J S S SEPARATION SCIENCE JOURNAL OF. Methods Chromatography Electroseparation. Applications Biomedicine Foods Environment

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1 J S S ISSN JSSCCJ 39 (7) (2016) Vol. 39 No. 7 April 2016 D JOURNAL OF SEPARATION SCIENCE 7 16 Methods Chromatography Electroseparation Applications Biomedicine Foods Environment

2 J. Sep. Sci. 2016, 39, Tao-fang Cheng Yu-ran Jia Zheng Zuo Xin Dong Ping Zhou Ping Li Fei Li State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, P. R. China Received November 17, 2015 Revised December 22, 2015 Accepted January 15, 2016 Research Article Quality assessment of traditional Chinese medicine herb couple by high-performance liquid chromatography and mass spectrometry combined with chemometrics This study was designed to develop a simple, specific and reliable method to overall analyze the chemical constituents in clematidis radix et rhizome/notopterygii rhizome et radix herb couple using high-performance liquid chromatography coupled with tandem mass spectrometry and multiple chemometric analysis. First, the separation and qualitative analysis of herb couple was achieved on an Agilent Zorbax Eclipse Plus C 18 column (250 mm 4.6 mm, 5 m), and 69 compounds were unambiguously or tentatively identified. Moreover, in quantitative analysis, eight ingredients including six coumarins and two triterpenoid sapogenins were quantified by high-performance liquid chromatography coupled with tandem mass spectrometry. In terms of good linearity (r ) with a relatively wide concentration range, recovery ( %) and repeatability ( %), the validation results suggested the proposed method was reliable, and successfully used to analyze ten batches of herb couple samples. Then, hierarchical cluster analysis and principal component analysis were used to classify samples and search significant ingredients. The results showed that ten batches of herb couple samples were classified into three groups, and six compounds were found for its better quality control. Keywords: Herb couples / Hierarchical cluster analysis / Mass spectrometry / Principal component analysis / Traditional Chinese medicine DOI /jssc Additional supporting information may be found in the online version of this article at the publisher s web-site 1 Introduction Traditional Chinese medicine (TCM), with notable effectiveness and few side effects, is gaining greater acceptance for preventing or healing a host of ailments worldwide. Generally, TCMs are composed of more than one herb that contains hundreds of chemical components [1], and the quality of each herbal medicine would differ according to cultivation region, harvest season, processing method, storage, and other factors [2]. Furthermore, the complex compositions of TCM could govern their clinical effects and safety, and separation and analysis of those chemical components are very important for TCM modernization [3]. However, identified and quantified Correspondence: Prof. Ping Li, State Key Laboratory of Natural Medicines, China Pharmaceutical University, No. 24 Tongjia Lane, Nanjing , P. R. China liping2004@126.com (P. Li) Fax: Abbreviations: CN, herb couple; CRR, clematidis radix et rhizome; HCA, hierarchical cluster analysis; NRR, Notopterygii rhizome et radix; PCA, principal component analysis; TCM, traditional Chinese medicine analytical markers or active markers of herbal medicines or formulations are limited, thus it is difficult to assess their quality, authenticity and even batch-to-batch consistency [4]. Clematidis radix et rhizome (CRR, Wei-Ling-Xian in Chinese), widely used as antioxidant [5], anti-inflammatory, against rheumatism, analgesic agent [6], antitumor [7], and antidiuretic [8] as a traditional medicine in Asia for a long time. Notopterygii rhizome et radix (NRR, Qiang-Huo in Chinese), is widely used for treating colds, inflammation, edema, arthritis, sores, ulcers [9] and as an analgesics [10], diaphoretics, and antifebrile agents [11]. Herb couple (two herbs ally), a basic composition of complicated Chinese herbal formulas, has special clinical significance and remain the basic therapeutic features of formulations according to TCM theory [12]. Additionally, herb couples are simpler and easier to prepare compared with complicated formulations. CRR/NRR herb couple (CN), is a well-known CN with remarkable clinical Additional corresponding author: Dr. Fei Li, State Key Laboratory of Natural Medicines, China Pharmaceutical University, No.24 Tongjia Lane, Nanjing , P. R. China lifeicpu@163.com (F. Li) Fax:

3 1224 T.-f. Cheng et al. J. Sep. Sci. 2016, 39, Table 1. The 10 batches of clematidis radix et rhizome notopterygii rhizome et radix herb couple from different origins for analysis Number Clematidis radix et rhizome Notopterygii rhizome et radix Cultivation region Voucher number Cultivation region Voucher number 1 Sichuan P Hebei P Sichuan Jilin Sichuan Anhui Qinghai Neimenggu Xizang Anhui Sichuan Jilin Sichuan Jilin Sichuan Liaoning Sichuan P Yunnan Gansu Heilongjiang Figure 1. The typical total ion chromatograms of CN herb couple by HPLC Q-TOF-MS in positive ion mode (A) and negative ion mode (B). efficacy. Several articles have reported that CRR contains triterpenoid saponins, alkaloids, phenolic glycosides, macrocyclic glucosides, flavonoid and lignans [8, 13 16], and NRR contains volatile oil, coumarins, phenethyl ferulate, falcarindiol, guaiane sesquiterpenes, polyacetylenes and derivatives [10, 17 19] using HPLC or UHPLC with ESI-MS, however, those methods were labor intensive, low sensitivity, or time and organic solvents consuming [20, 21]. In China pharmacopeia (2015 edition), notopterol and isoimperatorin are the analytical marker for NRR, and oleanolic acid is the analytical marker for CRR, respectively. In a word, few of them focus on comprehensive analysis of chemical constituents in CN to date. Chemometrics are effective methods for multivariate statistical analysis. Principal component analysis (PCA), a commonly used unsupervised dimension-reduction technique, can represent multivariate data sets in a low-dimensional plane and extract principal component of dependent and independent variables [3,22]. And as a conventional and unsupervised chemometrics technique, hierarchical cluster analysis (HCA) usually is used to assign samples into groups by creating a cluster tree based on the similarity of different samples [23]. Consequently, comprehensive analysis of chemical constituents is of importance to control quality of CN, and thus ensure its clinical effects and safety. In this study, a LC Q- TOF-MS method was developed for qualitative analysis of constituents, and HPLC MS was utilized for quantitative analysis in CN. Additionally, the classification and the importance of analytical markers in CN were further explored using HCA and PCA. Totally, 69 compounds in CN were unambiguously assigned or tentatively identified, and eight compounds, including six coumarins (originated from NRR) and two triterpenoid sapogenins (originated from CRR), were also quantified. Moreover, ten batches of CN samples were classified into three groups and six compounds were found for better QC in herbal couple compared with those makers stated in the China pharmacopeia for single herbal medicines, respectively, which could provide fundamental and supporting information for the industrial QC and clinical application of CN. 2 Materials and methods 2.1 Materials and chemicals Reference standards of chlorogenic acid (4), ferulic acid (15), nodakenin (17), bergaptol (25), bergapten (37), notopterol (53),isoimperatorin (58), hederagenin (62), bergamottin (68),

4 J. Sep. Sci. 2016, 39, Liquid Chromatography 1225 oleanolic acid (69), praeruptorin A (internal standard, IS1), and ruscogenin (IS2) were all purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). They were stored at 4 C before use. Ethanol was purchased from Tianjin concord technology (Tianjin, China). Acetonitrile was obtained from TEDIA (HPLC MS grade, Fairfield, Ohio, USA). Formic acid (HPLC grade) was from ROE (Neward, New Castle, USA). The deionized water (18 M ) was prepared using a Milli-Q water purification system (Millipore, Bedford, MA, USA). Ten batches of CN samples were purchased from different origins (Table 1). 2.2 Preparation of standard solutions The standard stock solutions of chlorogenic acid, ferulic acid, nodakenin, bergaptol, bergapten, notopterol, isoimperatorin, bergamottin, hederagenin, and oleanolic acid were dissolved in ethanol at the concentration of 1 mg/ml, respectively, for qualitative analysis. A certain amount of the ten aforementioned standard stock solutions were mixed and then diluted in ethanol to obtain standard mixture solution (about 50 g/ml for each standard). The solution was centrifuged at rpm for 10 min before HPLC Q-TOF-MS analysis. Accurately weighed amounts of eight reference compounds were dissolved in ethanol to prepare the mixed standard stock solution. Internal standard stock solutions of praeruptorin A (IS1) and ruscogenin (IS2) were prepared in ethanol at concentrations of and g/ml, respectively. The working solutions of these standards were obtained by adding different volumes of the two internal standards and diluting the mixed stock solutions with ethanol to appropriate concentrations. The final concentrations of IS1 and IS2 for all working standard solutions were 3.34 and 4.30 g/ml, respectively. All the above solutions were stored at 4 C. 2.3 Preparation of sample solutions for qualitative and quantitative analysis The dried powder of CRR and NRR stems (10 g, respectively, 40-mesh) were extracted with 95% ethanol for three times (3 100 ml, 1 h each) using heat reflux extraction, and the extracts were merged and concentrated under partial vacuum to 0.5 g/ml to obtain the concentrated liquid, and then diluted with ethanol at 1: 99 v/v ratio and centrifuged at rpm for 10 min to obtain the sample solutions. 2.4 Qualitative analysis by HPLC Q-TOF-MS The samples analysis were performed on Agilent 1100 series (Santa Clara, California, USA) equipped with an Agilent Table 2. Calibration curves, R 2, test ranges, LODs, LOQs, precision, repeatability, stability of the 8 compounds in CN herb couple No. Analytes Calibration curves R 2 Test ranges LODs LOQs Precision(RSD,%) Repeatability Stability Spiked recovery ( g/ml) ( g/ml) ( g/ml) (n = 6, RSD%) (n = 6, RSD%) (n = 6, RSD%) Intra-day(n = 3) Inter-day(n = 3) High Medium Low High Medium Low 17 Nodakenin y = x Bergaptol y = x Bergapten y = x Notopterol y = x Isoimperatorin y = x Hederagenin y = x Bergamottin y = x Oleanolicacid y = x

5 1226 T.-f. Cheng et al. J. Sep. Sci. 2016, 39, Figure 2. Chemical structures of 69 components identified from CN herb couple. zorbax plus C 18 analytical column (250 mm 4.6 mm, 5 m). The mobile phases consisted of deionized water containing 0.1% v/v formic acid (A) and acetonitrile containing 0.1% v/v formic acid (B), and the elution program was set as follows: 0 91 min, 1 77% B. The elution rate and column temperature were maintained at 1 ml/min and 40 C, respectively. The injection volume was set at 0.5 L. The qualitative analysis of mass detection was carried out on Agilent 6530 Q-TOF-MS equipped with ESI interface. The optimal parameters were as follows: drying gas (N 2 )flow rate was 11 L/min; drying gas temperature was set at 350 C; nebulizer gas pressure was 45 psig; fragmentor voltage was 135 V, and capillary voltage was set at 3500 V in negative ion mode and 4000 V in positive ion mode, respectively. The mass range was from m/z 100 to All the samples were analyzed both in negative and positive ion mode. 2.5 Quantitative analysis by HPLC MS The quantitative analysis were performed on Agilent 1100 series LC/MSD Trap system (Santa Clara, California, USA). The optimal separation of the eight markers in CN was carried on a Kromasil (150 mm 4.6 mm, 3.5 m) column using deionized water containing 0.1% v/v formic acid (A) and acetonitrile containing 0.1% v/v formic acid (B) as the mobile phases. The gradient elution condition was as follows: 0 9 min, 19 55% B; 9 32 min, 55% B; min, 55 82% B; min, 82% B. The elution rate and column temperature were maintained at 1 ml/min and 30 C, respectively, and injection volume was set at 2 L. The optimal MS parameters were as follows: positive ion mode; drying gas (N 2 ) flow rate was 10 L/min; drying gas temperature and nebulizer gas pressure were 350 C and

6 J. Sep. Sci. 2016, 39, Liquid Chromatography 1227 Figure 3. The proposed fragmentation pathway of Isoimperatorin (A) and Clematichinenoside AR (B). 45 psig, respectively; capillary voltage and fragmentor voltage were 4000 and 135 V, respectively. Analyte detection was performed in selective ion monitoring (SIM) mode by monitoring the molecular ions [M+H] +,[M+Na] +,or[m+h H 2 O] +. Supporting Information Table S1 shows the specifically selected ions for eight markers in the CN herb couple. RSD values. The recovery was used to evaluate the accuracy of the aforementioned method. A known amounts of references were added in the CN herb couple samples in sextuplicate. The spike recoveries were calculated by the following formula: Spiked recovery (%) = (observed amount original amount)/spiked amount 100%. 2.6 Method validation To improve quantification precision and repeatability, ruscogenin and praeruptorin A were chosen as internal standards for the quantitative determination of eight markers in CN. All the aforementioned stock solutions were all diluted to appropriate concentrations for calibration curves preparation, and the calibration curves were calculated using the ratio of the peaks area (analyte/is) and the concentrations of eight markers. The stock solutions were diluted with ethanol to a series of concentrations, and injected into HPLC MS for analysis. LODs and LOQs were determined at S/N = 3and 10, respectively. The CN herb couple samples were prepared and analyzed for five replicates to determine the repeatability of the aforementioned method that was represented by RSD value of the ratio of the peaks area (analyte/is). The intraand inter-day precisions, expressed by the RSDs of the data, were used to determine the precision of the aforementioned method. Three different concentrations of working solutions were analyzed for six replicates in a single day to evaluate the intra-day precision, and the same solutions were determined in successive three days to evaluate the inter-day precision for six replicates as well. The CN herb couple samples were analyzed over the period of 0, 2, 4, 6, 8, and 12 h to evaluate the stability of these samples that were represented by the 2.7 Application to quantitative determination of herb couple samples Ten batches of CN herb couple samples were diluted 100-fold with ethanol in a volumetric flask for the analysis of the eight markers, and the solutions were centrifuged at rpm for 10 min before analysis. 2.8 Chemometric analysis To explore the variability of the eight compounds among ten batches of CN samples, PCA and HCA were performed using the SIMCA-P 11.5 (Umetrics AB, Umea, Sweden) and Mev 4.6 (TM4, MA, USA) software, respectively. 3 Results and discussion 3.1 Qualitative analysis of herb couple samples by HPLC Q-TOF-MS These CN herb couple samples were analyzed by gradient reverse HPLC and ESI-Q-TOF-MS detection with both in positive and negative ion modes. The result showed that

7 1228 T.-f. Cheng et al. J. Sep. Sci. 2016, 39, Figure 4. The selected ion monitoring chromatograms of CN herb couple by HPLC MS in positive ion mode for eight marker compounds. 17, Nodakenin; 25, Bergaptol; 37, Bergapten; 53, Notopterol; 58, Isoimperatorin; 62, Hederagenin; 68, Bergamottin; 69, Oleanolic acid. the detection signals in positive ion mode were better than the negative ion mode according to sensitivity and selectivity. The developed method showed high precision with all the mass accuracy of less than 5 ppm. In this study, 69 compounds including 27 coumarins, 11 triterpenoid saponins, six lignans, two flavonoid, two polyacetylenes, one alkaloids, and 21 other compounds were observed, in which ten compounds were unambiguously assigned and 59 compounds were tentatively identified by comparing the retention time, UV spectra, fragmentation pathways, and MS/MS spectra data with previous literatures. The representative total ion currents (TICs) chromatograms with numbered peaks are illustrated in Fig. 1, and qualitative information are summarized in Supporting Information Table S2. Additionally, the structures of these identified compounds are shown in Fig. 2. In this study, coumarins were found to have higher mass response in positive ion mode. The coumarins, the main constituents in NRR, can be divided into three groups: furocoumarins, O-hemiterpenoidal coumarins, and pyranocoumarins. Twenty coumarins (peak 6, 11, 12, 17, 19, 22, 24, 25, 26, 27, 37, 38, 39, 44, 51, 53, 56, 58, 60, 69) were classified as furocoumarins. Six coumarins (peak 13, 23, 41, 48, 61, 65) were characterized as O-hemiterpenoidal coumarins. Lomatin (peak 16) was identified as pyranocoumarins. The characteristic fragmentation ions were traced by losing of H 2 O (18 Da), CO (28 Da), CO 2 (44 Da), C 3 H 6 (42 Da), C 4 H 8 (56Da),C 5 H 8 (68 Da), or Glc (162 Da) [24 27]. The proposed fragmentation pathways of representative coumarins are shown infig 3. Isoimperatorin (peak 58, t R = min) gave rise to ions at m/z , , , , , , , corresponding to [M+H] +,[M+H C 5 H 8 ] +, [M+H C 5 H 8 CO] +, [M+H C 5 H 8 CO 2 ] +, [M+H C 5 H 8 2CO] +,[M+H C 5 H 8 CO 2 CO] +,[M+H C 5 H 8 3CO] +,and [M+H C 5 H 8 CO 2 2CO] +, respectively. The fragmentation pathways of other coumarins types are shown in Supporting Information Fig. S1. Eleven triterpenoid saponins (peak 28, 29, 30, 32, 33, 34, 35, 43, 47, 50, 54) were tentatively identified from the pseudomolecular ions ([M H] or [M+Cl] )andms/msproduct ion profiles. In negative ion mode, the simultaneous or successive losses of Glc (162 Da), and/or Rha (146 Da), and/or Ara (132 Da), and/or Xyl (132 Da), and/or Rib (132 Da) were the characteristic pathways of triterpenoid saponins in CRR [28, 29]. Clematichinenoside AR was exemplified to characterize the fragmentation pathways of triterpenoid saponins (Fig. 3). The pseudomolecular ion [M+Cl] and [M H] were found at m/z , , respectively. The most abundant fragment peak at m/z was produced by simultaneous losing of one rhamnose and two glucose units in the C-28 position. The product ions at m/z , , , corresponded to [M H 3Glc 2Rha], [M H 4Glc 2Rha Rib], [M H 4Glc 3Rha Rib], [M H 4Glc 3Rha Rib Ara], respectively. In addition, five lignans (peak 9, 14, 18, 21, 31), two flavonoid (peak 20, 36), two polyacetylenes (peak 52, 63), one alkaloids (peak 5), and 21 other compounds (peak 1, 2, 3, 4, 7, 8, 10, 15, 40, 42, 45, 49, 55, 57, 59, 64, 66, 67, 70) were tentatively identified by comparing the retention time, UV spectra, fragmentation pathways, and MS/MS spectra data [30, 31]. 3.2 Quantitative analysis of the eight marker compounds in herb couple by HPLC MS Because of the unsatisfied sensitivity and accuracy of HPLC DAD or HPLC ELSD methods for the trace ingredients, HPLC MS was used to perform the quantification of CN samples in this study. The method validation of quantitative analysis was carried out using the aforementioned HPLC analytical conditions. Every calibration curve was performed with seven different concentrations in triplicate, and the result showed that

8 J. Sep. Sci. 2016, 39, Liquid Chromatography 1229 Table 3. The contents of the 8 compounds in the 10 batches of CN herb couple ( g/ml, mean ± SD, n = 3) No. Name Nodakenin ± ± ± ± ± ± ± ± ± Bergaptol ± ± ± ± ± ± ± ± ± ± Bergapten 55.24± ± ± ± ± ± ± ± ± Notopterol ± ± ± ± ± ± ± ± Isoimperatorin 12.76± ± ± ± ± ± ± ± ± ± Bergamottin 5.52± ± ± ± ± ± ± ± 0.62 Total Hederagenin 7.46 ± ± Oleanolic acid 0.71 ± ± ± ± ± ± ± ± 0.95 Total all calibration curves had wonderful linearity (r ) within the sample test ranges. The LODs and LOQs of the eight compounds were and g/ml, respectively. The intra- and inter-day precisions result show that RSD values for intra- and inter-day precisions were and %, respectively. The stability results indicated that these sample solutions were stable within 12 h at room temperature, and the RSD values were in the range of %. The recovery tests result showed that the recoveries were ranged from to %. The aforementioned results (Table 2) are considered to be valid for the analysis of all the CN samples. The aforementioned HPLC MS quantitative methods were applied to simultaneously quantify the eight marker compounds in a total of ten batches of different CN herb couple samples. The typical chromatograms of CN samples are shown in Fig. 4, and the quantification results were provided in Table 3, which indicated that the contents of the eight makers varied significantly in ten batches of samples. Additionally, coumarins and saponins were two kinds of predominant ingredients in CN. However, the results also showed that the hederagenin only existed in two batches of CN samples, and there were still two batches of CN samples without notopterol, bergamottin, and oleanolic acid, respectively, which indicated that the quality of CN samples circulating in the market should be strictly controlled to ensure their clinical efficacy and effectiveness. 3.3 Chemometric analysis of the eight compounds in ten batches of herb couple samples In this study, ten batches of CN samples were analyzed using HCA according to the content of all the eight analytical markers by Mev 4.6. Complete linkage clustering was chosen and the Euclidean distance was selected to assess the resemblance of the ten batches of CN samples, and then classify. The result showed that three well defined clusters were obtained in Fig. 5A. Batches 2, 5, 8, and 9 were categorized into cluster I; 3, 6, 1, and 7 were categorized into cluster II, while the rest were put into cluster III. Moreover, the contents of the eight compounds in CN samples were set as variables, and the ten batches samples were set as observations. Then, the variables were centered and scaled to Unit Variance before performing the PCA by SIMCA-P. The result showed that the first principal component (t1) explained 52.81% of the total variance, and the second principal component (t2) explained 31.36% (Fig. 5B). Moreover, the ten batches of CN samples collected from different stores were also divided into three groups: group I (2, 5, 8, and 9), group II (3, 6, 1, 4, and 7), and group III (10). The results obtained from HCA and PCA were in accord. Additionally, 2D loading plots (p1/p2 loading plot) could provide useful information to distinguish some important characterizations within the first and second PC dimensions. In this study, nodakenin, notopterol, bergamottin, oleanolic acid, isoimperatorin, and bergapten demonstrated the

9 1230 T.-f. Cheng et al. J. Sep. Sci. 2016, 39, Figure 5. (A) The cluster analysis of ten samples (the distance was calculated by Euclidean distance); (B) The score plot of PCA (t1/t2); (C) The loading plot of PCA (p1/p2).

10 J. Sep. Sci. 2016, 39, Liquid Chromatography 1231 importance of the original feature in the p1/p2 dimension with the larger loading coefficients (Fig. 5C). According to China Pharmacopeia (2015 Edition), notopterol and isoimperatorin are the analytical marker for NRR, and oleanolic acid is the analytical marker for CRR, respectively. However, barely to determine the aforementioned three analytical markers to control the quality of CN sample is lack of specificity and comprehensiveness, especially for the QC of CN in Chinese medicinal formulae. Therefore, besides notopterol, isoimperatorin and oleanolic acid, nodakenin, bergamottin, and bergapten also should be taken into consideration for CN QC. 4 Concluding remarks In this study, HPLC Q-TOF-MS and HPLC MS analytical methods were successfully developed for the comprehensive qualification and quantification of multiple constituents in CN herb couple samples. The most remarkable features of the developed method were simplicity, reliability, and specificity. Due to the aforementioned advantages, 69 compounds were unambiguously identified or tentatively assigned for the first time, and the eight major analytical or active markers were quantified for the first time. Furthermore, HCA and PCA were used to classify and evaluate the different batches of CN samples, and four important ingredients were chose according to PCA loading efficiencies. In conclusion, the proposed strategy was developed to globally assess the quality of CN herb couple or other herbal medicine, which may provide supporting data for their clinical use and future studies. This work was financially supported by the Natural Science Foundation of Jiangsu province (No. BK ), the National Natural Science Foundation of China (No ), the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (Grant No ) and the Fundamental Research Funds for the Central Universities. The authors declare that there are no conflicts of interest. 5 References [1] Zhang, X., Ning, Z., Ji Chen, Y., Mao, C., Lu, T., J. Sep. Sci. 2015, 38, [2] Yang, L. W., Wu, D. H., Tang, X., Peng, W., Wang, X. R., Ma, Y., Su, W. W., J. Chromatogr. A. 2005, 1070, [3] Yao, W., Dai, J., Zheng, C., Bao, B., Cheng, H., Zhang, L., Ding, A., Li, W., J. Sep. Sci. 2015, 38, [4] Li, F., Cheng, T. F., Dong, X., Li, P., Yang, H., J. Pharmaceut. Biomed. 2015, 117, [5] Zhang, R., Fang, W., Han, D., Sha, L., Wei, J., Liu, L., Li, Y., Planta Med. 2013, 79, [6] Djafoua, Y. M., Mouokeu, R. S., Tume, C., Kamtchueng, M. O., Kuiate, J. R., J. Complement. Integr. 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