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

“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.


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.
Molecules 2015, 20, 20518-20537; doi:10.3390/molecules201119712 www.mdpi.com/journal/molecules 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 R 1 , ginsenosides Rg 1 , Re, and Rb 1 ) 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.

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.

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.

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

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 (Figures 1 and 2).

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

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 (Figures 1 and 2).

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.
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).

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 ( Figures 3A, 4A and 5A). These characteristics could be explained by the existence of the space effect.

Identification of PPD Type Saponins
In Figure 3A

Identification of PPD Type Saponins
In Figure 3A   From the diagnostic ions at m/z 459, the aglycone could be deduced to be PPD-A type (Figures 1 and 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.

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 (Figures 1 and 5B). 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 (Figures 1 and 5C). According to the above elucidation and literature data, 9 could be tentatively identified as majonoside R 1 [26,43] . Compounds 10, 11, 16 /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 (Tables 1 and 2).    [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-S 1 (Glc-Glc-Glc). Similar diagnostic fragment ions were observed in 22 (Tables 1 and 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-5C 18 (Tables 1 and 2).
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 (Figures 7 and 8).

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.