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Article

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

1
Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, China
2
Tianjin Key Laboratory of TCM Chemistry and Analysis, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, China
*
Author to whom correspondence should be addressed.
Molecules 2015, 20(11), 20518-20537; https://doi.org/10.3390/molecules201119712
Submission received: 5 September 2015 / Revised: 27 October 2015 / Accepted: 6 November 2015 / Published: 18 November 2015
(This article belongs to the Section Natural Products Chemistry)

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.

Graphical Abstract

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. A diagram for rapid classification and identification of saponins by characteristic ions in MS and MS/MS mode.
Figure 1. A diagram for rapid classification and identification of saponins by characteristic ions in MS and MS/MS mode.
Molecules 20 19712 g001

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).

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.
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 ModeDiagnostic Ions of Aglycone Types in Positive Mode
1965.51981011.52780.1131.0312, 191.0668439.3453, 457.3560
2965.52371011.52910.15131.0310, 191.1745439.3459, 457.3559
3979.53871025.54510.2205.0648421,1231, 439.1502
4979.54071025.54640.2145.0321, 205.0667439.3480, 457.3572
5801.4635847.46620.1221.0687407.3257, 425.3360
6815.4807861.48810.14221.0117421.3436, 439.3547
7947.5170993.52300.18131.0287, 191.0432, 221.0223421.3586, 457.3660
8815.4807861.48810.14221.0654421.3446, 439.3581
9815.4669861.48170.02323.0378421.3324, 439.3512, 457.2314
10815.4814861.48200.09323.0665421.3477, 439.3594, 457.3701
11815.4701861.48360.1323.0656421.3577, 439.3659, 457.3730
12817.4872863.49820.8323.2112423.3736, 441.3848
13799.4312845.459820221.0599405.3408, 423.3536, 441.3938
14961.53321007.53820.1145.0595, 205.0690421.3660, 439.3758, 457.3886
15961.53281007.53330.15205.4532421.3748, 439.3861, 457.3971
16815.4673861.48150.1323.0454439.3907, 457.4193
17815.4668861.48170.15323.0452421.3840, 439.3967, 457.4080
18813.4677859.47390.1421.3640, 439.3767, 457.4070
19961.53231007.53660.2205.9385421.3975, 439.4145, 457.4258
20959.51941005.52200.25221.0431421.3841, 439.3967, 457.4081
21813.4591859.46520.1421.4140, 439.4235, 457.4046
22959.51581005.52050.3221.0446421.3880, 439.4228
231093.57341139.577140221.0995, 323.0662405.4047, 423.4172, 441.4303, 325.1562, 295.1434
241093.57191139.576714221.0676405.3985, 423.4120, 441.4245, 325.1519, 295.1366
25961.54091007.54601.5221.0670405.3973, 423.4204, 441.4223, 325.1495
261093.57051139.576110191.0538, 221.0668405.3913, 423.4036, 441.3970
27961.53051007.53410. 1221.0597405.3926, 423.4051, 441.4172
281107.58701153.59090.5205.0700, 221.0662405.3926, 423.4051, 441.4132
29961.53031007.53610.8221.0663405.3910, 423.4035, 441.4157, 325.1456
301107.57891153.59080.5205.0684, 221.0661405.3886, 423.4016, 441.4128, 309.1526
31961.52941007.52780.3221.0662405.3874, 423.4002, 441.4121
32961.52891007.53350.3221.0650405.3848, 423.3977, 441.4089
33931.5210977.52700.2131.0349, 191.0570405.3806, 423.3936, 441.4050
34961.52971007.53500.4221.0661405.3794, 423.3909, 441.4028
35961.52951007.53240.4221.0663405.3898, 423.3911, 441.4001
36931.5179977.52400.1191.0561, 221.0598405.3765, 423.3878, 441.3995
37961.52771007.53280.75221.0660405.3738, 423.3857, 441.3973
381121.56391167.565510221.0599407.3630, 425.3740, 443.3849
39959.12311005.12530.35221.0664407.3597, 425.3721, 443.3820
40799.4673845.48470.1405.3730, 423.3859, 441.3968
41945.5324991.53860.2145.0346, 205.1696405.3655, 423.3810, 441.3920, 309.1347
421255.62011301.625410131.0295, 191.0599, 221.0661405.3766, 423.3890, 441.3995, 295.1264, 325.1313
431141.58751187.59213221.0661423.3764, 441.3898
44979.53521025.5410100221.0665423.3764, 441.3898
451123.57211169.57635221.0660, 323.0961405.3629, 423.3588
46901.5177947.50630.88191.0660405.3629, 423.3588, 441.3587
47769.3977815.40320.15131.0231, 191.0623405.3629, 423.3588
48901.5008947.50500.8191.0601405.3629, 423.3588, 441.3887
49769.4713815.46610.2191.0600405.3587, 423.3712, 441.3816
50959.50321005.50820.4221.0672421.3554, 439.3660, 457.3768
511109.54961155.55672.5221.0670421.3541, 439.3654
52769.4673815.46100.15191.0643405.3584, 423.3694
53961.51081007.51590.4221.0667405.3558 , 423.3683, 441.3798
541123.56101169.56432.5221.0661405.3558 , 423.3683, 441.3798
55769.4412815.45370.1131.0329405.3565, 423.3679, 441.3781
56915.5043961.512360131.331, 205.0690405.3565, 423.3679, 441.3781
571123.54561169.54954221.0653423.3781, 441.3878, 325.1177
581123.54271169.54754221.0644, 323.0924405.3552, 423.3661, 441.3768
591121.52261167.52425221.0655, 323.0970421.3487, 439.3613, 457.3717, 325.1176
601125.54761171.55094221.0657, 323.0960407.3671, 425.3739
611123.53061169.53264221.0651405.3552, 423.3649, 441.3748
62797.4781843.42760.1421.3488, 439.3595, 457.3695
631123.51541169.51643221.0645405.3554, 423.3641, 441.3708
64947.4713993.46330.25191.0598, 221.0661421.3447, 439.3612, 457.3701
65961.47231007.47530.3221.0644407.3661, 425.3773
66781.4321827.422250.1405.3531, 423.3637, 441.3738
67961.46871007.47040.2221.0660405.3523, 423.3623, 441.3754
68961.46231007.46346221.0664405.3534, 423.3665, 441.3771
69799.4215845.42320.3221.0612405.3503, 423.3621, 441.3719
70961.46201007.46170.2221.0618405.1917, 423.3524, 441.3701
71961.46301007.46350.7221.0660405.3501, 423.3633, 441.3721
72769.4185815.42070.35131.0304, 191.0624405.3499, 423.3609, 441.3706
73799.4331845.43040.8221.0588, 323.0889405.3489, 423.3597, 441.3700, 325.1119
74959.45371005.45540.22221.0580439.3546, 457.3632, 325.1057
751371.58951417.5705100131.0298, 221.0645, 293.0659, 323.0926407.3640, 425.3740, 443.3839, 325.1109, 295.1006
76901.4601947.46210.8191.0599407.3629, 4253724, 443.3830
77901.4614947.46550.6131.0334, 191.0621407.3621, 4253733, 443.3843
78783.4448829.44930.1145.0247, 205.0579405.3472, 423.3587, 441.3686
791239.57321285.571515221.0642, 293.0883, 323.1087407.3631, 425.3733, 443.3838, 325.1097, 295.0995
80637.3796683.40540.1405.3461, 423.3562, 441.3665
811269.58941315.580950221.0468, 323.0734407.3614, 425.3722, 443.3799
821239.58921315.587985131.0342, 191.0660, 221.0661, 323.0979407.3624, 425.3726, 443.3827
831105.53571151.54010.1221.0664405.3459, 423.3565, 441.3661
841105.54251151.54490.1221.0596405.3421, 423.3554, 441.3667
851239.23311285.237740131.0121, 191.0366, 221.0663407.3665, 425.3779, 443.3760
861107.56431153.566210221.0488, 323.0729407.3613, 425.3719, 443.3813
871107.56321153.56436.6221.0599407.3601, 425.3677, 443.3805
881077.56961123.57463.3131.0257, 191.0466, 221.0540407.3583, 425.3682, 443.3781
891077.57051123.57613.3131.0265, 191.0453, 221.0536407.3583, 425.3690, 443.3800
90943.5121989.51838221.0662405.3433, 423.3545, 441.3794
91945.5351991.53980.3221.5863407.3622, 425.3730, 443.3826, 325.1087
92945.5458991.55160.35221.0664, 323.0721407.3829, 425.3941, 443.4048, 325.1278
93619.4279665.42390.1405.4064, 423.4219, 441.4347
94619.4255665.42450.1405.4064, 423.4202, 441.4332
Figure 2. The structures of different types of saponin aglycone.
Figure 2. The structures of different types of saponin aglycone.
Molecules 20 19712 g002

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.

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.
Figure 3. 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 3. 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.
Molecules 20 19712 g003
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).

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.
Table 2. Identification of compounds in ZSYXST.
Table 2. Identification of compounds in ZSYXST.
Comp.Rt (min)MS/MS m/zArea%Identification
17.54965.5198785.9465 [M − H − 180], 653.3969 [M − H − 180 − 132], 491.3843 [M − H − 180 − 132 − 162], 415.33500.019OCO-A-S1(Glc)-S2(Glc-Xyl/Ara) *
28.611965.5237785.9489 [M − H − 180], 653.3794 [M − H − 180 − 132], 491.3654 [M − H − 180 − 132 − 162], 415.32290.006isomer of OCO-A-S1(Glc)-S2(Glc-Xyl/Ara) *
38.811979.5387799.4633 [M − H − 180], 635.3669 [M − H − 180 − 146], 491.3370 [M − H − 180 − 146 − 162], 415.33010.016OCO-A-S1(Glc)-S2(Glc-Rha) [14]
410.189979.5407799.4532 [M − H − 180], 635.3868 [M − H − 180 − 146], 491.3569 [M − H − 180 − 146 − 162], 415.32770.006isomer of OCO-A-S1(Glc)-S2(Glc-Rha) [14]
512.992801.4635639.4097 [M − H − 162], 477.3605 [M − H − 162 − 162]0.006ginsenoside C-Y1 [22]
613.699815.4807653.4212 [M − H − 162], 491.3770 [M − H − 162 − 162], 391.28720.032OCO-B-S1(Glc-Glc)-S2(OH) [23]
713.852947.5170785.4339 [M − H − 162], 623.4062 [M − H − 162 − 162], 491.4131 [M − H − 162 − 162 − 132], 415.33100.032vinaginsenoside R5 or yesanchinoside C [24,25]
814.151815.4801653.4276 [M − H − 162], 491.3788 [M − H − 162 − 162], 391.28780.032isomer of OCO-B-S1(Glc-Glc)-S2(OH)
914.252861.4892653.4263 [M − H − 162], 491.3777 [M − H − 162 − 162], 415.32530.010majonoside R1 [12,19]
1014.582815.4814653.4162 [M − H − 162], 491.3716 [M − H − 162 − 162], 415.35330.019isomer of majonoside R1 [12,19]
1115.03815.4701653.4162 [M − H − 162], 491.3716 [M − H − 162 − 162], 415.35190.151isomer of majonoside R1 [12,19]
1215.948817.4872655.4462 [M − H − 162],493.3934 [M − H − 162 − 162]0.048PPT-C-S1(Glc-Glc)-S2(H) [26]
1316.431799.4312799.4678 [M − H − 162], 637.4793 [M − H − 162 − 162], 475.3866 [M − H − 162 − 162 − 162], 391.29040.013ginsenoside Rf [27]
1416.714961.5332799.4679 [M − H − 162], 653.4002 [M − H − 162 − 146], 491.3304 [M − H − 162 − 146 − 162], 391.27720.032OCO-B-S1(Glc-Rha-Glc)-S2(CH3) [26]
1517.597961.5328799.5297 [M − H − 162], 653.4002 [M − H − 162 − 146], 491.3693 [M − H − 162 − 146 − 162], 391.89430.032isomer of OCO-B-S1(Glc-Rha-Glc)-S2(CH3)
1618.398815.4868653.4263 [M − H − 162], 491.3777 [M − H − 162 − 162], 415.33610.515isomer of majonoside R1 [12,19]
1718.092815.4673653.4373 [M − H − 162], 491.3788 [M − H − 162 − 162], 415.34150.032isomer of majonoside R1 [12,19]
1819.481859.4723651.4111 [M − H − 162], 489.3579 [M − H − 162 − 162]0.019OCO-E-S1(Glc-Glc) *
1920.247961.5323799.4824 [M − H − 162], 653.4002 [M − H − 162 − 146], 491.3558 [M − H − 162 − 146 − 162], 391.34410.016isomer of OCO-B-S1(Glc-Rha-Glc)-S2(CH3) [21]
2021.142959.5194797.4207 [M − H − 162], 635.3567 [M − H − 162 − 162], 473.3460 [M − H − 162 − 162 − 162]0.016OCO-D-S1(Glc-Glc-Glc) *
2122.39859.4721651.4113 [M − H − 162], 489.3635 [M − H − 162 − 162]0.023isomer of OCO-E-S1(Glc-Glc) *
2223.909959.5158797.4575 [M − H − 162], 635.4076 [M − H − 162 − 162], 473.3611 [M − H − 162 − 162 − 162]0.016isomer of OCO-D-S1(Glc-Glc-Glc) *
2326.9361093.5734961.5389 [M − H − 132], 637.4333 [M − H − 132 − 162 − 162], 475.3832 [M − H − 132 − 162 − 162 − 162], 391.28720.093sanchirhinoside A6
2428.3141093.5719961.5374 [M − H − 132], 637. 4382 [M − H − 132 − 162 − 162], 475.3753 [M − H − 132 − 162 − 162 − 162], 391.28830.064isomer of sanchirhinoside A6 [19,28]
2528.962961.5374799.4883 [M − H − 162], 637.4389 [M − H − 162 − 162], 475.3826 [M − H − 162 − 162 − 162], 391.28720.328notoginsenoside R3
2629.7511093.5705961.5272 [M − H − 132], 637.4367 [M − H − 132 − 162 − 162], 475.3753 [M − H − 132 − 162 − 162 − 162], 391.28790.028isomer of sanchirhinoside A6 [19,28]
2730.175961.5305799.4817 [M − H − 162], 637.4185 [M − H − 162 − 162], 475.3757 [M − H − 162 − 162 − 162], 391.29010.187notoginsenoside N
2830.1751107.5870961.5311 [M − H − 146], 799.4749 [M − H − 146 − 162], 637.4310 [M − H − 146 − 162 − 162], 475.3794 [M − H − 146 − 162 − 162 − 162], 391.2892yesanchinoside E [17]
2930.599961.5303637.4328 [M − H − 162 − 162], 475.3781 [M − H − 162 − 162 − 162], 391.29120.170notoginsenoside R6
3030.9991107.5789961.5299 [M − H − 146], 799.4747 [M − H − 146 − 162], 637.4224 [M − H − 146 − 162 − 162], 475.3760 [M − H − 146 − 162 − 162 − 162], 391.29020.026isomer of yesanchinoside E [17]
3131.788961.5294799.4784 [M − H − 162], 637.4286 [M − H − 162 − 162], 475.3782 [M − H − 162 − 162 − 162], 391.28990.71520-O-glucoginsenoside Rf
3232.259961.5289799.4824 [M − H − 162], 637.4403 [M − H − 162 − 162], 475.3832 [M − H − 162 − 162 − 162], 391.28760.052isomer of 20-O-glucoginsenoside Rf
3333.661931.5210637.4369 [M − H − 132 − 162], 475.3824 [M − H − 132 − 162 − 162],391.327013.39notoginsenoside R1
3434.296961.5297799.4803 [M − H − 162], 637.4268 [M − H − 162 − 162], 475.3785 [M − H − 162 − 162 − 162], 391.29130.151notoginsenoside M or N [19,21]
3534.991961.5295799.4811 [M − H − 162], 637.4194 [M − H − 162 − 162], 475.3634 [M − H − 162 − 162 − 162], 391.29200.067isomer of notoginsenoside M or N [19,21]
3635.392931.5179637.4281 [M − H − 132 − 162], 475.3762 [M − H − 132 − 162 − 162], 391.29010.138gypenoside LXIV [29]
3735.957961.5277799.4883 [M − H − 162], 637.4255 [M − H − 162 − 162], 475.3787 [M − H − 162 − 162 − 162], 391.28770.041isomer of notoginsenoside R3 or isomer of notoginsenoside R6 [17,19]
3836.311121.5639959.3973 [M − H − 162], 797.4040 [M − H − 162 − 162], 473.3085 [M − H − 162 − 162 − 162 − 162]0.229quinquenoside IV [27]
3936.31959.1231797.3990 [M − H − 162], 635.4110 [M − H − 162 − 162], 473.3675 [M − H − 162 − 162 − 162]notoginsenoside G
4036.781799.4673637.4352 [M − H − 162], 475.3815 [M − H − 162 − 162], 391.293027.59ginsenoside Rg1 [27]
4137.3945.5324783.4775 [M − H − 162], 637.4309 [M − H − 162 − 146], 475.3791 [M − H − 162 − 146 − 162], 391.30017.670ginsenoside Re
4237.6761255.62011123.1027 [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], 391.29000.032PPT-A-S1(Glc-Glc-Xyl/Ara)-S2(Glc-Glc) [21]
4339.3491141.5875817.4870 [M − H − 162 − 162], 655.4351 [M − H − 162 − 162 − 162], 493.3991 [M − H − 162 − 162 − 162 − 162]0.222quinquenoside L16 [18]
4439.349979.5352817.4912 [M − H − 162], 655.4429 [M − H − 162 − 162], 493.4046 [M − H − 162 − 162 − 162]PPT-C-S1(Glc)-S2(Glc-Glc) [30]
4541.411123.5721961.5210 [M − H − 162], 799.4757 [M − H − 162 − 162], 637.4238 [M − H − 162 − 162 − 162], 475.3770 [M − H − 162 − 162 − 162 − 162], 391.28700.248PPT-A-S1(Glc-Glc)-S2(Glc-Glc) [31]
4641.41901.5177637.4241 [M − H − 132 − 132], 475.3827 [M − H − 132 − 132 − 162], 391.2881chikusetsusaponin L5 [27]
4741.41769.3977637.3997 [M − H − 162], 475.3876 [M − H − 162 − 132], 391.2851pseudoginsenoside Rt3
4842.717901.5008637.4230 [M − H − 132 − 132], 475.3744 [M − H − 132 − 132 − 162], 391.29010.077isomer of chikusetsusaponin L5 [27]
4943.223769.4713637.4342 [M − H − 132], 475.3828 [M − H − 132 − 162], 391.28730.090notoginsenoside R2 [19]
5043.8959.5032797.4575 [M − H − 162], 635.4076 [M − H − 162 − 162], 473.3611 [M − H − 162 − 162 − 162]0.145isomer of OCO-D-S1(Glc-Glc-Glc)*
5144.3771109.5496785.4931 [M − H − 162 − 162], 623.4047 [M − H − 162 − 162 − 162], 491.3606 [M − H − 162 − 162 − 162 − 132], 391.86540.032OCO-B-S1(Xyl-Glc-Glc-Glc)-S2(OH)*
5244.79769.4673637.4342 [M − H − 132], 475.3828 [M − H − 132 − 162], 391.28900.045isomer of notoginsenoside R2 [19]
5345.108961.5108799.4836 [M − H − 162], 637.4239 [M − H − 162 − 162], 475.3783 [M − H − 162 − 162 − 162], 391.29030.174isomer of 20-O-glucoginsenoside Rf [32,33]
5445.1081123.5610961.5215 [M − H − 162], 799.4657 [M − H − 162 − 162], 637.4338 [M − H − 162 − 162 − 162], 475.3770 [M − H − 162 − 162 − 162 − 162], 391.2891isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) [31]
5546.344769.4412637.4341 [M − H − 132],475.3807 [M − H − 132 − 162],391.28910.196sanchirhinoside A3 [19]
5646.344915.5043783.4801 [M − H − 132], 637.4801 [M − H − 132 − 146], 475.3756 [M − H − 132 − 146 − 162], 391.3001PPT-A-S1(Rha-Xyl)-S2(Glc) [31]
5746.9091123.5456961.5244 [M − H − 162], 799.4729 [M − H − 162 − 162], 637.4283 [M − H − 162 − 162 − 162], 475.3721 [M − H − 162 − 162 − 162 − 162], 391.29110.051isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) [31]
5847.1691123.5427961.5227 [M − H − 162], 799.4758 [M − H − 162 − 162], 637.4274 [M − H − 162 − 162 − 162], 475.3728 [M − H − 162 − 162 − 162 − 162], 391.28930.012isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) [31]
5947.841121.5226959.5057 [M − H − 162], 797.4549 [M − H − 162 − 162], 635.4088 [M − H − 162 − 162 − 162], 473.3567 [M − H − 162 − 162 − 162 − 162]0.077OCO-D-S1(Glc-Glc-Glc-Glc) *
6048.171125.5476963.5411 [M − H − 162], 801.4900 [M − H − 162 − 162], 635.4389 [M − H − 162 − 162 − 162], 477.3889 [M − H − 162 − 162 − 162 − 162]0.066PPD-B-S1(Glc-Glc)-S2(Glc-Glc) [34]
6148.5941123.5306961.5171 [M − H − 162], 799.4745 [M − H − 162 − 162], 637.4232 [M − H − 162 − 162 − 162], 475.3754 [M − H − 162 − 162 − 162 − 162], 391.28910.035isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) [31]
6248.864797.4781635.4190 [M − H − 162], 473.3695 [M − H − 162 − 162]0.039PPT-d-S1(Glc-Glc) [21]
6349.631123.5254961.5203 [M − H − 162], 799.4698 [M − H − 162 − 162], 637.4431 [M − H − 162 − 162 − 162], 475.3701 [M − H − 162 − 162 − 162 − 162], 391.29730.097isomer of PPT-A-S1(Glc-Glc)-S2(Glc-Glc) [31]
6449.63947.4713815.3567 [M − H − 132], 653.3778 [M − H − 132 − 162], 491.3608 [M − H − 132 − 162 − 162], 391.8554OCO-B-S1(Glc-Glc-Xyl/Ara)-S2(OH) [35]
6550.101961.4723799.4722 [M − H − 162], 637.4220 [M − H − 162 − 162], 475.3720 [M − H − 162 − 162 − 162]0.1160vina−ginsenoside R4
6650.254781.4321619.3533 [M − H − 162], 457.3661 [M − H − 162 − 162]0.032sanchirhinoside B
6750.619961.4687799.4598 [M − H − 162], 637.4037 [M − H − 162 − 162], 475.3770 [M − H − 162 − 162 − 162], 391.28660.058isomer of 20-O-glucoginsenoside Rf [32,33]
6851.762961.4623781.4714 [M − H − 180], 637.4232 [M − H − 180 − 144], 475.3801 [M − H − 180 − 144 − 162], 391.29090.019isomer of notoginsenoside R3 or isomer of notoginsenoside R6 [17,19]
6952.221799.4215637.4209 [M − H − 162], 475.3722 [M − H − 162 − 162], 391.28770.068isomer of ginsenoside Rg1 [36,37]
7053.552961.4620799.4434 [M − H − 162], 637.4263 [M − H − 162 − 162], 475.4024 [M − H − 162 − 162 − 162], 391.29070.039isomer of 20-O-glucoginsenoside Rf [32,33]
7154.376961.4630799.4695 [M − H − 162], 637.4074 [M − H − 162 − 162], 475.3638 [M − H − 162 − 162 − 162], 391.28810.019isomer of notoginsenoside R3 or isomer of notoginsenoside R6 [17,19]
7255.185769.4795637.4336 [M − H − 132], 475.3814 [M − H − 132 − 162], 391.28691.572isomer of notoginsenoside R2
7355.813799.4331637.4356 [M − H − 162], 475.4315 [M − H − 162 − 162], 391.28720.206notoginsenoside U
7456.461959.4537797.4608 [M − H − 162], 635.4138 [M − H − 162 − 162], 473.3590 [M − H − 162 − 162 − 162]0.026ginsenoside III or vinaginsenoside R20 [38]
7556.7671371.58951107.5948 [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]0.296notoginsenoside D [17]
7657.992901.4601769.4200 [M − H − 132], 637.4355 [M − H − 132 − 132], 475.4321 [M − H − 132 − 132 − 162]0.171chikusetsusaponin L5 [39]
7758.392901.4614769.4231 [M − H − 132], 637.4352 [M − H − 132 − 132], 475.4335 [M − H − 132 − 132 − 162]0.602notoginsenoside Rw1 [21]
7858.569783.4448621.3941 [M − H − 146], 475.3495 [M − H − 146 − 162], 391.28960.754ginsenoside Rg2
7959.1461239.57321107.6034 [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]2.087chikusetsusaponinVI [40]
8060.406637.3796475.3437 [M − H − 162],391.29010.103ginsenoside F1 [27]
8160.8181269.58941107.5013 [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]0.045ginsenoside Ra0 or quinquenoside V [19,21]
8261.5721239.58921107.5974 [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]2.622notoginsenoside Fa
8362.1021105.5357943.5543 [M − H − 162], 781.4996 [M − H − 162 − 162], 619.3441 [M − H − 162 − 162 − 162], 457.3654 [M − H − 162 − 162 − 162 − 162]0.941PPT-B-S1(Glc-Glc-Glc-Glc) [38]
8463.5621105.5425943.5521 [M − H − 162], 781.5010 [M − H − 162 − 162], 619.3501 [M − H − 162 − 162 − 162], 457.2601 [M − H − 162 − 162 − 162 − 162]0.161isomer of PPT-B-S1(Glc-Glc-Glc-Glc) [38]
8563.5621239.23311077.4231 [M − H − 162], 915.4635 [M − H − 162 − 162], 621.3862 [M − H − 162 − 162 − 294], 459.3452 [M − H − 162 − 162 − 294 − 162]0.019ginsenoside Ra3 [39]
8664.1981107.5642945.4624 [M − H − 162], 783.4239 [M − H − 162 − 162], 621.3852 [M − H − 162 − 162 − 162], 459.3475 [M − H − 162 − 162 − 162 − 162]32.21ginsenoside Rb1
8766.4831107.5632945.4631 [M − H − 162], 783.4197 [M − H − 162 − 162], 621.3800 [M − H − 162 − 162 − 162], 459.3452 [M − H − 162 − 162 − 162 − 162]0.019isomer of ginsenoside Rb1 [12,40]
8868.7911077.5696945.4812 [M − H − 132], 783.4405 [M − H − 132 − 162], 621.4014 [M − H − 132 − 162 − 162], 459.3421 [M − H − 132 − 162 − 162 − 162]0.110notoginsenoside L [12,27]
8969.6391077.5705945.4888 [M − H − 132], 783.4465 [M − H − 132 − 162], 621.4008 [M − H − 132 − 162 − 162], 459.3544 [M − H − 132 − 162 − 162 − 162]0.080isomer of notoginsenoside L [12,27]
9070.487943.5121781.4300 [M − H − 162], 619.3881 [M − H − 162 − 162],457.3656 [M − H − 162 − 162 − 162]0.058PPT-B-S1(Glc-Glc-Glc) *
9173.384945.5495783.4954 [M − H − 162], 621.4422 [M − H − 162 − 162],459.3887 [M − H − 162 − 162 − 162]3.134ginsenoside Rd
9277.824945.5520783.4964 [M − H − 162], 621.4415 [M − H − 162 − 162],459.3887 [M − H − 162 − 162 − 162]0.370isomer of ginsenoside Rd [9,12]
9383.312619.4279457.3652 [M − H − 162]0.019ginsenoside Rk3 [41,42]
9485.608619.4255457.3686 [M − H − 162]0.067ginsenoside Rh4 [41,42]
*: new compounds.
Figure 4. 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 4. 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.
Molecules 20 19712 g004
[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).
Figure 5. 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.
Figure 5. 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.
Molecules 20 19712 g005
[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. The typical TOF-MS spectra of compounds 18 (A); 25 (B); 92 (C) in negative mode (1: MS spectra; 2: MS/MS spectra).
Figure 6. The typical TOF-MS spectra of compounds 18 (A); 25 (B); 92 (C) in negative mode (1: MS spectra; 2: MS/MS spectra).
Molecules 20 19712 g006
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.
Figure 7. The PHPLC chromatogram of sample 1 (0: Notoginsenoside R1; 1: Ginsenoside Rg1 and Re; 2: Ginsenoside Rb1).
Figure 7. The PHPLC chromatogram of sample 1 (0: Notoginsenoside R1; 1: Ginsenoside Rg1 and Re; 2: Ginsenoside Rb1).
Molecules 20 19712 g007
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 8. The total ion chromatograms of UHPLC-Q-TOF/MS in negative mode.
Figure 8. The total ion chromatograms of UHPLC-Q-TOF/MS in negative mode.
Molecules 20 19712 g008

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.

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

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MDPI and ACS Style

Wang, L.-L.; Han, L.-F.; Yu, H.-S.; Sang, M.-M.; Liu, E.-W.; Zhang, Y.; Fang, S.-M.; Wang, T.; Gao, X.-M. 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. Molecules 2015, 20, 20518-20537. https://doi.org/10.3390/molecules201119712

AMA Style

Wang L-L, Han L-F, Yu H-S, Sang M-M, Liu E-W, Zhang Y, Fang S-M, Wang T, Gao X-M. 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. Molecules. 2015; 20(11):20518-20537. https://doi.org/10.3390/molecules201119712

Chicago/Turabian Style

Wang, Lin-Lin, Li-Feng Han, He-Shui Yu, Mang-Mang Sang, Er-Wei Liu, Yi Zhang, Shi-Ming Fang, Tao Wang, and Xiu-Mei Gao. 2015. "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" Molecules 20, no. 11: 20518-20537. https://doi.org/10.3390/molecules201119712

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