Chemical Investigation of Saponins in Different Parts of Panax notoginseng by Pressurized Liquid Extraction and Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry

A pressurized liquid extraction (PLE) and high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method was developed for the qualitative determination of saponins in different parts of P. notoginseng, including rhizome, root, fibre root, seed, stem, leaf and flower. The samples were extracted using PLE. The analysis was achieved on a Zorbax SB-C18 column with gradient elution of acetonitrile and 8 mM aqueous ammonium acetate as mobile phase. The mass spectrometer was operated in the negative ion mode using the electrospray ionization, and a collision induced dissociation (CID) experiment was also carried out to aid the identification of compounds. Forty one saponins were identified in different parts of P. notoginseng according to the fragmentation patterns and literature reports, among them, 21 saponins were confirmed by comparing the retention time and ESI-MS data with those of standard compounds. The results showed that the chemical characteristics were obviously diverse in different parts of P. notoginseng, which is helpful for pharmacological evaluation and quality control of P. notoginseng.


HPLC-UV-MS Analysis of Reference Compounds
Under the optimized conditions, 21 reference compounds were analyzed by HPLC-UV-MS. The total ion chromatogram (TIC) of mixed standards was in good agreement with the HPLC-UV chromatogram ( Figure 2). The majority of reference compounds were well-separated in 50 min. Pseudo-ginsenoside F 11 , an ocotillol type triterpene, lacked suitable chromophore and showed very poor UV absorption, so its corresponding peak was hardly detected at 203 nm ( Figure 2A). However, it was readily detected using mass spectrometry, a universal, non-specific detector ( Figure 2B). Although several pairs of saponins, including ginsenoside Rg 1 and Re, ginsenoside Rf and notoginsenoside R 4 , chikusetsusaponin L 5 and notoginsenoside Fa, notoginsenoside R 2 and didehydroginsenoside Rb 1 , and ginsenoside Ra 3 and Rb 1 , have identical retention times (Figure 2A), simultaneous determination could be accomplished by reference to the different MS of their molecular ions. For example, ginsenosides Rg 1 and Re were co-eluted at the retention time of 13.5 min and failed to be distinguished with the UV detector ( Figure 2A), but they were easily discriminated using extracted ion chromatograms (EIC) of their deprotonated molecular ions at m/z 799 and m/z 945, respectively ( Figure 3A).  [17]. However, since chlorine-containing solvents were not employed during the whole HPLC-MS analysis, the presence of adduct ions [M+Cl] − was considered to be generated from contamination of the ion source [25,26]. In the ESI-MS spectra of ginsenoside Rg 1 and Re, their deprotonated molecular ions In the CID spectra of ginsenoside Re, the parent ion at m/z 945 and six main fragment ions at m/z 799, 783, 765, 637, 619 and 475 were observed ( Figure 3D). The mass differences between the parent ion and fragment ions m/z 799 and 783 were 146 and 162, which corresponded to loss of one rhamnose unit and one glucose unit, respectively. The simultaneous loss of two sugar units indicated the existence of two different terminal residues of the glycosidic moieties in its chemical structure. The fragment ions at m/z 637 corresponded to the loss of a disaccharide consisting of the one rhamnose and one glucose unit. The fragment ions m/z 765 and 619 were the daughter ions produced from m/z 945, corresponding to the loss of one or two sugar unit and one unit of H 2 O molecule. The fragment ion at m/z 475, corresponding to 20(S)-protopanaxatriol aglycon moiety, was the result of a subsequent loss of all linked glucose unit. Thus, the mass spectra of the selected ginsenoside was characterized by the observation of the two main types of fragment ions, the ions produced from the consecutive loss of saccharide unit and the ions resulting from loss of the H 2 O molecule. According to the information of ESI-MS and MS/MS, the saponins were easily identified by mass spectrometry.
However, the MS-based structural analysis faced difficulties in some cases. Ginsenoside Rc, Rb 2 and Rb 3 , belonging to the (20S)-protopanaxadiol-type saponins, have the same molecular weight and fragment ions. In their parent ion CID spectrum (Figure 4), the four mass intervals of 162, 132, 162 and 162 were also observed, which indicated the presence of three glucose unit and one pentose unit. However, the pentose unit could be xylose, arabinose (pyranose) and arabinose (furanose), which are present in ginsenosides Rc, Rb 2 and Rb 3 respectively. These three isomers could be distinguished by their retention times, 31.5 min, 34.3 min and 35.0 min for ginsenoside Rc, Rb 2 and Rb 3 respectively, in the total ion chromatogram (TIC).

HPLC-UV-MS Analysis of Different Parts of P. notoginseng
The molecular mass could be clearly identified in their ESI-MS, and the type of ginsenoside and its sugar moiety could be further obtained using MS/MS. Therefore, those saponins that could not be identified from their retention times, due to lack of their chemical standards, could also be identified by their MS/MS spectral characteristics and by comparison with literature data. The MS data of characteristic peaks for identification are summarized in Table 1.   Figure 6A). The mass differences between the parent ion and fragment ions indicated the existence of four glucose units and one xylose unit in their structures. In addition, the fragment ion at m/z 459 corresponding to the protopanaxadiol aglycon moiety was observed in the CIDs of their parent ions ( Figure 6B). Hence, the three peaks were finally identified as notoginsenoside R 4 , ginsenoside Fa and Ra 3 , respectively, which was confirmed by spiking with their chemical standards.     (4) and 21.9 min (12), respectively. The fragment ion at m/z 475 corresponding to the 20(S)-protopanaxatriol aglycon moiety was visible in CID spectra. Hence, the four peaks were tentatively identified as notoginsenoside R 3 , R 6 , N or 20-O-glucoginsenoside Rf, which possess the same molecular weight of 962 and belong to the 20(S)-protopanaxatriol type saponins [27,28]. Similarly, peaks 17 and 23 were identified as notoginsenoside Q or S in the leaf and flower of P. notoginseng, which were isomers with molecular weight of 1,342 and belonged to 20(S)-protopanaxadiol type saponins [28].
The pseudoginsenoside F 11 , an ocotillol-type saponin isolated from P. quinquefolius, was not detected in any part of P. notoginseng. Thus, pseudoginsenoside F 11 could be a marker to distinguish P. quinquefolius from Panax genus plants [11]. Liu et al reported that the malonylginsenosides Rb 1 , Rd and Rg1 were identified in the root of P. notoginseng using HPLC-ESI-MS [29]. In this study, no malonylginsenoside was detected. The discrepancy between Liu's report and our study may be caused by the low contents of these compounds and their thermally sensitive degradation in the PLE extraction utilized in the present study. Several minor saponins were detected in the underground parts and tentatively identified as yesanchinoside H (1), notoginsenosides E (10), A (11), G (13), I (22), L (36) and didehydroginsenoside Rd (33), by comparing their MS spectra with literature data [27,28,[30][31][32].
The results showed that identical chemical ingredients presented in the underground parts of P. notoginseng, including root, fibre root and rhizome. However, the saponins were obviously different among the underground parts, seed, stem, leaf and flower. In total 36 peaks were identified in the underground parts of P. notoginseng (root, fibre root and rhizome), while 11, 17, 18 and 20 saponins were identified in the seed, stem, leaf and flower, respectively. Furthermore, the components of peaks 1-4, 6, 9-13, 18-19, 21, 24, 27, 29, 33, 34 and 40 were unique to the underground parts, notoinsenoside Q/S, ginsenoside Fc/Ra1/Ra2 were only found in flowers, and the notoginsenoside O/P were specifically contained in the leafs and flowers.

Chemicals, Standards and Samples
The samples of different parts from P. notoginseng, including rhizome, root, fibre root, seed, stem, leaf and flower, were derived from same batch of plants, obtained from Wenshan region, Yunnan Province, China. The peeled seed was used in this study. The botanical origins of materials were identified by Dr. Xiu-Ming Cui, Wenshan Prefecture Sanqi Research Institute, Yunnan Province. The voucher specimens were deposited at the Institute of Chinese Medical Sciences, University of Macau, Macao, China.
Notoginsenoside R 1 was supplied by the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China); pseudo-ginsenoside F11, ginsenosides Rg 1 , Re, Rf, Rb 1 , Rg 2 , Rh 1 , Rc, Rb 2 , Rb 3 , Rd and Rg 3 were purchased from International Laboratory (South San Francisco, CA, USA); Chikusetsusaponin L 5 , ginsenoside R 2 , F 1 , Ra 3 , notoginsenoside K, R 4 , Fa and didehydroginsenoside Rb 1 were previously isolated and purified from the root of P. notoginseng by repeated silica gel, medium pressure liquid chromatography (MPLC) and preparative high performance liquid chromatography (prep-HPLC). Their structures were elucidated by comparison of their spectral data (MS, 1 H-NMR and 13 C-NMR) with corresponding references (Figure 1). Didehydroginsenoside Rb 1 was a novel triterpene saponin we isolated from P. notoginseng, and its chemical structure was previously reported [32]. Their purities were determined to be higher than 97% by normalization of the peak areas detected by HPLC-UV. HPLC-grade methanol and acetonitrile were products of Merck (Darmstadt, Germany), and the deionized water was purified by a Milli-Q purification system (Millipore, Bedford, MA, USA). Ammonium acetate was purchased from Riedel-de Haën (Seelze, Germany).

Pressurized Liquid Extraction
Sample preparation was performed with a pressurized liquid extraction (PLE), which was operated on a Dionex ASE 200 system (Dionex Corp., Sunnyvale, CA, USA) under optimized conditions reported before [33]. In brief, dried sample powder (0.5 g) was placed in an 11 mL stainless steel extraction cell. The optimized conditions were: particle size, 0.3-0.45 mm; solvent, methanol; temperature, 150 °C; static time, 15 min; pressure, 6.895 × 10 3 MPa; static cycles, 1, and number of extractions, 1. PLE extract was transferred into a 25 mL volumetric flask which was brought up to its volume with the same solvent and filtered through a 0.45 μm Econofilter (Agilent Technologies, Palo Alto, CA, USA) prior to injection into the HPLC system.

HPLC-ESI-MS/MS Analysis
An LC-MSD trap VL mass spectrometer with electrospray ionization source (Agilent Technologies,) was coupled to the HPLC system described in Section 3.3. The chromatographic conditions for the HPLC-MS were the same as those used for the HPLC-UV, except that the water (A) of mobile phase was replaced by 8 mM aqueous ammonium acetate. The ESI-MS conditions were as follows: negative-ion mode; capillary voltage, 3.5 kV; dry gas N 2 , 12 L·min −1 ; temperature, 350 °C; pressure of nebulizer, 40 psi. The ESI-MS/MS was set with compounds stability of 100%, fragment amplification of 2.0 V and isolation width of 4. Scan range of both ESI-MS and ESI-MS/MS was fixed at m/z 200-1,400 U.

Conclusions
In conclusion, an HPLC-ESI-MS/MS and PLE method was developed for the extraction and qualitative determination of saponins in different parts of P. notoginseng, including the rhizome, root, fiber root, seed, stem, leaf and flower. A total 41 saponins were identified and the chemical profiles of the different parts were obviously diverse. The developed HPLC-ESI-MS/MS method provided a reliable means of distinguishing the saponins in different parts of P. notoginseng, which should be helpful for the pharmacological evaluation and quality control of P. notoginseng.