The Chemical Characterization of Eleutherococcus senticosus and Ci-wu-jia Tea Using UHPLC-UV-QTOF/MS

Eleutherococcus senticosus Maxim. belongs to the Araliaceae family. Phytochemical studies reveal that E. senticosus leaves contain triterpene glycosides along with organic acid derivatives and flavonoid compounds. It is believed that E. senticosus is similar to ginseng because they come from same family and both contain triterpene saponins. E. senticosus leaves have been developed as a functional beverage called ci-wu-jia tea in recent years. Triterpene glycosides are difficult to identify by ultraviolet (UV) detection and contents of these compounds are low in E. senticosus leaves. In this study, a sensitive ultra-high performance liquid chromatographic (UHPLC) method combining UV and tandem mass spectrometry (MS/MS) was developed to characterize the triterpene glycosides from E. senticosus leaves and related commercial products. Fragmentation patterns of three sub-groups of triterpene glycosides in E. senticosus leaves were investigated. Additionally, fragmentation pathways and UV characteristics of organic acid derivatives and flavonoids were also characterized. A compound screening library, including 241 compounds reported in the literature, was created and used to confirm the compounds in the samples. In this study, a total of 24 samples, including 13 plant samples of E. senticosus and 11 ci-wu-jia tea products, were analyzed. Out of the 11 commercial products, three products were discovered to contain green tea (Camellia sinensis) that was considered to be an adulterant since it was not an ingredient on the labels. The developed UHPLC-UV-MS/MS analytical method combined with the UNIFI processing method can simultaneously characterize organic acid derivatives, flavonoids, and triterpene saponins from E. senticosus. It provides a simple and sensitive way to perform quality control of E. senticosus and related ci-wu-jia tea products.


Introduction
Eleutherococcus senticosus Maxim. (syn. Acanthopanax senticosus Harms) is a species of the Araliaceae family. It usually grows in forests or thickets, where it is elevated from hundreds to above 2000 m in altitude in China. Globally, this plant is distributed in Russia and East Asia, including China,

Results and Discussion
The chemical constituents of E. senticosus leaves include hydrophilic compounds, such as organic acid derivatives, flavonoids, as well as triterpene glycosides. It can be a challenge to retain and separate these compound classes on most of C18 reversed phase columns. The high strength silica (HSS) column enables polar compounds to more readily access the pore structure of the solid material and increases their retention time. In this work, the developed UHPLC-UV-MS/MS method is optimized with UHPLC columns, column temperature, mobile phase, gradient elution method, flow rate, and MS responses on a UHPLC system coupled to a quadrupole time-of-flight mass spectrometer with electrospray ionization. As a result, acetonitrile-water with 0.05% formic acid combined with the optimized gradient elution on an UPLC HSS T3 column (1.8 µm, 2.1 × 100 mm i.d.) afforded the best separation and MS response in the positive mode to simultaneously identify different classes of compounds, such as organic acid derivatives, flavonoids, and triterpene glycosides, in a single injection analysis. This is the first method to focus on full characterization of the main components in E. senticosus leaves.
Organic acid derivatives, such as chlorogenic acid and 3,5-dicaffeoylquinic acid, have been reported from E. senticosus. As the phenolic group conjugates with the α,β-unsaturated acid unit in caffeic acid or ferulic acid molecules, the organic acid derivatives have characteristic UV absorption around 218, 243, 295 (shoulder), and 327 nm. A peak of λ max at 327 nm with a shoulder at 295 nm is a unique signature of organic acid derivatives and can be used to identify this family from other classes of compounds in E. senticosus. In addition, MS and MS/MS spectra of organic acid derivatives contain common fragment ions at m/z 163 or 177 Da corresponding to the loss of caffeic acid or ferulic acid, respectively. In E. senticosus leaves, 3,5-dicaffeoylquinic acid elutes at 8 (Table 1). and separate these compound classes on most of C18 reversed phase columns. The high strength silica (HSS) column enables polar compounds to more readily access the pore structure of the solid material and increases their retention time. In this work, the developed UHPLC-UV-MS/MS method is optimized with UHPLC columns, column temperature, mobile phase, gradient elution method, flow rate, and MS responses on a UHPLC system coupled to a quadrupole time-of-flight mass spectrometer with electrospray ionization. As a result, acetonitrile-water with 0.05% formic acid combined with the optimized gradient elution on an UPLC HSS T3 column (1.8 µm, 2.1 × 100 mm i.d.) afforded the best separation and MS response in the positive mode to simultaneously identify different classes of compounds, such as organic acid derivatives, flavonoids, and triterpene glycosides, in a single injection analysis. This is the first method to focus on full characterization of the main components in E. senticosus leaves.
Organic acid derivatives, such as chlorogenic acid and 3,5-dicaffeoylquinic acid, have been reported from E. senticosus. As the phenolic group conjugates with the α,β-unsaturated acid unit in caffeic acid or ferulic acid molecules, the organic acid derivatives have characteristic UV absorption around 218, 243, 295 (shoulder), and 327 nm. A peak of λmax at 327 nm with a shoulder at 295 nm is a unique signature of organic acid derivatives and can be used to identify this family from other classes of compounds in E. senticosus. In addition, MS and MS/MS spectra of organic acid derivatives contain common fragment ions at m/z 163 or 177 Da corresponding to the loss of caffeic acid or ferulic acid, respectively. In E. senticosus leaves, 3,5-dicaffeoylquinic acid elutes at 8.72 min with the typical UV absorption of organic acid derivatives and showed pronated molecular ions at m/z 517.    Flavonoids are another group of compounds in E. senticosus leaves with typical UV absorptions around 203, 255, and 354 nm that are related to flavonoid core quercetin or kaempferol. These compounds contain key fragment ions at m/z 303 or 287 Da corresponding to aglycone quercetin or kaempferol, respectively, in the MS and MS/MS spectra. Further, a neutral loss of 162 or 146 Da related to the loss of hexose and deoxyhexose, respectively, are commonly found for flavonoid glycosides. In E. senticosus leaves, rutin eluted at 7.22 min and is confirmed by comparing the retention time, UV, MS, and MS/MS spectra with that of a standard compound. Key UV absorption and MS/MS fragments of rutin are listed in Table 1 along with the other six flavonoid glycosides.
Triterpene glycoside compounds are key constituents in E. senticosus leaves, but show weak UV absorption due to a lack of obvious chromophore on the triterpene aglycone. Investigation of the MS/MS fragmentation pattern provides a tool to characterize this class of compounds. Typical sub-groups of triterpene glycosides were classified according to the difference of substitution or oxidization at C-20 ( Figure 2). Oleanolic acid and akebonoic acid are two classical core skeletons of triterpene glycosides found in E. senticosus leaves [3,8]. As shown in Figure 2, αand β-sugar chains connect at C-28 and C-3, respectively. The sugar chains, α-S1 and α-S2, both contain the moiety glucose-glucose-rhamnose, but α-S1 has an extra acetyl group connecting at C-6 of the second glucose [3]. MS/MS spectra of α-S1 and α-S2 show fragments of m/z 513 and 471, respectively, corresponding to dissociation of α-S1 or α-S2 from C-28. Following that, α-S1 produces fragments at Flavonoids are another group of compounds in E. senticosus leaves with typical UV absorptions around 203, 255, and 354 nm that are related to flavonoid core quercetin or kaempferol. These compounds contain key fragment ions at m/z 303 or 287 Da corresponding to aglycone quercetin or kaempferol, respectively, in the MS and MS/MS spectra. Further, a neutral loss of 162 or 146 Da related to the loss of hexose and deoxyhexose, respectively, are commonly found for flavonoid glycosides. In E. senticosus leaves, rutin eluted at 7.22 min and is confirmed by comparing the retention time, UV, MS, and MS/MS spectra with that of a standard compound. Key UV absorption and MS/MS fragments of rutin are listed in Table 1 along with the other six flavonoid glycosides.
Triterpene glycoside compounds are key constituents in E. senticosus leaves, but show weak UV absorption due to a lack of obvious chromophore on the triterpene aglycone. Investigation of the MS/MS fragmentation pattern provides a tool to characterize this class of compounds. Typical subgroups of triterpene glycosides were classified according to the difference of substitution or oxidization at C-20 ( Figure 2). Oleanolic acid and akebonoic acid are two classical core skeletons of triterpene glycosides found in E. senticosus leaves [3,8]. As shown in Figure 2, α-and β-sugar chains connect at C-28 and C-3, respectively. The sugar chains, α-S1 and α-S2, both contain the moiety glucose-glucose-rhamnose, but α-S1 has an extra acetyl group connecting at C-6 of the second glucose [3]. MS/MS spectra of α-S1 and α-S2 show fragments of m/z 513 and 471, respectively, corresponding to dissociation of α-S1 or α-S2 from C-28. Following that, α-S1 produces fragments at m/z 367 and 205, respectively, corresponding to ions of     Figure S1), a key fragment at m/z 439.3 Da indicates that ciwujianoside C4 contains the aglycone of sub-group II saponins. In addition, fragments at m/z 513.1, 367.1, and 205.0 Da correspond to the loss of sugars of the α-S1 sugar chain at C-28 ( Figure 3); fragments at m/z 1101.6 and 969.5 Da are related to the loss of rhamnose and arabinose from C-3, respectively. Therefore, the fragmentation pathway of ciwujianoside C4 is proposed in Figure 3 in which ions of m/z 351.1, 315.1, 309.1, 279.1, and 273.0 Da are essential fragments related to the α-S1 sugar chain. Reviewing results of all the samples, tea products of E. senticosus EPS-3 to EPS-7 and EPS-9 to EPS-11 have identical profiles with that of authentic E. senticosus leaves. Their confirmed plots and tables are same as samples of E. senticosus leaves (Figure 4). However, profiles of samples EPS-1, EPS-2, and EPS-8 are different with that of authentic plant material ( Figure 5). Major components between 2 and 8 min are noteworthy in these E. senticosus tea products. The compound at 3.78 min displays a UV absorption of λmax at 205 and 272 nm and molecular ions of m/z 195.0873 ([C8H11N4O2] + , calc. 195.0882). In addition, compounds at 3.26, 5.01, 5.19, and 7.48 min contain common key fragments of m/z 139 Da and similar UV absorption around 205, 230 (shoulder), and 274 nm. Using the developed personal library, which contains compounds identified from Eleutherococus species and green tea as well as the built-in UNIFI TCM library, the compound at 3.78 min is identified as caffeine. The components at 3.26, 5.01, 5.19, and 7.48 min were determined to be epigallocatechin, epicatechin, epigallocatechin gallate, and epicatechin gallate, respectively, by using the in-house created UNIFI library and confirmed using injections of reference standards. An authentic green tea sample is prepared and tested by the developed UHPLC-UV-MS/MS method. Products EPS-1, EPS-2, and EPS-8 are identified as green tea products mixed with E. senticosus leaves. However, labels of products EPS-1, EPS-2, and EPS-8 do not list green tea as an ingredient. Therefore, the green tea in products EPS-1, EPS-2, and EPS-8 is considered an adulterant that would enhance the tasting flavor and reduce the products' cost. Saponins in sub-group I(a), I(b), and III present very similar fragmentation patterns as sub-group II triterpene glycosides, such as ciwujinoside C4. Determination of sugar chains at C-3 and C-28 can follow the same rule as the ciwujianoside C4 example in Figure 3. The key point is to identify the core skeleton of saponins' aglycone. MS and MS/MS spectra of sub-group I(a) usually shows ion pairs at m/z 441.3 and 423.3 Da, but sub-group I(b) only shows ions at m/z 423.3 Da. In sub-group III saponins, ions at m/z 455.3 Da are fragments indicating the existence of the sub-group III core ( Figure 2). In summary, the characterization of key fragments of 441/423 pair, 423, 439, and 455 Da can differentiate compounds in sub-group I(a), I(b), II, and III. Sugar chains at C-28 (α-S1 and α-S2) easily break down completely and the newly forming α-sugar chains fragments are more likely to yield sugar fragments step by step as shown in Figure 3. The sugar chain at C-3 (β-sugar chains, Figure 2) usually dissociates one-by-one from the far end of sugar chains. Therefore, C-3 and C-28 can be determined on the basis of MS and MS/MS along with reference standards and known values from the literature. In this work, a total of 30 saponins are characterized in Table 1.
To create a processing method for the quality control analysis of E. senticosus and related products, MS and MS/MS raw data of authentic plant samples, reference standards, and testing samples are imported into an LC-UV-MS, the screening platform, UNIFI, for characterization. A personal library with 241 entries of compounds identified from Eleutherococus species and green tea is compiled and used for the screening of 13 of E. senticosus plant material and 11 of ci-wu-jia tea products. To ensure the processing method contained all the specific parameters required to screen E. senticosus and related products, several iterations of method development were performed. In the preliminary UNIFI processing method, non-pecific parameters, such as peak processing settings using 3D peak apex, target by mass of targeted screen settings, common fragment and neutral loss parameters of discovery settings, and adducts and lock mass of analysis specific settings, were set up. When reviewing the processed data, the reference standards for E. senticosus were confirmed by screening these compounds in the authentic E. senticosus leaves. Information, such as the accurate mass, MS/MS fragments, UV absorption, fragmentation pattern, and retention time of the reference standards, were determined experimentally as well as referencing previous literature findings in authentic E. senticosus leaves [3][4][5]. Following preliminary processing, the method was updated using specific parameters. Reviewing results of all the samples, tea products of E. senticosus EPS-3 to EPS-7 and EPS-9 to EPS-11 have identical profiles with that of authentic E. senticosus leaves. Their confirmed plots and tables are same as samples of E. senticosus leaves (Figure 4) In addition, compounds at 3.26, 5.01, 5.19, and 7.48 min contain common key fragments of m/z 139 Da and similar UV absorption around 205, 230 (shoulder), and 274 nm. Using the developed personal library, which contains compounds identified from Eleutherococus species and green tea as well as the built-in UNIFI TCM library, the compound at 3.78 min is identified as caffeine. The components at 3.26, 5.01, 5.19, and 7.48 min were determined to be epigallocatechin, epicatechin, epigallocatechin gallate, and epicatechin gallate, respectively, by using the in-house created UNIFI library and confirmed using injections of reference standards. An authentic green tea sample is prepared and tested by the developed UHPLC-UV-MS/MS method. Products EPS-1, EPS-2, and EPS-8 are identified as green tea products mixed with E. senticosus leaves. However, labels of products EPS-1, EPS-2, and EPS-8 do not list green tea as an ingredient. Therefore, the green tea in products EPS-1, EPS-2, and EPS-8 is considered an adulterant that would enhance the tasting flavor and reduce the products' cost.

Instrumentation and Chromatographic Conditions for UHPLC-UV-MS Analysis
All samples were analyzed by using a Waters Acquity UPLC HSS T3 column (100 × 2.1 mm i.d.,
The high-resolution ESI-MS experiments were carried out on a Xevo G2-S QToF mass spectrometer that was connected to the UHPLC system via an ESI interface. The ESI source was operated in the positive ionization mode with the following settings of the parameters: 3.5 kV capillary voltage; 35 V cone voltage; 85 and 450 • C for ion source and desolvation temperature, respectively; and 50 and 900 L/h for cone and desolvation gas flows, respectively. Mass accuracy of the parent and major fragments in this study was limited within 5 ppm, but a few minor fragment ions were tolerated up to 10 ppm when considering its limited peak intensity in the analysis. Leucine-enkephalin was used for the lock mass at a concentration of 2 ng/mL and flow rate of 5 µL/min. Ions [M + H] + (m/z 556.2771 Da) and a fragment ion (m/z 278.1141 Da) of leucine-enkephalin were employed to ensure mass accuracy during the MS analysis. The lock spray interval was set at 30 s, and the data was averaged over three scans. The mass spectrometer was programmed to step between low (10 eV) and elevated (15-35 eV) collision energies on the gas cell, using a scan time of 0.1 s per function over a mass range of m/z 100-1500 Da.

Chemicals and Reagents
Methanol, acetonitrile, and formic acid were HPLC grade and purchased from Fisher Scientific. Water for the HPLC mobile phase was purified using a Millipore Synergy UV Water Purification System (Millipore SAS, Molsheim, France).

Plant Material and Confiscated Products
E. senticosus plant samples were collected in China by Dr. Yonghai Meng. E. senticosus tea products were purchased from local stores in Heilongjiang, China. The information of these samples is listed in Table 2. Specimens of all samples are deposited at the Repository of Botanicals, National Center for Natural Products Research, University of Mississippi, University, Mississippi, USA.

Sample Preparation
Extraction method was optimized in preliminary studies to ensure that the recoveries of major components were above 95%. The fine powder of the plant material or tea product (1 g) was accurately weighed and added into a 15 mL centrifuge tube. The sample was extracted with 2.5 mL of methanol in an ultrasonic water bath for 30 min, then followed by centrifugation at 959× g for 15 min. The supernatant was transferred to a 10 mL volumetric flask. The procedure was repeated three more times and the respective supernatants were combined. The final volume was adjusted to 10 mL with methanol. Prior to LC analysis, the prepared sample was mixed thoroughly. An adequate volume of extract was passed through a 0.45 µm polytetrafluoroethylene (PTFE) filter and collected in an LC sample vial.

Data Process and Analysis
All LC-UV-MS data was processed, peak picked, and analyzed using the UNIFI informatics platform (Waters, Milford, MA, USA). A three-dimensional (3D) peak detection algorithm was used to detect the peak apexes of all the ion responses based on their 3D shapes to obtain cleaner spectra and more accurate peak volumes than 2D extracted ion chromatograms. For determination of the 3D peak apex, the retention time and intensity threshold of high and low energy were set as 0.5-25 min, 50 counts, and 100 counts, respectively. Ammonium ion (NH 4 + ) was add as an adduct cluster for the mass defect search.

Conclusions
An UHPLC-UV-MS/MS method was developed for the characterization of different classes of compounds, including organic acid derivatives, flavonoids, and triterpene glycosides, in E. senticosus leaves and related tea products. According to the characteristic UV spectra, accurate mass, and MS/MS fragmentation mechanisms, 13 of the organic acid derivatives, seven of the flavonoids, and 30 of the triterpene glycosides were identified from E. senticosus leaves. A personal library of 241 entries related to the Eleutherococcus genus and green tea extracts was created in the UNIFI informatics platform. Using the UNIFI processing method that was established on the basis of the characteristics of identified compounds in E. senticosus leaves and green tea extract, 13 of E. senticosus leaves and 11 Eleutherococcus tea products were analyzed. Out of 11 commercial products, three samples, EPS-1, EPS-2, and EPS-8, were found to be adulterated with green tea. The approach in this work, which determined the UV, MS, and MS/MS characteristics of different classes of compounds in authentic samples and established a specific processing method to process and quantify testing samples, provides a comprehensive, but effective way to routinely analyze the quality control of complex products, such as herbal medicines and dietary supplements.