162 − 162 − 162]−, 391.2881 0.019 isomer of notoginsenoside R3 or isomer of notoginsenoside R6 [17,19]
72 55.185 769.4795 637.4336 [M − H − 132]−, 475.3814 [M − H − 132 − 162]−, 391.2869 1.572 isomer of notoginsenoside R2
73 55.813 799.4331 637.4356 [M − H − 162]−, 475.4315 [M − H − 162 − 162]−, 391.2872 0.206 notoginsenoside U
74 56.461 959.4537 797.4608 [M − H − 162]−, 635.4138 [M − H − 162 − 162]−, 473.3590 [M − H − 162 − 162 − 162]− 0.026 ginsenoside III or vinaginsenoside R20 [38]
75 56.767 1371.5895 1107.5948 [M − H − 132 − 132]−, 945.5704 [M − H − 132 − 132 − 162]−, 783.4893 [M − H − 132 − 132 − 162 − 162]−, 459.5118 [M − H − 132 − 132 − 162 − 162 − 162 − 162]− 0.296 notoginsenoside D [17]
76 57.992 901.4601 769.4200 [M − H − 132]−, 637.4355 [M − H − 132 − 132]−, 475.4321 [M − H − 132 − 132 − 162]− 0.171 chikusetsusaponin L5 [39]
77 58.392 901.4614 769.4231 [M − H − 132]−, 637.4352 [M − H − 132 − 132]−, 475.4335 [M − H − 132 − 132 − 162]− 0.602 notoginsenoside Rw1 [21]
78 58.569 783.4448 621.3941 [M − H − 146]−, 475.3495 [M − H − 146 − 162]−, 391.2896 0.754 ginsenoside Rg2
79 59.146 1239.5732 1107.6034 [M − H − 132]−, 945.5396 [M − H − 132 − 162]−, 783.5018 [M − H − 132 − 162 − 162]−, 621.4381 [M − H − 132 − 162 − 162 − 162]−, 459.5110 [M − H − 132 − 162 − 162 − 162 − 162]− 2.087 chikusetsusaponinVI [40]
80 60.406 637.3796 475.3437 [M − H − 162]−,391.2901 0.103 ginsenoside F1 [27]
81 60.818 1269.5894 1107.5013 [M − H − 162]−, 945.4587 [M − H − 162 − 162]−, 783.4201 [M − H − 162 − 162 − 162]−, 621.3833 [M − H − 162 − 162 − 162 − 162]−, 459.5120 [M − H − 162 − 162 − 162 − 162 − 162]− 0.045 ginsenoside Ra0 or quinquenoside V [19,21]
82 61.572 1239.5892 1107.5974 [M − H − 132]−, 945.5267 [M − H − 132 − 162]−, 783.4965 [M − H − 132 − 162 − 162]−, 621.4475 [M − H − 132 − 162 − 162 − 162]−, 459.5121 [M − H − 132 − 162 − 162 − 162 − 162]− 2.622 notoginsenoside Fa
83 62.102 1105.5357 943.5543 [M − H − 162]−, 781.4996 [M − H − 162 − 162]−, 619.3441 [M − H − 162 − 162 − 162]−, 457.3654 [M − H − 162 − 162 − 162 − 162]− 0.941 PPT-B-S1(Glc-Glc-Glc-Glc) [38]
84 63.562 1105.5425 943.5521 [M − H − 162]−, 781.5010 [M − H − 162 − 162]−, 619.3501 [M − H − 162 − 162 − 162]−, 457.2601 [M − H − 162 − 162 − 162 − 162]− 0.161 isomer of PPT-B-S1(Glc-Glc-Glc-Glc) [38]
85 63.562 1239.2331 1077.4231 [M − H − 162]−, 915.4635 [M − H − 162 − 162]−, 621.3862 [M − H − 162 − 162 − 294]−, 459.3452 [M − H − 162 − 162 − 294 − 162]− 0.019 ginsenoside Ra3 [39]
86 64.198 1107.5642 945.4624 [M − H − 162]−, 783.4239 [M − H − 162 − 162]−, 621.3852 [M − H − 162 − 162 − 162]−, 459.3475 [M − H − 162 − 162 − 162 − 162]− 32.21 ginsenoside Rb1
87 66.483 1107.5632 945.4631 [M − H − 162]−, 783.4197 [M − H − 162 − 162]−, 621.3800 [M − H − 162 − 162 − 162]−, 459.3452 [M − H − 162 − 162 − 162 − 162]− 0.019 isomer of ginsenoside Rb1 [12,40]
88 68.791 1077.5696 945.4812 [M − H − 132]−, 783.4405 [M − H − 132 − 162]−, 621.4014 [M − H − 132 − 162 − 162]−, 459.3421 [M − H − 132 − 162 − 162 − 162]− 0.110 notoginsenoside L [12,27]
89 69.639 1077.5705 945.4888 [M − H − 132]−, 783.4465 [M − H − 132 − 162]−, 621.4008 [M − H − 132 − 162 − 162]−, 459.3544 [M − H − 132 − 162 − 162 − 162]− 0.080 isomer of notoginsenoside L [12,27]
90 70.487 943.5121 781.4300 [M − H − 162]−, 619.3881 [M − H − 162 − 162]−,457.3656 [M − H − 162 − 162 − 162]− 0.058 PPT-B-S1(Glc-Glc-Glc) *
91 73.384 945.5495 783.4954 [M − H − 162]−, 621.4422 [M − H − 162 − 162]−,459.3887 [M − H − 162 − 162 − 162]− 3.134 ginsenoside Rd
92 77.824 945.5520 783.4964 [M − H − 162]−, 621.4415 [M − H − 162 − 162]−,459.3887 [M − H − 162 − 162 − 162]− 0.370 isomer of ginsenoside Rd [9,12]
93 83.312 619.4279 457.3652 [M − H − 162]− 0.019 ginsenoside Rk3 [41,42]
94 85.608 619.4255 457.3686 [M − H − 162]− 0.067 ginsenoside Rh4 [41,42]
Open in a new tab

*: new compounds.

2.3.2. Identification of PPT Type Saponins

According to the quasi−molecular ions at m/z 769.4185 [M − H]−, 815.4207 [M + COOH]− and 793.4671 [M + Na] + , the molecular weight of compound 72 should be 770 (Figure 4A,B). The peak ratio of [M − H]− to [M + COOH]− was about 0.35, which indicated that there was less than one sugar linked at C-20 (Figure 4A). Furthermore, the aglycone type could be identified as PPT Type through diagnostic ions at m/z 405, 441, 423 (Figure 1 and Figure 4B). In the MS/MS spectrum of [M − H]− (Figure 4C), fragment ions at m/z 637.4336, 475.3814 could be deduced to represent neutral losses of xylose and glucose successively or simultaneously. Fragment ions at m/z 191 indicated there was a Glc-Xyl chain in 72. Characteristic ions at m/z 391 and 475 indicated the aglycone should be PPT-A type (Figure 1 and Figure 4C). According to the peak ratio rule of [M − H]− to [M + COOH]−, we could deduce that the Glc-Xyl chain should be linked at the C-6 position of the aglycone. As a result, compound 72 could be identified as notoginsenoside R2.

Compound 23 produced [M − H]− ions at m/z 1093.5734 in negative mode, while the peak ratio of [M − H]− to [M + COOH]− was 40. In MS/MS mode, characteristic fragment ions at m/z 961.5389 [M − H − 132]−, 637.4333 [M − H − 132 − 162 − 162]−, 475.3832 [M − H − 132 − 162 − 162 − 162]−, 391.2772 and Glc-Glc diagnostic fragment ions at m/z 221.0995, 323.0662 were obtained. In positive mode, it produced ions at m/z 405.4047, 423.4172, 441.4303 which could be used to identify it as PPT-A type. According to the retention time and MS fragment rules, 23 was unambiguously identified as sanchirhinoside A6. Similar diagnostic fragment ions were observed in compounds 24 and 26.

[M − H]− (m/z 961.5409) and [M + COOH]− (m/z 1007.5460) peaks of compound 25 were observed in the negative MS spectrum, and the peak ratio of [M − H]− to [M + COOH]− was 1.5, which indicated more than one sugar was located at the C-20 position. Diagnostic fragment ions at m/z 799.4883 [M − H − 162]−, 637.4389 [M − H − 162 − 162]−, 475.3826 [M − H − 162 − 162 − 162]−, 391.2872 as well as m/z 405.3973, 423.4204, 441.4223 were observed in negative and positive mode, respectively. As a result, compound 25 was identified as notoginsenoside R3, and compounds 37 and 71 were identified as isomers of 25 because similar fragment ions were observed in their spectra.

Compound 90 produced [M − H]− ions at m/z 943.5121 and [M + COOH]− ions at m/z 989.5183 in negative mode, while the peak ratio of [M − H]− to [M + COOH]− was 8, which indicated there are two or more sugars at the C-20 position. Diagnostic ions of PPT type were observed at m/z 405.3433, 423.3545, 441.3794, and diagnostic ions of PPT-A type were observed at m/z 457.3656. In the MS/MS spectrum, characteristic fragment ions at m/z 781.4300 [M − H − 162]−, 619.3881 [M − H − 162 − 162]−, 457.3656 [M − H − 162 − 162]− and Glc-Glc ions at m/z 221.0662 were obtained. Based on the MS cleavage rules (Table 1 and Table 2), literature data and a SciFinder database search, compound 90 was tentatively identified as PPT-B-S1(Glc-Glc-Glc).

The molecular weight of compound 83 could be supposed to 1106 through negative ions at m/z 1105.5357 [M − H]— and 1151.5401 [M + COOH]− (Table 1 and Table 2). There must be less than one sugar at C-20 position according to the peak ratio of [M − H]− to [M + COOH]−(0.1). Furthermore, diagnostic ions of PPT type were observed at m/z 405.3459, 423.3565, 441.3661. In the MS/MS spectrum, fragment ions at m/z 943.5543 [M − H − 162]−, 781.4996 [M − H − 162 − 162]−, 619.3441 [M − H − 162 − 162 − 162]−, 457.3654 [M − H − 162 − 162 − 162 − 162]− could be deduced to represent neutral losses of four glucose moieties successively from the precursor ions. Glc-Glc diagnostic ions at m/z 221.0664 could be detected. Characteristic ions at m/z 457 indicated the aglycone type of 83 should be PPT-B (Figure 1). According to the above elucidation and literature data, 83 could be tentatively identified as PPT-B-S1(Glc-Glc-Glc-Glc). Compound 84 with a similar fragmentation behavior could be tentatively identified as an isomer of 83 (Table 1 and Table 2).

Compound 12 produced [M − H]− ions at m/z 817.4872 and [M + COOH]− ions at m/z 863.4982 in the negative spectrum, while the peak ratio of [M − H]− to [M + COOH]− was 0.8, which indicated there is more than one sugar at C-20. In MS/MS mode, characteristic fragment ions at m/z 655.4462 [M − H − 162]−, 493.3934 [M − H − 162 − 162]− and 323. 2112 [Glc-Glc]− were obtained. In positive mode, it produced ions at m/z 423.3736 and 441.3848, which could be used to identify it as a PPT type, and in MS/MS mode, PPT-C type characteristic fragment ions at m/z 493.3934 was observed. According to retention time and MS fragment rules, compound 12 was tentatively identified as PPT-C-S1(Glc-Glc)-S2(H).

By utilizing MS cleavage rules, as well as comparison of the retention time to references, compounds 27, 29, 31, 33, 40, 41, 47, 55, 66, 73, 78, 93 and 94 were unambiguously identified as notoginsenoside N, notoginsenoside R6, 20-O-glucoginsenoside Rf, notoginsenoside R1, ginsenoside Rg1,ginsenoside Re, pseudoginsenoside Rt3, sanchirhinoside A3, sanchirhinoside B, notoginsenoside U, ginsenoside Rg2, ginsenoside Rk3, and ginsenoside Rh4, respectively (Table 1 and Table 2).

Similarly, by utilizing MS cleavage rules and literature data, another 27 PPT type saponins were identified or tentatively identified as ginsenoside Rf (13), yesanchinoside E (28), an isomer of yesanchinoside E (30), an isomer of 20-O-glucoginsenoside Rf (32), notoginsenoside M or N (34), an isomer of notoginsenoside M or N (35), gypenoside LXIV (36), an isomer of notoginsenoside R3 or notoginsenoside R6 (37), PPT-A-S1(Glc-Glc-Xyl/Ara)-S2(Glc-Glc) (42), quinquenoside L16 (43), PPT-C-S1(Glc)-S2(Glc-Glc) (44), PPT-A-S1(Glc-Glc)-S2(Glc-Glc) (45), chikusetsusaponin L5 (46), an isomer of chikusetsusaponin L5 (48), an isomer of notoginsenoside R2 (49), an isomer of notoginsenoside R2 (52), an isomer of 20-O-glucoginsenoside Rf (53), an isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) (54), PPT-A-S1(Rha-Xyl)-S2(Glc) (56), an isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) (57), an isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) (58), an isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) (61), an isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) (63), an isomer of 20-O-glucoginsenoside Rf (67), an isomer of notoginsenoside R3 or an isomer of notoginsenoside R6 (68), ginsenoside Rg1 (69), an isomer of 20-O-glucoginsenoside Rf (70), an isomer of notoginsenoside R3 or an isomer of notoginsenoside R6 (71), an isomer of notoginsenoside R2(72) and ginsenoside F1 (78), respectively (Table 1 and Table 2).

2.3.3. Identification of OCO Type Saponins

The molecular weight of compound 9 could be supposed to 816 through negative ions at m/z 861.4817 [M + COOH]− and 815.4669 [M − H]− (Figure 5A,B). There must be less than one sugar moiety at the C-20 position according to the peak ratio of [M − H]− to [M + COOH]−(0.02). Furthermore, diagnostic ions of OCO type was observed at m/z 421, 439, 457 (Figure 1 and Figure 5B). In the MS/MS spectrum of [M + HCOO]− (Figure 5C), fragment ions at m/z 653.4263, 491.3777 could be deduced to correspond to neutral losses of two glucoses successively from the precursor ions. Diagnostic ions at m/z 323 could be used for confirming a Glc-Glc chain located at the C-6 position. Characteristic ions at m/z 415 and 491 indicated the aglycone type of 9 should be OCO-C (Figure 1 and Figure 5C). According to the above elucidation and literature data, 9 could be tentatively identified as majonoside R1 [26,43]. Compounds 10, 11, 16, 17 had similar fragmentation behaviors at m/z 653.4263 [M − H − 162]−, 491.3777 [M − H − 162 − 162]−, 415.3253. Glc-Glc diagnostic ions at m/z 323 and diagnostic ions of OCO type was also observed at m/z 421, 439, 457, so compounds 10, 11, 16, 17 were tentatively identified as isomers of majonoside R1 since similar characteristic fragment ions were obtained (Table 1 and Table 2).

[M − H]− (m/z 947.4713) and [M + COOH]− (m/z 993.4633) of compound 64 were observed in the negative MS spectrum, and the peak ratio of [M − H]− to [M + COOH]− was 0.25. Diagnostic fragment ions at m/z 815.3567 [M − H − 132]−, 653.3778 [M − H − 132 − 162]−, 491.3608 [M − H − 132 − 162 − 162]−, 391.8554, as well as m/z 421.3447, 439.3612, 457.3701 were observed in negative and positive mode, respectively. As a result, 64 was identified as OCO-B-S1(Glc-Glc-Xyl/Ara)-S2(OH).

The molecular weight of compound 1 could be deduced as 966 according to quasi−molecular ions at m/z 965.5198 [M − H]− and 1011.5278 [M + COOH]−. The peak ratio of [M − H]− to [M + COOH]− was about 0.1, which indicated that there was one or less sugar at C-20. The aglycone type could be identified as OCO type through diagnostic ions at m/z 439, 457. In the MS/MS spectrum of [M − H]−, fragment ions at m/z 785.9465 [M − H − 180]−, 653.3969 [M − H − 180 − 132]−, 491.3843 [M − H − 180 − 132 − 162]−, 415.3350 and fragment ions at m/z 191 indicated there was a Glc-Xyl/Ara chain in compound 1. Characteristic ions at m/z 415 and 491 indicated the aglycone would be OCO-A type. According to the peak ratio rule of [M − H]− to [M + COOH]− and the structural features of OCO-A, we could deduce that the Glc-Xyl chain was linked at the C-6 position of the aglycone. As a result, compound 1 was tentatively identified as OCO-A-S1(Glc)-S2(Glc-Xyl/Ara), compound 2 was identified as an isomer of 1 because a similar MS cleavage pathway was observed.

Compound 18 could be supposed to have a molecular weight of 814 through negative ions at m/z 813.4677 [M − H]− and 859.4739 [M + COOH]− (Figure 6). There must be less than one sugar at the C-20 position according to the peak ratio (0.1) of [M − H]− to [M + COOH]−. Furthermore, diagnostic ions of OCO type was observed at m/z 421, 439, 457 (Figure 1). In the MS/MS spectrum, fragment ions at m/z 651.4111 [M − H − 162]− and 489.3579 [M − H − 162 − 162]− could be deduced as successive neutral losses of two glucose molecules (Figure 6). Characteristic ions at m/z 489 indicated the aglycone type should be OCO-E. According to the above elucidation and literature data, 18 could be tentatively identified as OCO-E-S1(Glc-Glc). Compound 21 with similar fragmentation behaviors at m/z 651.4113 [M − H − 162]−, 489.3635 [M − H − 162 − 162]−, was tentatively identified as an isomer of OCO-E-S1(Glc-Glc).

Figure 6.

Figure 6

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The typical TOF-MS spectra of compounds 18 (A); 25 (B); 92 (C) in negative mode (1: MS spectra; 2: MS/MS spectra).

In the negative MS spectrum of compound 20, [M − H]− ions at m/z 959.5194 and [M + COOH]− at 1005.5220 as well as their peak ratio (0.25) were observed. In the MS/MS spectrum, characteristic fragment ions at m/z 797.4207 [M − H − 162]−, 635.3567 [M − H − 162 − 162]−, 473.3460 [M − H − 162 − 162 − 162]− and 221.0431 [Glc-Glc]− were also observed. In the positive spectrum it produced ions at m/z 421, 439, 457 which could be used to identify an OCO type compound. In MS/MS mode, OCO-D type fragment ions at m/z 473 could be obtained. According to the MS fragment rules, 20 was tentatively identified as OCO-D-S1(Glc-Glc-Glc). Similar diagnostic fragment ions were observed in 22 (Table 1 and Table 2).

Similarly, with the help of MS cleavage rules and literature values, compounds 7, 14, 15, 19, 51 were tentatively identified as vinaginsenoside R5 or yesanchinoside C, OCO-B-S1(Glc-Rha-Glc)-S2(CH3), an isomer of OCO-B-S1(Glc-Rha-Glc)-S2(CH3), an isomer of OCO-B-S1(Glc-Rha-Glc)-S2(CH3) and OCO-B-S1(Xyl-Glc-Glc-Glc)-S2(OH) (Table 1 and Table 2).

3. Experimental Section

3.1. Reagents and Materials

HPLC grade acetonitrile (ACN) and formic acid were purchased from Merck Technologies Inc. (Darmstadt, Germany), and Tedia Company Inc. (Fairfield, OH, USA). Deionized water was obtained from a Millipore Milli-Q water system (Bedford, MA, USA). All other reagents were of analytical purity. ZSYXST sample was obtained from Guangxi Wuzhou Pharmaceutical (Group) Co., Ltd. (Wuzhou, China). Twenty-one reference compounds: sanchirhinoside A6 (23), notoginsenoside R3 (25), notoginsenoside N (27), notoginsenoside R6 (29), 20(S)-20-O-glucoginsenoside Rf (31), notoginsenoside R1 (33), notoginsenoside G (39), ginsenoside Rg1 (40), ginsenoside Re (41), pseudoginsenoside Rt3 (47), sanchirhinoside A3 (55), vina-ginsenoside R4 (65), sanchirhinoside B (66), notoginsenoside R2 (72), notoginsenoside U (73), ginsenoside Rg2 (78), notoginsenoside Fa (82), ginsenoside Ra3 (85), ginsenoside Rb1 (86), ginsenoside Rd (91), ginsenoside Rk3 (93), and ginsenoside Rh4 (94) were isolated from ZSYXST by the authors. Their structures were elucidated by 1D and 2D NMR spectra [28,44].

3.2. Sample Preparation and PHPLC Chromatography Conditions

ZSYXST (10 mg)was dissolved in 19% acetonitrile (1 mL)to obtain sample 1, which was then centrifuged for 10 min at 14,000 rpm, and the supernatant of sample 1 was applied to a Shimadzu LC-8A PHPLC system (Shimadzu, Kyoto, Japan), equipped with a binary pump, an UV detector and a fraction collector(FRC-10A). Chromatographic separation was achieved on a Cosmosil-5C18 column (20 × 250 mm, 5 µm), (NACALAI, Kyoto, Japan). The mobile phase consisted of water (A) and ACN (B), using 19%–20% B at 0–25 min, 20%–30% B at 25–35 min, 30%–35% B at 35–45 min, 35%–45% B at 45–70 min, 45%–90% B at 70–75 min, 90% B at 75–76 min, 90%–19% B at 76–80 min, the flow rate was 8 mL/min.

Utilizing an online collector simulation system, the major peaks 0–2 of ZSYXST were easily removed through the control of the collection parameters, set at level of 500 µV, peak slope of 1000 µV/s and the delay volume of 200 µL. Finally, sample 2 (1.3 mg) without major components was obtained, and the peaks of lower content ingredients became more obvious finally (Figure 7 and Figure 8).

Figure 7.

Figure 7

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The PHPLC chromatogram of sample 1 (0: Notoginsenoside R1; 1: Ginsenoside Rg1 and Re; 2: Ginsenoside Rb1).

Figure 8.

Figure 8

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The total ion chromatograms of UHPLC-Q-TOF/MS in negative mode.

3.3. UHPLC-Q-TOF/MS Conditions

Sample 2 (1 mg/mL) and ZSYXST sample (1 mg/mL) were analyzed on an Agilent 1290 Ultra high performance liquid chromatography system (Agilent, Palo alto, CA, USA), equipped with a T3 column (2.1 × 100 mm, 1.8 µm, Waters, Milford, MA, USA) under 40 °C. The mobile phase consisted of 0.1% formic acid water (A) and ACN (B), using 10% B at 0–10 min, 10−40% B at 10–90 min, 40%–100% B at 90–91 min, 100% B at 91–100 min, 100%–10% B at 100–100.1 min, 10% B at 100.1–105 min. The flow rate was 0.3 mL/min, and the sample volume injected was 2 µL.

The Q-TOF/MS analysis was performed on an Agilent 6520 Accurate-Mass Q-TOF/MS system. The conditions of the ESI source were: drying gas (N2) flow rate, 8.0 L/min; drying gas temperature, 350 °C; nebulizer, 30 psig; capillary voltage (Vcap), 3500V. Cone voltage was 120 V and 175 V in the positive and negative mode, respectively. Collision energy (CE) was dynamically adjusted from 45 to 70 V according to the m/z of precursor ions in the negative MS/MS mode. The mass analyzer scanned over m/z 100–2000. All the data were recorded and processed by Agilent Mass Hunter Workstation software (Version B.02.00).

4. Conclusions

In this work, a novel UHPLC-Q-TOF-MS combined with PHPLC method was established. The sensitivity of some minor components in ZSYXST could be enhanced significantly by this method. Combining the characteristic ions in positive and negative mode, the types of aglycone, saccharide, as well as the linkage positions of the saccharide chains of saponins were quickly determined. As a result, based on the exact mass, fragmentation behaviors, retention times and literature, 94 saponins, including 20 pairs of isomers, which could represent over 98% of the components in ZSYXST were identified or tentatively identified and ten of these saponins were identified as new compounds. This method could provide a powerful platform for profiling the compounds in ZSYXST and also could be useful for identification of saponins of P. notoginseng and P. ginseng.

Acknowledgements

This study was supported by Important Drug Development Fund, The National Science Fund for Distinguished Young Scholars (81125024), Ministry of Science and Technology of China (No. 2012ZX09101201-002, 2012ZX09304007), Program for Innovative Research Team in Universities of Tianjin (TD12-5033) and Program for New Century Excellent Talents in University (NCET-12-1069).

Author Contributions

Wang L-L and Han L-F made the same contribution on this research. Linlin Wang, Heshui Yu, and Mangmang Sang performed the experiment; Lifeng Han wrote the manuscript; Erwei Liu, Yi Zhang, Shiming Fang, and Tao Wang perfected language; Xiumei Gao designed the research. All authors read and approved the final version.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Sample Availability: Samples of the compounds are available from the authors.

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