MS

Accepted Manuscript Title: Simultaneous Qualitative and Quantitative Evaluation of Ilex kudingcha C. J. Tseng by using UPLC and UHPLC-qTOF-MS/MS Authors: Jie Zhou, Huan Yi, Zhong-Xiang Zhao, Xue-Ying Shang, Ming-Juan Zhu, Guo-Jun Kuang, Chen-Chen Zhu, Lei Zhang PII: DOI: Reference:

S0731-7085(17)31865-4 https://doi.org/10.1016/j.jpba.2018.02.037 PBA 11805

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

26-7-2017 16-2-2018 17-2-2018

Please cite this article as: Jie Zhou, Huan Yi, Zhong-Xiang Zhao, Xue-Ying Shang, Ming-Juan Zhu, Guo-Jun Kuang, Chen-Chen Zhu, Lei Zhang, Simultaneous Qualitative and Quantitative Evaluation of Ilex kudingcha C.J.Tseng by using UPLC and UHPLC-qTOF-MS/MS, Journal of Pharmaceutical and Biomedical Analysis https://doi.org/10.1016/j.jpba.2018.02.037 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Simultaneous Qualitative and Quantitative Evaluation of Ilex kudingcha C. J. Tseng by using UPLC and UHPLC-qTOF-MS/MS

Jie Zhoua, Huan Yia, Zhong-Xiang Zhaoa, Xue-Ying Shanga, Ming-Juan Zhua, Guo-Jun

College of Chinese Traditional Medicine, Guangzhou University of Traditional Chinese

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a

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Kuangb, Chen-Chen Zhua, Lei Zhanga,*

Medicine, Guangzhou 510006, Guangdong, P. R. China

Guangzhou Institute for Drug Control, Guangzhou 510160, Guangdong, P. R. China

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b

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College of Chinese Traditional Medicine

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Corresponding author*：Lei Zhang, Professor

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Guangzhou University of Traditional Chinese Medicine

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232 East of Waihuan Road, University Town

Guangzhou 510006, Guangdong, P. R. China

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Tel：+86-20-39358081

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E-mail address: * [email protected]

Highlights

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•

Firstly

present

a

strategy

based

on

the

UHPLC-qTOF-MS/MS-oriented

characterizition and UPLC-DAD-based quantification for systematic assessment of the quality homogeneity of Ilex kudingcha C. J. Tseng.

•

A total of 55 compounds in Ilex kudingcha were firstly identified.

•

18 major compounds in Ilex kudingcha samples were simultaneously detected for the 31

first time, which is meaningful for sample discrimination.

ABSTRACT In this study, a systematic method was established for the holistic quality control of Ilex kudingcha C. J. Tseng, a popular functional drink for adjuvant treatment of diabetes,

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hypertension, obesity and hyperlipidemia. Both qualitative and quantitative analyses were conducted. For qualitative analysis, an ultra high performance liquid chromatography (UHPLC) coupled with an electrospray ionization quadrupole time-of-flight mass

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spectrometry (ESI-qTOF-MS) method was established for rapid separation and structural

identification of the constituents in Ilex kudingcha. Samples were separated on an ACQUITY UPLC HSS T3 C18 column (2.1 mm × 100 mm, 1.8 μm) by gradient elution using 0.1% (v/v)

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formic acid (solvent A) and acetonitrile (solvent B) as mobile phases at a flow rate of 0.25

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mL·min-1. The chromatographic profiling of Ilex kudingcha by UHPLC-qTOF-MS/MS

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resulted in the characterization of 53 compounds, comprising 18 compounds that were

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unambiguously identified by comparison with reference standards. For quantitative analysis, 18 major compounds from 15 batches of Ilex kudingcha samples were simultaneously

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detected by UPLC-DAD at wavelengths of 210 nm, 260 nm, and 326 nm. The method was validated with respect to precision, linearity, repeatability, stability, accuracy, and so on. The

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contents of the 18 target compounds were applied for hierarchical clustering analysis (HCA)

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and principal component analysis (PCA) to differentiate between the samples. The results of HCA and PCA were consistent with each other. Sample No. 1 differed significantly based on HCA and PCA, and the differentiating components were confirmed to originate from different

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batches of samples. Phenolic acids and triterpenes were found to be the main ingredients in Ilex kudingcha. This strategy was effective and straightforward, and provided a potential approach for holistic quality control of Ilex kudingcha.

Keywords: Ilex kudingcha C. J. Tseng 32

UPLC UHPLC-qTOF-MS/MS Quality evaluation Identification

1. Introduction

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Ilex kudingcha (Kuding tea), the dried leaf of the Ilex kudingcha C.J. Tseng belonging to the family Aquifoliaceae, is a popular functional tea for quenching thirst, removing phlegm,

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refreshing the mind and improving eyesight for a millennium [1]. Drinking Ilex kudingcha has

been reported to prevent various diseases, such as cardiovascular disease, diabetes, and metabolic syndrome [2-3].. Meanwhile, the different

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hypertension, obesity, cancer

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extracts and active components from Kuding tea, including triterpenoid saponins, phenolic

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acids and flavonoids, have been reported to possess significant antioxidative [4], antiobesity

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[5.6], anti-inflammatory [7], antidiabetic [8] and anticancer activities [9,10] in vitro or in vivo. Moreover, the contents of these components also contribute to the diversified function and

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tastes of different species of tea [11, 12]. Although chemical constituents of Ilex kudingcha have been intensively studied by phytochemistry [2,13-14], the chemical quantitative profile

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of this natural health drink remains unfamiliar and quality evaluation has been limited to the

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determination of 2-6 phenolic acids [15,16], 1-2 flavanoids [17,18], or 2-5 triterpenes [19,20], independently. The quality control of Ilex kudingcha is essential owing to its multiple substitutes being used in some areas. It is universally acknowledged that multiple components

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of the natural products act on multiple targets or interact with different biochemical pathways to synergistically perform the overall health promoting functions [21]. It is therefore vital to establish an analytical method that will enable holistic quality control of the Ilex kudingcha qualitatively and quantitatively. Ultra-performance liquid chromatography coupled with DAD has been one of the most

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promising developments for rapid, sensitive, and stable chromatographic quantification [22]. On the contrary, the well-developed UHPLC-TOF-MS/MS system launches a more comprehensive chemical profiling by providing accurate precursor and/or product ions information with a mass error of less than 5 ppm, which substantially enhances the reliability of components characterization [23-26]. Additionally, multivariate statistical analyses by

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classic pattern recognition segregate the chemical groups and rapidly identify discriminating components [27-29]. The integrated use of LC-qTOF-MS/MS qualitative strategy,

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LC-DAD-based quantification and chemometrics has been demonstrated to be a powerful tool for analysis of natural products [30,31].

In this study, the major goal was to analyze the chemical components in Ilex kudingcha

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qualitatively and quantitatively. UPLC-DAD for quantitative analysis was conducted for

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chromatographic profiling and determination of the contents of 18 components in Ilex

A

kudingcha, which was used for chemometric analysis and discrimination of varying samples.

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UHPLC coupled with electrospray ionization quadrupole time-of-flight mass spectrometry

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(ESI-qTOF-MS) was carried out for quality analysis. In sum, this study presented a strategy based on the UHPLC-qTOF-MS/MS-oriented characteristics and UPLC-DAD-based

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quantitative components data set for providing a rapid solution for multi-quantitative assessment of the quality homogeneity. To the best of our knowledge, this is the most

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comprehensive and systematic quality evaluation of Ilex kudingcha. 2. Experimental

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2.1. Reagents and chemicals HPLC grade acetonitrile (Merck, Darmstadt, Germany), formic acid (Reo Science Inc.,

USA), phosphoric acid (Kemiou Chemical Reagent Co. Ltd, Tianjin) and ultra-pure water (Huaren Yibao drinks Co, Hong Kong, China) were used in the mobile phase. Methanol (A.R.) for sample preparation was bought from Sinopharm Chemical Reagent Co. Ltd. (Shanghai,

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China). Protocatechuic acid (3,4-DA, R3), rutin (R4), chlorogenic acid (3-CQA, C2) and caffeic acid (CA, C4) were purchased from National Institute for Food and Drug Control (Beijing, China). Neochlorogenic acid (5-CQA, C1), cryptochlorogenic acid (4-CQA, C3), isochlorogenic acid B (3,4-diCQA, C5), isochlorogenic acid A (3,5-diCQA, C6) and isochlorogenic acid C (4,5-diCQA, C7) were bought from Weikeqi Biological Technology Co., (Sichuan,

China).

The

reference

substances

of

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Ltd

6-hydroxy-7.7α-dihydrobenzofuran-2(6H)-one (R1), hydroxyl-casein (R2), latifoloside G

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(K1), kudinoside G (K2), kudinoside A (K3), kudinoside E (R5), kudinside D (R6),

ilekudinoside T (K4), and latifoloside H (K5) were isolated from Ilex kudingcha, and their

of these reference substances were tested above 98%.

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2.2. Plant materials

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structures were fully characterized by chemical and spectroscopic methods [32]. The purities

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Fifteen commercial herbal samples of Ilex kudingcha were purchased from Guangzhou

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stores or markets (with serial batch numbers 1-15) in 2015, and were authenticated by

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Professor Jin-Song Zhou from Guangzhou University of Traditional Chinese Medicine. The voucher specimens were deposited at the Herbarium at Guangzhou University of Traditional

a desiccator.

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Chinese Medicine. The air-dried samples were ground into powder (40 meshes) and stored in

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2.3. UHPLC-qTOF-MS/MS for qualitative analysis The high-accuracy mass spectrometric data was recorded on a 5600+ quadrupole

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time-of-flight mass spectrometer (AB SCIEX, USA ), equipped with an Shimadzu UHPLC system, which included a LC-30AD pump, a SIL-30AC autosampler, and a CTO-20AC column oven. Chromatographic separation was achieved on an ACQUITY UPLC HSS T3 C18 column (2.1 mm × 100 mm, 1.8 μm) with 0.1% (v/v) formic acid (solvent A) and acetonitrile (solvent B) as mobile phases, using a gradient elution of 6% B at 0-1 min, 6-25% B at 1-7 min, 25% B

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at 7-10 min, 25-32% B at 10-11 min, 32-52% B at 11-18 min, and 52-90% B at 18-22 min. The chromatographic conditions were optimized to acquire a good separation within the duration of 25 min. The flow rate was set at 0.25 mL·min-1 and the temperature maintained at 30°C. Re-equilibration duration was set at 3 min between individual runs. After injection of 1 μL of sample, the column effluent was directly introduced into the heated electrospray (ESI)

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source and analysis was performed in negative ion mode. The ESI source parameters were as

follows: the ion spray voltage floating (ISVF), −4.5 kV; turbo spray temperature (TEM), 550°C;

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nebulizer gas (gas 1), 55 psi ; heater gas (gas 2) , 55 psi; curtain gas (CUR), 35 psi; collision

energy (CE), -5.0 eV for MS1 and -45 eV for MS2; and declustering potential (DP), -180 eV. Mass spectra were recorded in full-scan MS mode from m/z 100 to m/z 2000 with a 250 ms ion

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accumulation time after an external mass calibration was performed and data acquisition was

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controlled by MultiQuant 3.0 software (AB SCIEX, USA). The full scans were run with

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dynamic background subtract (DBS) and information dependent acquisition (IDA), which

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was used to trigger the acquisition of MS/MS analysis of low concentration constituents. For

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the IDA criteria, the 8 most intense fragment ions of each analyte that exceeded 100 cps were selected for a production scan. In addition, an automated calibration delivery system (CDS)

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could regulate the MS and the MS/MS every 3 h automatically. 2.4. UPLC-UV for quantification

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Quantitative analysis was performed on an ACQUITY UPLC H-Class system (Waters, USA),

R HSS T3 C18 (2.1 mm × 100 mm, 1.8 μm) column. The mobile using an ACQUITY UPLC○

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phase consisted of 0.05% aqueous phosphoric acid (A) and acetonitrile (B), using a gradient elution. The detection wavelengths were set at 210 nm, 260 nm, and 326 nm for recording the absorption maxima of different ingredients. The other chromatographic conditions, such as the gradient program, flow rate, column temperature, and injection volume were the same as those mentioned in section 2.3.

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2.5. Preparation of sample solutions Approximately 0.2 g of finely ground sample powder (40 mesh) was accurately weighed in a brown stoppered flask, accurately added 20.0 mL of methanol (A.R) and weighted, extracted using ultrasonication in an ice bath for 30 min after immersed at room temperature (25 ± 3°C) for 30 minutes. The extraction solution was allowed to cool and weighted again,

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the loss of solvent was replenished with methanol and mixed well. The extract was filtered through a 0.22-μm filter membrane prior to injection.

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2.6. Preparation of standard solutions

The appropriate amounts of reference substances of R1, R2, R3, 5-CQA, 3-CQA, 4-CQA, C4, R4, 3,4-diCQA, 3,5-diCQA, 4,5-diCQA, K1, K2, K3, R5, R6, K4, and K5 were

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accurately weighed and dissolved in methanol to prepare the reference standard test solutions.

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Meanwhile, a mixed stock solution of all eighteen reference compounds with concentrations

A

of 62.0 μg·mL-1, 380 μg·mL-1, 10.13 μg·mL-1, 101.7 μg·mL-1, 755 μg·mL-1, 101.0 μg·mL-1,

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106.0 μg·mL-1, 31.6 μg·mL-1, 106.6 μg·mL-1, 1365 μg·mL-1, 485 μg·mL-1, 1045 μg·mL-1, 915

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μg·mL-1, 1065 μg·mL-1, 60.0 μg·mL-1, 505 μg·mL-1, 39.0 μg·mL-1, and 99.8 μg·mL-1, respectively was prepared and then diluted to the appropriate concentration ranges for the

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calibration curves. All test solutions were stored at 4°C prior to analysis. 2.7. Establishment of the in-house library of Ilex kudingcha

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To ensure the reliability of peak identification, an in-house library that covered the known

major components in Ilex kudingcha was established by researching the databases of TCM @

Taiwan

(http://tcm.cmu.edu.tw),

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Database

(http://www.ncbi.nlm.nih.gov/pccompound),

CHEM-TCM

PubChem

Database

(http://www.chemtcm.com),

ChemSpider database (http://www.chemspider.com), MassBank (http://www.massbank.jp) data source, and other literatures [32-34]. 2.8. Data processing

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Information on more than 200 compounds, including trivial names, molecular formulas, and molecular weights was added into the Peak View Software

TM

V.1.1 (AB SCIEX, Foster

City, CA). The “match” function of the Peak View Software identified the compounds that matched the determined molecular formula as potential candidates. The eighteen peaks were positively confirmed by comparison of their retention times and fragmentation patterns with

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standards or reported literatures. Others that belong to the existing in-house formula database were tentatively identified without reference compounds by the following aspects:

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comparison to a clear extracted ion chromatogram, determining accurate mass with an error of 5 ppm, judging reasonable fragmentation pathway corresponding to the structure, and similar compounds to validate the correctness.

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The relative retention time (RTR) of the 18 components peaks grouped by detection

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wavelength versus the corresponding reference peaks [R4 (λmax = 260 nm), C6 (λmax = 326

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nm) and K3 (λmax = 210 nm)] was calculated. The HCA analysis was realized by SPSS 19.0

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software. Between-group linkage method was applied, and euclidean distance was selected as

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a measurement. Dendrogram resulting from the content of the 18

components was derived

from the UPLC profiles of the tested samples. The PCA analysis was done by SIMCA-P 13.0 components in 15 batches of samples

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software. In this study, content of the detected 18

composed a data matrix with 15 rows and 18 columns, which was used for the PCA analysis.

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3. Results and Discussion

3.1. Optimization of the extraction procedure

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According to the literature, sonication with an aqueous solution of methanol was the

preferred extraction method [35, 36]. To optimize the effects of other variables on the extraction efficiency of the 18 components involved in this procedure, an univariate test was employed in terms of different solid-liquid ratio

(30-fold, 50-fold and 100-fold), extraction

time (15 min, 30 min, 45 min and 60 min), and extraction frequency (one time, twice and

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three times). The extraction efficiency was assessed by comparing the sum-content of each category components, respectively, and the most efficient extraction was considered as the one which extracted the highest amount of these components. As for the single factor cycle experiment, 100-fold of methanol solvent volume, and one time extraction frequency were tentatively chosen as procedure to evaluate the influence of extraction duration (factor 1).

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Following this way, we in turn investigated the solvent multiple and extraction frequency. One complete extraction experiment counted as one run. There were 30 extraction experiments in

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total. As a result (supplementary Fig. 1S.), the extraction procedure, namely ultrasonication

under an ice bath using 100-fold of methanol for 30 min once, turned out to be sufficient for complete extraction of the target compounds.

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3.2. Optimization of UPLC-UV and UHPLC-QTOF-MS conditions

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In order to achieve proper separation for determination of multiple ingredients, several

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parameters were varied: elution programs, elution solvent systems (water-acetonitrile and

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water-methanol), and concentrations of phosphoric acid in the solvent (0.02%, 0.05%, and

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0.1%). From the results of linear gradient elution, water (containing 0.05% phosphoric acid) and acetonitrile showed the best resolution. Acetonitrile showed better separation efficiency,

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reduced background noise, and a gentler baseline drift with comparation to methanol as expected. Additionally, the addition of phosphoric acid had a substantial effect on reducing

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peak tailing of organic acids, and its lack of UV absorption made it suitable for gradient elution and analysis of the components with detection wavelength close to the end-absorption.

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Different columns, including Waters Acquity HSS T3 UPLC column (2.1 mm × 100 mm, 1.8 μm) and YMC-Triart C18 UPLC column (2.0 mm × 100 mm, 1.9 μm) were evaluated. Different column temperatures (25°C, 30°C and 35°C) and flow rates of mobile phase (0.2 mL·min-1, 0.25 mL·min-1 and 0.3 mL·min-1) were also compared. The chromatographic condition of Waters Acquity HSS T3 UPLC column (2.1 mm × 100 mm, 1.8 μm), 30°C

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column temperature and flow rate of 0.25 mL·min-1 was chosen for optimum separation. The detection wavelengths were set at 260 nm, 326 nm and 210 nm, in accordance with the maximum absorption values of the analytes. Different MS parameters, such as detection mode, collision energy, and declustering potential (DP) were investigated. The results showed that more information on active

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ingredients was obtained in the negative-ion ESI spectrum than in the positive-ion ESI spectrum. By comparing the mass response of representative phenolic acids and triterpenoid

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saponins versus DP value ranging from -60 eV to -200 eV, it was found that the signal abundance of most triterpenoid saponins was improved with an increasing DP value, whereas

an opposite trend was observed for most phenolic acids. Thus, the MS conditions with DP

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values of -60 eV and -180 eV were chosen for independent collection of TIC chromatograms.

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Increasing the CE for MS2 would heighten the signal abundance of triterpenoid saponins, so

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the CE of MS2 was set at -45 eV. The other MS condition parameters, including ion spray

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voltage floating (ISVF), turbo spray temperature (TEM) , nebulizer gas (gas 1), heater gas (gas

recommended values.

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2), curtain gas (CUR), and CE for MS1 were adopted according to a general rule of

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3.3. Characterization of the chemical constituents of Ilex kudingcha Detailed clarification of the chemical constituents in Ilex kudingcha is essential for holistic

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quality control. In order to accomplish comprehensive characterization, an in-house library that covered the major constituents in the Ilex kudingcha was established, and input into the

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Peak View Software TM V.1.1 (AB SCIEX, Foster City, CA). In order to understand better the MS fragmentation patterns of the constituents in Ilex kudingcha, 18 authentic compounds, including

saponins,

phenolic

acids,

and

flavonoids

were

investigated

by

UHPLC-qTOP-MS/MS. In the full scan mass spectra, most of the authentic compounds exhibited [M-H]- in negative mode. Owing to the presence of formic acid in the mobile phase,

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ions of [M-H+HCOOH]- were observed for most saponins. Multiple approaches, including database search, reference standard comparison, and qTOF-MS and MS/MS data analysis were employed for structural characterization of the constituents of Ilex kudingcha. The chemical formula of an unknown structure was deduced based on the high-accuracy [M-H]-/[M+HCOO]- (in the negative ion mode) precursor ion. The compound that matched

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the determined chemical formula and that was considered to have undergone the observed

MS/MS fragmentation was selected as the final identity. Fig.1 showed the representative

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negative total ion chromatogram (TIC) of Ilex kudingcha. Among 60 labeled peaks, a total of 53 compounds, including 16 phenolic acids, 28 terpenoids, and 3 flavonoids were identified or partially characterized. Among these components, 18 compounds were unambiguously

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identified by comparison with reference standards in terms of the tR and MS/MS product

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fragments. Information regarding the characteristic constituents, such as tR (min), molecular

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formula, observed m/z values, mass error (in ppm), and MS2 product fragment ions was

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summarized in Table 1. Their structures were shown in Fig. 2.

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3.3.1. Flavonoids

Three common flavonoids were identified from Ilex kudingcha. The negative ion MS/MS

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fragmentations of flavonoid aglycones are characterized by the Retro-Diels-Alder (RDA) cleavage of ring C (1,3A- and

1,3

B-) and multiple neutral loss pathway, whereas sequential

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elimination of sugar residues, combined with aglycone fragments are diagnostic for the characterization of glycosidic flavonoids [37,38]. For the flavone reference compound of

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quercetin, the product fragments observed at m/z of 151 ([C7H3O4]-) and 149 ([C8H5O3]-) corresponded to its

1,3

A- and

1,3

B- ions. In the case of rutin, the liquiritigenin ion at m/z 301

(aglycone ion) was obtained after eliminating the (-rha-glu) residue, which further fragmented into product ions m/z 151 ([C7H3O4]-) and 149 ([C8H5O3]-), consistent with the typical

1,3

A-

and 1,3B- fragments, respectively. A series of regular neutral losses, such as 28 Da, 44 Da, and

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56 Da, and the observation that flavones with multi-hydroxy substitution could yield ions like [M-H-C3O2]-, [M-H-C2H2O]-, or [M-H-CH2O]- contributed to confirmation of the components [39]. These MS/MS cleavage features assisted in identification of the unknown compound 13. The [M-H]- precursor ion at m/z 593.1508 indicated the possible molecular formula C27H30O15, which matched nicotifloroside on the basis of in-house data library search. Its MS2

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fragmentation produced three major product ions at m/z 285 ([M-H-rutinoside]-), 255 ([M-Hrutinoside-CH2O]-), 229 ([M-H-rutinoside-2CO]-), which further confirmed the identity of

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compound 13 as a flavonoid. 3.3.2. Identification of saponins

Triterpenoids were another major group of bioactive components from Ilex kudingcha.

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Most of them were pentacyclic triterpenoids, as shown in Fig. 2. As summarized in Table 1, a

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total of 28 saponins were identified and tentatively characterized, based on their mass spectra

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and literature reports [40]. Triterpenoids were difficult to be fragmented, and additional

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complex mass fragmentation could not be observed in the MS/MS spectra, except

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predominant ions of [M+HCOO]-, [M-H]-, and [M-H-saccharide groups]-. Therefore, most unknown triterpenoids were characterized based on accurate mass measurements (error ≤

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5ppm) and available references mentioned above. 3.3.3. Identification of chlorogenic acids (CGAs)

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This study provided a much higher mass resolution and accuracy, which facilitated the identification of known and unknown constituents in Ilex kudingcha. Three caffeoylquinic

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acids (CQA, 5-7) and three dicaffeoylquinic acids (diCQA, 14-16) were unambiguously identified in the extracted ion chromatograms and assigned as neochlorogenic acid (5), chlorogenic acid (6), cryptochlorogenic acid (7), isochlorogenic acid B (14), isochlorogenic acid A (15), and isochlorogenic acid C (16) by comparison with reference standards, in terms of the tR and MS/MS product fragment ions. -In the previous study, diagnostic product ions of

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chlorogenic acids have been summarized based on the high-resolution and low-resolution MS data acquired [41-43]. Here, we took the isomeric forms of diCQA as examples to demonstrate their structural characterization. They yielded [M-H]-/[diCQA-H]- ions at m/z 515.1178 (C25H23O12). In the negative ion mode, CID of [M-H]- led to sequential eliminations of caffeoyl units to generate an MS2 base peak ion at m/z 353.0867 ([M-H-caffeoyl]-/[CQA-H] ,) and an abundant product fragment ion at m/z 191.0559 ([M-H- 2caffeoyl]-/[quinic acid-H]).

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-

This was further cleaved into 173.0445 ([M-H-2Caffeoyl-H2O]-/[quinic acid-H-H2O]). m/z

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179, representing a ([caffeic acid-H]-) fragment, yielded a relatively abundant product ion at m/z 135 of ([caffeic acid-H-CO2]-). Thus, neutral loss of 1~2 caffeoyl units, transitions from

[M-H]- to [caffeic acid-H]-, and subsequent neutral loss of CO2 provide diagnostic information

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regarding caffeoylquinic acid [44]. It is also reported that the linkage position of caffeoyl

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groups on quinic acid could be determined according to the relative intensities of ESI-MS2

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base peak ion and dominant product ions [43]. Based on the HR-MS information, the

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differentiated retention behavior and fragmentation patterns in the literarue [43], except for

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these six caffeoylquinic acids, another three potential chlorogenic acids (feruloylquinic acids type, FQA) were tentatively identified as 3-FQA, 5-FQA and 4-FQA, respectively.

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3.3.4. Identification of organic acids

Only two asiatic acid-type of organic acids (Peak 57, 58) were characterized in this study.

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The [M-H]- precursor ion for 57 appearing at 453.3356 indicated the molecular formula to be C30H46O3, consistent with Ilelic acid C in the in-house database. Further evidence was

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obtained from the negative MS/MS fragmentation ion at m/z 407 ([M-H-CH2O2]-). The neutral loss of CH2O2 was considered a diagnostic fragment for the asiatic acid-type of ingredients and was readily observed for both peaks 57 and 58 [45]. For compound 58, only the [M-H]- parent ion was detected at m/z 487.3410 for C30H48O5 (-3.7 ppm). Upon CID, it generated m/z 453 ([M-H-2OH]-) and 407 ([M-H-2OH-CH2O2]-. These fragments were consistent with the

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previous report of asiatic acid [46]. 3.4. UPLC-DAD quantification 3.4.1. Matrix effect and selectivity The degree of interference by endogenous substances in extraction solvent was assessed by inspection of chromatograms derived from processed blank methanol and the samples. The

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selectivity was estimated by comparing the consistency of retention time and UV spectrum of

each analyte between a sample and its corresponding reference standard. Fig.3 showed the

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typical separation of blank solvent sample, a standard mixture (A)-(C) and Ilex kudingcha extracts (A)’-(C)’ under the optimized chromatographic conditions. No detectable interference was found. The 18 detected components were well resolved from background peaks, and

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showed a good resolution of adjacent peaks within the duration of analysis. Fig.3D and D’

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showed that the UV spectra of the eighteen analytes in Ilex kudingcha were identical to those

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of the standards, indicating that the specificity met the requirements of the analysis.

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Meanwhile, peak purity detection function of PDA was applied, which confirmed the

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acceptable purity of the eighteen analytes peaks in the sample chromatograms. 3.4.2 .Calibration curves and LOQ

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A stock solution, as described in section 2.5, containing eighteen reference standards, was prepared and diluted with methanol to appropriate concentration ranges for preparing the

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calibration curves. Each calibration curve was performed with six different concentrations in duplicates by plotting the peak area (y) versus concentration (x). All calibration curves

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showed good linearity with correlation coefficients (r2) no less than 0.9994. The limits of quantification (LOQ) under the chromatographic conditions were determined at a signal-to-noise ratio (S/N) of 10. It was found that the LOQs for most components, except for triterpenes of K1, K2, K3, K4and K5, could reach a concentration of 0.2~0.8 µg/mL, which might be due to the high column efficiency and the determination of these analytes at their

44

maximum UV absorption. The regression equations, linear range, and LOQ were summarized in Table 2. The RSD values of RTR were less than 1.61%, which suggested that RTR obtained on the same instrument and column at different concentrations were highly reproducible. 3.4.3. Precision, repeatability, stability, and accuracy Precision, repeatability, stability, and accuracy of the method were validated for each

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analyte. The precision was determined from the intra- and inter-day variations. For the

intra-day variability test, a sample solution, prepared as described in Section 2.5, was

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analyzed for six replicates within one day, while for the inter-day variability test; the sample was examined in duplicates for three consecutive days. The relative standard deviation (RSD)

for peak area was calculated as the measure of precision. To confirm repeatability, six Ilex

U

kudingcha samples from the same batch were extracted using the same preparation protocol

N

outlined in section 2.5 and analyzed. The stability was tested with a sample solution that was

A

stored at 10°C and analyzed at 0 h, 2 h, 4 h, 6 h, 8 h, and 12 h. Relative standard deviations

M

(RSD) of intra- and inter- day precision, repeatability, and stability were less than 4.3%, 4.6%,

ED

5.7% and 4.8%, respectively (Table 3). The accuracy was validated by analyzing spiked samples. A known amount of the standards at medium concentration levels were added to the

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samples (0.1 g) and then extracted and analyzed following the same procedure. Six replicates were performed for the test, and the ratios of measured versus added amounts were calculated

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to reflect the recoveries. As shown in Table 3, the recoveries for eighteen components varied between 93.3% and 108.2% with RSDs less than 6.5%. The above data were considered

A

satisfactory for subsequent analysis of the samples. 3.4.4. Robustness In order to introduce the quantification method into different laboratories, the robustness test was performed. Four experimental conditions were slightly varied, including column temperature (±2°C), flow rate (±0.02 mL·min-1), wavelength (±1.0 nm) and concentration

45

of acid (±0.01%). The RSD value of each analyte’s content calculated by the external standard method at three levels of the above experimental conditions were used to determine the robustness of the method. As Table 1S illustrated, a slight variation of the flow rate (± 0.05), column temperature (±2°C), wavelength (±1.0 nm) and concentration of acid (±

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0.01%) had no obvious influence on the content of each detected component with RSD less than 5.35%. Moreover, judging from RSD, it seemed that variation of flow rate at 0.05 interval had relatively greater influence on analytes content compared with other experimental

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conditions. Therefore, the flow rate should be consistent for inter-lab using. Fortunately, modern primary UPLC instruments could generally meet the requirement.

U

3.5. Sample analysis

N

The validated method was employed to assay eighteen analytes in 15 batches of Ilex

A

kudingcha samples. The concentrations were calculated using the external standard method,

M

and the results were summarized in Table 4 and Fig.4A. Outliers were picked out by the box plot and marked as dots. It was found that the first and twelfth batches were the main outliers

ED

among the fifteen batches of Ilex kudingcha. The total contents of each type of compounds were calculated, and the results were summarized in Table 4 and Fig.4B. It was obvious that

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phenolic acids and triterpenoid saponins with the total content range of 65.4-169.0 mg/g and

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41.3-117.2 mg/g, respectively, were the most abundant constituents among the analytes. Several flavonoids isolated from this plant, such as keampferol, quercetin and apigenin, could not be detected in all the samples using a DAD detector. Therefore, phenolic acids and

A

triterpenoid saponins were considered the main characteristic ingredients in Ilex kudingcha. 3.6. Discrimination of Ilex kudingcha samples by chemometric analysis All the 18 components could be found in collected 15 samples. Although the chemical constituents of 15 certified samples from different geographical regions were similar, the content of each compound varied greatly. The discrimination of these samples by visualization 46

could be extremely difficult and time-consuming. In contrast, chemical pattern recognition is acknowledged as a more objective and effective method for identifying particular herbs from related species and evaluating the similarities and differences among medicinal materials. In this study, hierarchical clustering analysis (HCA) and principal component analysis (PCA) was performed to assess the quality of samples collected from different places in China and to

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explore the chemical markers contributing to their classification. In the HCA analysis,

‘Euclidean distance’ was selected for measurement, and the method of ‘Within-group Linkage’

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was applied. As shown in Fig.5A, at the first clustering level, the tested fifteen populations

were classified into two main groups (I and II) based on their data contents. Sample No. 1 from Hainan with significant content differences in each type of component was clearly

U

distinguished from other original herbal samples. At the second clustering level, sample No.

N

12, 10, and 9 in group I could clearly be categorized into another group. In terms of PCA

A

analysis, the first three principal components (PCs) cumulatively accounted for 72.7% of total

M

variance, which indicated that the three principal components could be focused on for

ED

comprehensively reflecting the overall information. Through a visual analysis of Fig.5, the consistent distinguishing trend with HCA was compared. The 15 batches of samples could be

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discriminated into three zones by the unsupervised method and S1 scattering on the boundary of the circle was easily distinguished from the other samples. In particular, the distance of the

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other zones to the central sphere reflected the degree of discrepancy between different original samples. Besides the quantitative 18 chemical markers, the other detectable chemical markers

A

had also been added for chemometric analysis by using their absolute peak areas, and the same HCA and PCA classification results were obtained, which proved that these 18 chemical markers were the common characteristic peaks and could be applied for sample discrimination. Factor loading matrix was obtained after orthogonal rotation and the data was listed in Table 2S. From the result, seven components (R6, K4, C7, K1, K3, K2, R4) mostly

47

contributed to the first principal component and five components (R2, C6, C3, C5, R1) accounted for the second one. Based on these comparisons, it could be stated that the integrated use of chemometric analysis proved to be a powerful and easy strategy that could be used for the qualitative and quantitative assessment of Ilex kudingcha samples. 4. Conclusion

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As an integral aspect of a long-term project aimed at the exploration of new strategies for

applying integrated techniques to the contemporary quality control of complex nature

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products, the present study has outlined a detailed approach based on the characteristic

components data set (CCDS), multi-quantification, and pattern recognition chemometrics for the quality evaluation of Ilex kudingcha. Using eighteen components, a standard operational

U

procedure comprising of four successive steps was established to qualitatively and

N

quantitatively evaluate the qualities of Ilex kudingcha. The chromatographic profiling of Ilex

A

kudingcha by UHPLC-ESI-qTOF-MS/MS in a negative ionization mode resulted in the

M

characterization of 53 compounds covering different structural subtypes. The present study

ED

suggests that the integrated use of characteristic components data set and chemometric analysis is not only a powerful tool that can be applied for holistic quality control of complex studies of Ilex

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TCMs, but also will provide a basis for spectrum-clinical effect correlation

Kudingcha. Last but not least, the established HUPLC-QTOF-MS method can provide a

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helpful reference for efficient separation and identification of the components in other plants of the same genus.

A

Conflict and interest The authors declare no conflict of interest.

Acknowledgments This research work was supported by the Science and Technology Planning Project of Guangdong province (No. 2016A020226031 and 2017A020213022), the Program of Science

48

and Technology of Guangzhou (No: 201607010334) and the National Natural Science Foundation of China (Nos. 81673565 and 81270054). References . .

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Figure list: Fig. 1. The TIC of Ilex kudingcha in negtive ion mode obtained by UHPLC-qTOF-MS/MS. Fig. 2. Chemical structures of identified compounds in ilex kudingcha. 54

Fig. 3. Specificity test. A-A’: UPLC chromatograms at 210 nm; B-B’: UPLC chromatograms at 260 nm; C-C’; UPLC chromatograms at 326 nm; D-D’: UV spectrum of standards and analytes in sample solution. Fig. 4. The box plot of the 18 compounds` contents (A) and the histogram of category

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contents (B) of samples from different batches. Fig. 5. Results of hierarchical clustering analysis (A) and principal components analysis (B)

A

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PT

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M

A

N

U

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of the 15 samples of Ilex kudingcha C. J. Tseng.

55

I N U SC R A M ED PT CC E A

Fig. 1. The TIC of Ilex kudingcha in negtive ion mode obtained by UHPLC-qTOF-MS/MS.

56

57

A ED

PT

CC E A

M

N U SC R

I

58

A ED

PT

CC E A

M

N U SC R

I

I N U SC R A M ED

Fig. 2. The chemical structures of the identified components in Ilex kudingcha C. J. Tseng.

A

CC E

Samples

Standards

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Fig. 3.

59

ED

PT

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Standards

A

Samples

Standards

Samples

D’

60

A M

N U SC R

Samples

Standard s

DD

I

I N U SC R

Fig. 3. Specificity test. A-A’: UPLC chromatograms at 210 nm; B-B’: UPLC chromatograms at 260 nm; C-C’; UPLC chromatograms at 326 nm;

A

CC E

PT

ED

M

A

D-D’: UV spectrum of standards and analytes in sample solution.

61

Fig. 4.

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PT

ED

M

A

N

U

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A

A

Fig. 4. The Box plot of the 18 compounds` contents (A) and the histogram of category contents (B) of samples from different batches.

62

A ED

PT

CC E

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U

N

A

M

Fig. 5.

I

63

II

Fig. 5. Results of hierarchical clustering analysis (A) and principal components analysis (B) of the 15 sample

UPLC-qTOF-MS/MS in negative ion mode. Table 2. The results of linearity, LOQ and retention time (tR).

U

Table 3. Method validation test of UPLC-UV.

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Table 1. Identification of the chemical constituents of Ilex kudingcha by

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Table list

A

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M

A

N

Table 4. Contents of the 18 major constituents in 15 Ilex kudingcha samples (mg/g).

64

I N U SC R

(min)

M.F

Theoretical

Experimental weight of negative

M.W (Da)

qTOF-MS/error (ppm)

M

tR

MS/MS fragments (m/z)

Identification

503.1765[M+HCOO]-/(-1.0)

[503]: 221, 191, 179, 161, 89

Ilexperphenoside A*

152.0473

197.0451 [M+HCOO]-/(2.5)

[197]: 163

6-hydroxy-7,7a-dihydrobenzofuran-2(6 H)-one ( R1)

316.1158

361.1136 [M+HCOO]-/(1.9)

[361]: 343, 315, 297, 269, 253, 217, 201, 175, 161, 135,131, 101, 71

Hydroxyl-casein (R2)

C7H6O4

154.0266

153.0196 [M-H]-/(2.0)

[153]: 109,91,81, 65

Protocatechuic acid (R3)

C16H18O9

354.0951

353.0888 [M-H]-/(2.8)

[353]:191,179, 135

neochlorogenic acid (5-CQA, C1)

1.037

C21H30O11

2

3.621

C8H8O3

3

3.814

C14H20O8

4

4.280

5

4.902

458.1788

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PT

1

ED

No.

A

Table 1 Identification of the chemical constituents of Ilex kudingcha by UHPLC-qTOF-MS/MS in negative ion mode.

5.902

C16H18O9

354.0951

353.0886 [M-H]-/(2.3)

[353]: 191,179

Chlorogenic acid (3-CQA, C2)

7

6.098

C16H18O9

354.0951

353.0886 [M-H]- / (2.3)

[353]: 191,179,173, 135, 93

Cryptochlorogenic acid (4-CQA, C3)

[M-H]-/(-3.4)

[179]: 135

Caffeic acid (C4)

8

6.592

C9H8O4

180.0423

179.0338

9

7.330

C17H20O9

368.1107

367.1034 [M-H]-/(-0.1)

[367]: 301,193,161,135,133

3-FQA

A

6

10

7.821

C17H20O9

368.1107

367.1030 [M-H]-/(-1.4)

[367]: 301,191,161, 135, 133

5-FQA

11

8.043

C27H30O16

610.1534

609.1457 [M-H]-/(-0.7)

[609]: 301,271, 255, 151, 149

Rutin

12

8.202

C21H22O10

434.1213

433.1153 [M-H]-/(2.8)

[433]:271,179,161, 133,109

Vanillic acid 4-O-β-D-6-Obenzoylglucopyranoside

13

8.678

C27H30O15

594.1585

593.1508 [M-H]- /(-0.5)

[593]: 285, 255,229

nicotifloroside

14

8.789

C25H24O12

516.1268

515.1200 [M-H]-/(1.0)

[515]: 353,335,191, 179,173,161,135

Isochlorogenic acid B (3,4-diQCA, C5)

65

(R4)

I N U SC R

9.160

C25H24O12

516.1268

515.1197 [M-H]- /(0.4)

[515]: 353,191,179,173, 135

Isochlorogenic acid A (3,5-diQCA, C6)

16

9.601

C25H24O12

516.1268

515.1203 [M-H]- /(1.6)

[515]: 353,191,179, 135

Isochlorogenic acid C (4,5-diQCA, C7)

17

11.177

C17H20O9

368.1107

367.1023 [M-H]-/(-3.0)

[367]: 161,135,133

Unidentifed

Theoretical

Experimental weight of negative

tR (min)

M.F

M.W (Da)

Identification

367.1023 [M-H]- /(-3.0)

[367]:193,173,155,137,134,93,67

4-FQA

529.1369 [M-H]-/(3.4)

[529]:367,349,179,161,135

Macroantoin G

1382.6718

1427.6679 [M+HCOO]- /(-0.8)

[1427]: 1103, 1057, 937,791,749

Kudinoside O

1220.6190

1265.6182 [M+HCOO]-/(1.7)

[1265]: 1219, 911,749,471

latifoloside G (K1)

302.0427

301.0353 [M-H]-/(3.3)

[301]:151,149, 121,107,93,83,65

Quercetin

1088.5403

1133.5365 [M+HCOO]-/ (-1.8)

[1133]:1087,925, 701, 555

kudinoside C

912.5083

957.5081 [M+HCOO]-/ (1.8)

[957]: 911,749

latifoloside D

11.922

C17H20O9

368.1107

19

12.850

C26H26O12

530.1424

20

13.061

C65H106O31

21

13.187

C59H96O26

22

13.300

C15H10O7

23

13.471

C53H84O23

24

13.686

C47H76O17

PT

ED

18

CC E

MS/MS fragments (m/z)

qTOF-MS/error (ppm)

M

No.

A

15

13.749

C53H86O22

1074.5611

1119.5619 [M+HCOO]- /(2.3)

[1119]: 1073,911, 749, 603, 471

Kudinoside G (K2)

26

13.831

C42H66O15

810.4402

855.4346 [M+HCOO]- /(-4.3)

[855]: 809

Unidentifed

27

13.894

C53H86O21

1058.5662

1103.5677 [M+HCOO]-/(3.1)

[1103]:1057,937,791,749,731

ilekudinoside A

28

13.958

C47H76O19

944.4981

989.4959 [M+HCOO]-/( 0.7)

29

13.981

C49H78O19

970.5137

1015.5143 [M+HCOO]-/ (3.3)

30

14.326

C47H76O19

944.4981

989.4959 [M+HCOO]-/(0.7)

31

14.389

C47H74O18

926.4875

971.4868 [M+HCOO]-/(2.2)

[971]: 925,763,701, 555, 493

Kudinoside A (K3)

32

15.170

C59H96O25

1204.6241

1249.6218 [M+HCOO]-/(0.5)

[1249]:1203,895,733,455

latifoloside Q

A

25

latifoloside I-isomer [1015]: 926, 764

Chikusetsusaponin V methyl ester latifoloside I- isomer

66

I N U SC R

15.294

C47H76O19

944.4981

989.4959 [M+HCOO]-/(0.7)

[989]:911.5,62.0

Latifoloside

34

15.395

C53H82O22

1070.5298

1115.5309[M+HCOO]-/(3.6)

[1115]:1069,907, 745, 555

Kudinoside E*# (R5)

35

15.457

C47H76O19

944.4981

989.49589 [M+HCOO]-/(0.7)

[989]:911.5,62.0

latifoloside I- isomer

Theoretical

Experimental weight of negative

No.

tR (min)

M.F

A

33

M.W (Da)

MS/MS fragments (m/z)

qTOF-MS/error (ppm)

Identification

[987]: 939,,777

latifoloside J

953.4769 [M+HCOO]-/(2.9)

[953]:907,745, 555

Kudinoside D (R6)

764.4347

809.4326 [M+HCOO]-/(0.4)

[809]: 763,701,555

ilekudinoside B

776.4347

821.4318 [M+HCOO]- /(0.0)

[775]: 629

ilekudinoside K

618.3768

663.3850 [M+HCOO]- /(1.7)

[663]: 617,599,555,529,461,131

kudinoside I

C35H54O9

618.3768

663.3765[M+HCOO]- /(3.9)

[663]: 617,599,555,543, 501,483,439

kudinoside I- isomer

16.651

C47H74O17

910.4926

909.4890 [M-H]-/ (4.1)

[909]: 909,747,601

Ilexgenin B

43

16.719

C49H78O19

970.5137

969.5044 [M-H]-/ (-2.1)

[969]: 923, 761

Chikusetsusaponin V methyl ester

43’

16.718

C47H74O17

910.4926

955.4902 [M+HCOO]- /(0.4)

[955]:909, 745

Ilekudinoside T (K4)

43’

16.717

C47H76O18

928.5032

973.50043 [M+HCOO]- /(0.1)

[973]:

Ilekudinoside E or latifoloside I

44

17.223

C48H78O18

942.51882

941.5116 [M-H]-/ (0.1)

[941]: 895, 733

latifoloside J

44’

17.224

C47H76O17

912.5083

957.5085 [M+HCOO]- /(3.2)

[957]: 911, 749

45

17.340

C41H66O14

782.4453

781.4072 [M-H]- /(-0.9)

[781]: 745

latifolosideA or latifoloside B or latifolosideD Ilexsaponin C

46

17.353

C42H64O14

792.4296

791.4255 [M-H]-/(4.0)

[791]: 745

ilekudinoside N

46’

17.838

C41H66O13

766.4503

811.4450 [M+HCOO]- /(-3.2)

C48H78O18

942.5188

37

15.743

C47H72O17

908.4769

38

15.861

C41H64O13

39

15.934

C42H64O13

40

16.348

C35H54O9

41

16.462

42

PT

CC E

A

ED

15.601

M

987.51484 [M+HCOO]-/(-1.2)

36

Kudinoside H 67

I N U SC R

18.491

C47H74O16

894.4977

893.4915 [M-H]-/ (1.2)

[893]: 731,585,453

latifoloside H (K5)

48

19.961

C30H48O6

504.3451

503.3365 [M-H]-/(-2.6)

503.3

(+)-Arjungenin

49

20.220

C30H46O5

486.3345

531.3315 [M+HCOO]-/(-0.4)

[531]: 485,467,423,397, 381,329

β-Kudinlactone

Theoretical

Experimental weight of negative

M.W (Da)

qTOF-MS/error (ppm)

tR (min)

M.F

21.086

C35H56O8

604.3975

51

21.228

C30H46O5

486.3345

52

21.446

C30H48O4

53

21.547

C30H44O4

54

21.712

C29H42O4

55

21.933

C32H40O8

56

22.220

C35H58O6

MS/MS fragments (m/z)

Identification

[649]: 469,179,161,135, 106

Ilexoside A or Ilexoside B

485.3259 [M-H]-/ (-2.7)

[485]: 423,383,353,87, 57

β-Kudinlactone

472.3553

471.34751 [M-H]-/ (-0.8)

[471]: 441

Pomolic acid

468.3240

513.3196 [M+HCOO]-/(-2.9)

[513]: 468, 277,253

α-Kudinlactone

454.3083

453.30284 [M-H]-/(3.9)

[453]: 255, 195

ilekudinnol A

552.2723

551.2637 [M-H]-/ (-2.4)

[551]: 483,437,255,227, 171,153,79

Handelin

574.4233

619.4202 [M+HCOO]-/ (-0.5)

[619]: 453,163,119

Clerosterol

PT

CC E

649.3954 [M+HCOO]-/(1.1)

ED

50

M

No.

A

47

22.381

C30H46O3

454.3447

453.3356 [M-H]- /(-4.0)

[453]: 407, 405, 255,79

Ilelic acid C

58

22.374

C30H48O5

488.3502

487.3410 [M-H]-/(-3.7)

[487]: 453,407

asiatic acid

A

57

68

I N U SC R

Table 2 The results of linearity, LOQ and retention time (tR). Component

LOQ (μg/ml)

tR

RSD %

3.10-62.0

0.14

3.535

1.6

R² = 0.9999

19.00-380.0

0.18

4.098

1.4

y = 23615 x - 6410.9

R2

y = 16141 x - 38156

R3

y = 8772.1 x - 691.0

R² = 0.9996

0.51-10.2

0.37

4.309

0.95

C1

y = 11769 x - 9493

R² = 1

5.09-101.8

0.22

4.780

0.72

y = 10931 x – 258.4

R² = 0.9999

37.75-755.0

0.42

6.002

0.28

y = 10922 x - 13148

R² = 0.9999

5.05-101.0

0.34

6.252

0.22

y = 18876 x - 25782

R² = 0.9998

5.30-106.0

0.16

6.742

0.22

R4

y = 6416.7 x - 1738.3

R² = 0.9998

1.58-31.6

0.73

8.359

0.08

C5

y = 19677 x - 28546

R² = 0.9999

5.33-106.6

0.16

9.214

0.05

C6

y = 10343 x - 26275

R² = 0.9995

68.25-1365

0.28

9.570

0.05

C7

y = 15277 x - 74560

R² = 0.9999

24.25-485.0

0.22

10.061

0.06

K1

y = 580.9 x - 1834.2

R² = 0.9999

52.25-1045

2.53

13.767

0.06

K2

y = 200.3 x + 877

R² = 0.9997

45.75-915

5.91

14.317

0.07

K3

y = 1357.3 x - 6721.2

R² = 1

53.25-1065

1.21

15.004

0.07

R5

y = 6912.6 x - 3842.5

R² = 0.9999

3.00-60.00

0.46

16.240

0.08

R6

y = 7064.3 x - 44617

R² = 0.9999

25.25-505.0

0.62

16.647

0.08

K4

y = 1186.8 x – 82.35

R² = 0.9999

1.95-39.0

1.01

17.836

0.07

CC E

M

PT

C4

ED

C3

A

R1

C2

A

Liner range (μg/ml)

Standard curve

R² = 1

69

I N U SC R

y = 507.8 x -866.7

R² = 1

4.99-99.8

A

CC E

PT

ED

M

A

K5

70

3.19

18.053

0.06

I N U SC R

Precision

precision

Repeatability

Stability

Intra-day (RSD%, n = 6)

Inter-day (RSD%, n = 3)

(RSD%, n = 6)

(RSD%, n = 6)

x/%

RSD%

R1

2.1

2.7

3.5

2.6

97.7

4.6

R2

1.8

A

Table 3 Method validation test of UPLC-UV.

2.5

4.3

4.8

99.2

4.0

R3

3.5

4.3

5.2

3.2

101.5

2.8

C1

2.3

3.4

3.6

0.8

104.1

1.8

C2

1.7

1.9

3.4

1.7

95.7

3.5

C3

1.8

3.7

4.8

1.1

99.7

4.0

2.2

3.2

5.1

3.7

94.7

4.8

3.1

4.2

4.6

2.2

107.5

6.4

C5

1.8

2.3

5.2

2.3

96.5

2.4

C6

1.9

4.3

5.7

1.2

93.3

1.2

C7

1.5

2.2

5.0

1.6

95.6

2.0

K1

2.2

4.5

5.7

1.3

96.6

3.4

K2

4.3

3.2

5.6

2.5

105.1

4.9

K3

1.9

2.6

4.3

3.0

100.8

2.0

R5

4.1

4.6

3.8

3.6

96.4

2.3

R6

1.9

3.5

4.0

1.4

98.6

1.3

K4

4.1

3.9

6.1

3.9

107.5

5.9

A

ED

CC E

R4

PT

C4

M

Comp.

71

Recovery

I 3.3

N U SC R

K5

4.5

5.0

4.5

108.2

6.5

Table 4 Contents of the 18 major constituents in 15 Ilex kudingcha samples (mg/g). 2

3

4

5

R1

1.74

0.31

0.52

0.87

R2

34.3

5.46

2.74

8.54

R3 C1

0.18 4.14

0.16 6.63

0.41 4.2

C2

17.4

25.4

13

C3

0.81

5.56

C4

2.37

0.58

R4 C6

0.61 1.54 123.8

C7

K1

Peak

6

7

8

9

10

11

12

13

14

15

A

1

0.46

0.25

0.82

0.46

2.46

2.67

0.66

1.25

0.82

0.24

1.21

98.2%

2.78

1.82

4.04

2.54

1.01

12.88

5.75

10.97

7.01

1.92

7.02

97.8%

0.51 4.57

0.33 3.58

0.28 2.47

0.21 3.9

0.47 2.01

0.65 1.89

0.35 4.11

0.22 5.08

0.27 2.77

0.29 3.46

0.24 7.53

98.6% 100.0%

18.3

12.3

10.4

11.3

15.7

5.58

5.42

19.2

18.6

11.3

19.0

20.1

100.0%

5.41

3.6

4.76

3.08

3.73

3.92

1.79

2.03

4.15

4.71

3.65

4.31

5.36

100.0%

1.59

0.89

1.51

1.12

0.98

0.45

0.49

0.6

3.27

0.49

1.45

1.53

0.91

100.0%

0.88 3.22 37.0

0.42 4.81 34.9

0.61 4.14 58.9

0.32 3.88 36.7

0.35 2.86 28.5

0.47 3.87 30.9

0.25 4.14 28.2

0.24 4.3 71.2

0.26 2.92 76.4

1.17 3.69 43.6

1.72 4.51 70.1

0.76 4.62 38.5

0.33 5.54 50.9

1.18 4.56 58.0

98.0% 100.0% 100.0%

18.7

12.4

11.2

16.8

16.4

9.08

13.0

8.93

21.5

22.0

24.9

26.4

19.9

24.1

25.0

100.0%

31.4 30.3

11.65 9.81

6.38 7.03

15.43 11.8

9.84 6.65

7.45 4.12

13.9 13.32

11.0 4.42

18.7 12.92

16.3 15.9

28.0 23.6

39.2 33.2

22.4 25.7

18.2 11.0

18.1 12.3

100.0% 100.0%

R5

31.4 0.3

28.7 0.34

22.6 0.38

22.2 0.44

22.5 0.47

21.3 0.38

19.1 0.45

23.9 0.46

22.5 3.27

28.2 0.28

34.9 0.67

36.1 0.9

21.3 0.65

40.9 0.49

35.4 0.61

100.0% 97.4%

R6

3.9

3.34

3.86

3.04

3.15

2.71

3.51

3.19

2.78

3.23

6.39

6.53

4.99

5.02

4.03

100.0%

K4

0.28

0.23

0.19

0.13

0.44

0.21

0.12

0.3

0.33

0.26

0.96

0.77

0.49

0.46

0.47

99.9%

K5

0.57

0.6

0.87

0.42

0.74

0.7

0.53

0.57

0.72

0.5

0.28

0.39

0.38

1.06

0.63

100.0%

Sum-P.A

169.0

91.0

75.5

107.8

75.8

58.9

66.5

65.4

107.3

111.9

103.3

130.1

82.4

109.1

121.7

Sum-S

98.2 36.7

54.6 6.65

41.3 3.68

53.4 10.0

43.7 3.56

36.9 2.02

51.0 5.33

43.8 3.25

61.3 3.71

64.8 15.8

94.8 7.58

117.2 13.9

75.8 8.59

77.1 2.49

71.5 9.41

K2

A

K3

Sum-F&O

0.17 4.95

ED

PT

CC E

C5

M

Compounds

72

purity

I N U SC R

A

CC E

PT

ED

M

A

*Sum-P.A: sum content of Phenolic acids; Sum-S: sum content of Saponins; Sum-F&O : sum content of flavonoids and others.

73