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Quantification Analysis of 13 Organic Components and 8 Inorganic Elements in Angelica Sinensis Radix and Its Different Parts Combined with Chemical Recognition Pattern

Quantification Analysis of 13 Organic Components and 8 Inorganic Elements in Angelica Sinensis... Hindawi Journal of Analytical Methods in Chemistry Volume 2020, Article ID 8836184, 11 pages https://doi.org/10.1155/2020/8836184 Research Article Quantification Analysis of 13 Organic Components and 8 Inorganic Elements in Angelica Sinensis Radix and Its Different Parts Combined with Chemical Recognition Pattern 1 2 3 4 1 5 Xi Li, Yixin Yao , Xiaoxiao Wang, Chang An, Shanshan Gao, Fangtao Xiang, and Yangli Dong Sichuan Institute for Food and Drug Control, Chengdu 611730, China Kangmei Pharmaceutical Co., Ltd., Shenzhen 518000, China Deyang Food and Drug Safety Inspection and Testing Center, Deyang 618000, China School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China Affiliated Hospital, Leshan Normal University, Leshan 614004, China Correspondence should be addressed to Yixin Yao; 14759155387@163.com Received 16 April 2020; Revised 4 August 2020; Accepted 19 August 2020; Published 31 August 2020 Academic Editor: Giuseppe Ruberto Copyright © 2020 Xi Li et al. ,is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Angelica Sinensis Radix (Danggui, DG) is one of the most commonly prescribed traditional Chinese medicines. ,e organic components include phthalides and phenolic acids. Meanwhile, inorganic elements play an important role in clinical effect. DG and its different parts have different effects. ,ere is no relevant report on the analysis of organic compounds and inorganic elements among them. ,erefore, ultra-high-performance liquid chromatography coupled with triple quadrupole mass spec- trometry was developed for the simultaneous determination of 13 organic components (8 phthalides and 5 phenolic acids), and 8 inorganic elements were determined by inductively coupled plasma mass spectrometry. ,e contents of 32 samples were analyzed by orthogonal partial least squares discrimination analysis, hierarchical cluster analysis, and least-significant difference of one-way analysis of variance. ,e results showed that the differences were significant among DG and its different parts. 11 difference markers (Ca, Z-ligustilide, Mg, Mn, Fe, Na, K, Cu, Zn, coniferyl ferulate, and senkyunolide A) were obtained by variable importance for the project. ,ese difference markers were some different among DG and its different parts, especially Z-ligustilide, coniferyl ferulate, Mg, Zn, the differences were significant. ,is study can provide a reference for DG research. components mainly consist of phthalides and phenolic acids. 1. Introduction Phthalides include senkyunolide I, senkyunolide H, Z-lig- Angelica Sinensis Radix (Danggui, DG), the dried root of ustilide, senkyunolide A, etc. Phenolic acids comprise ferulic Angelica sinensis (Oliv) Diels. (Umbelliferae), is one of the acid, chlorogenic acid, caffeic acid, and coniferyl ferulate [5–7]. In recent years, numerous studies have found that the most commonly prescribed traditional Chinese medicines (TCM). DG is commonly used to enrich blood and regulate efficacy of TCM is not only related to the organic compo- menstruation and employed in the treatment of blood de- nents but also closely related to inorganic elements. In the ficiency and chlorosis, vertigo and palpitation, irregular participation and regulation of metabolism, inorganic ele- menstruation, amenorrhea and dysmenorrhea, asthenia cold ments also represent an important factor for the exertion of abdominalgia, intestinal dryness, and constipation [1]. pharmacological effects [8, 9]. Mn-superoxide dismutase Modern studies have shown that DG can increase red blood (Mn-SOD) has a strong correlation with epithelial ovarian cells and hemoglobin, improve hemorheology and acute tumors [10]. ,e low ratio of Cu to Zn is prone to hyper- myocardial infarction, etc [2–4]. ,e organic bioactive lipidemia and coronary heart disease, and Cu deficiency is an 2 Journal of Analytical Methods in Chemistry important risk factor of coronary heart disease [11, 12]. In butylidenephthalide, neocnidilide, and levistilide A 2+ ischemic diseases, the excessive increase of [Ca ] in (purities≥ 98% by HPLC) were purchased from Chengdu myocardium will lead to calcium overload and cell death. Pufei De Biotech Co., Ltd. (Chengdu, China). Calibration 2+ −1 Ca in cardiomyocytes is mainly involved in the excitation solutions of 1,000 mg·L of Na, Mg, K, Ca, Mn, Fe, Cu, and 2+ contraction coupling of myocardium. Meanwhile, Ca Zn were all purchased from Agilent Technologies Inc. homeostasis is regulated by a variety of proteins, including (USA). Internal standard elements, consisting of + 2+ 2+ −1 73 115 209 Na /Ca exchanger, etc [13, 14]. ,e content of serum Mg 1,000 mg·L of Ge, In, and Bi, were obtained from 2+ and Ca is low in patients with cerebral infarction [15, 16]. National Institute of Metrology (China). All experimental Fe enriches blood and is related with heart failure [17]. solutions were prepared with ultrapure water −1 Modern research indicates that the efficacy of DG is strongly (18.2 MΩ·cm ), which was produced by a purification related with uterus disease and cardiovascular and cere- system (Milli-Q Gradient, Millipore, USA). brovascular diseases [18]. It indicates that these inorganic elements are also the bioactive components of DG. 2.2. Apparatus. BSA224S Precision electronic balance was ,ere are different parts of DG: head (H), body (B), and purchased from Beijing Sartorius Scientific Instrument Co., tail (T). Whole DG (W) and its different parts have different Ltd. (Beijing, China). KQ-500VDE double frequency digital effects in TCM [19]. Recent research shows that the contents ultrasonic cleaning instrument was purchased from Kun- of bioactive components and pharmacological actions are shan Ultrasonic Instrument Co., Ltd. (Kunshan, China). different in DG and its different parts [20–23]. However, Chromatographic analysis was performed on a Waters there is no study of DG and its different parts based on Acquity UHPLC system (Waters, Corp., Milford, MA, USA), organic constituents and inorganic elements at the same consisting of a binary pump solvent management system, an time. online degasser, and an autosampler. Mass spectrometry Ultra-high-performance liquid chromatography coupled detection was performed using a Xevo Triple Quadrupole with triple quadrupole mass spectrometry (UHPLC–MS/ MS (Waters Corp., Milford, MA, USA) equipped with an MS) has the characteristics of high accuracy, high sensitivity electrospray ionization source (ESI). ,e ESI-MS spectra and rapid analysis and is suitable for the quantitative de- were acquired by using multiple reaction monitoring termination of minor compounds with complex matrix and (MRM). Inorganic elements analysis was performed on serious interference. It had been widely used in the analysis Agilent 7800 ICP-MS system (Agilent Technologies Inc., research of TCM [5, 6]. Inductively coupled plasma mass USA). CEM MARS6 microwave digestion apparatus was spectrometry (ICP-MS) is a rapid development of element purchased from BERGHOF Co., Ltd. (CEM MARS6, Ber- analysis technology in recent years, with the advantages of ghof Co., Germany). rapid, accurate, and simultaneous determination of multi- element and it is widely used in the inorganic elements of TCM [8, 9]. 2.3. Samples Collection. DG samples (8 samples, 2-year- ,erefore, the UHPLC-MS/MS method was developed olds) were collected from Minxian, Gansu province, and to simultaneously determine 13 organic components further were identified as dried radix of Angelica sinensis (chlorogenic acid, caffeic acid, vanillin, ferulic acid, sen- (Oliv.) Diels. by chief pharmacist Liu Maogui, director of kyunolide I, senkyunolide H, coniferyl ferulate, Z-ligustilide, quality management department, Kangmei Pharmaceutical butylphthalide, senkyunolide A, butylidenephthalide, neo- Co., Ltd. DG samples were divided into H, B, and T. All cnidilide, and levistilide A) in different parts of DG. 8 in- samples were deposited in the traditional Chinese Medicine organic elements, including Na, Mg, K, Ca, Mn, Fe, Cu, and Laboratory of Puning Production Base of Kangmei Zn, were simultaneously quantified by inductively coupled Pharmaceutical. plasma mass spectrometry (ICP-MS). ,e results were an- alyzed by hierarchical cluster analysis (HCA), orthogonal partial least squares discrimination analysis (OPLS-DA), 2.4. Determination of Organic Components by and least-significant difference (LSD) of one-way analysis of UHPLC-MS/MS variance (ANOVA). It provides useful information for DG research. 2.4.1. Condition of UHPLC-MS/MS. ,e column was an Agilent Eclipse Plus C18 column (1.8 μm, 50 × 2.1 mm, Agilent), and the column temperature was kept at 35 C. ,e 2. Materials and Methods −1 flow rate was set at 0.3 mL·min . ,e injection volume was 2.1. Materials and Reagents. Acetonitrile (HPLC grade) was 2 μL. 0.1% formic acid (V/V) was selected as mobile phase A, obtained from Fisher Corporation (Waltham, MA, USA). and acetonitrile was selected as mobile phase B. ,e linear Glacial acetic acid was analytical grade and acquired from gradient elution of A was performed as follows: 5%A at Guangzhou Chemical Reagent Factory (Guangzhou, 0–2 min, 5%–25% A at 2–5 min, 24%–45% A at 5-6 min, China). Nitric acid 67% was MOS grade and purchased 45%–70% A at 6–12 min, and 70%–100% A at 12–15 min. from Tianjin Kemiou Chemical Reagent Co., Ltd. (Tianjin, ,e ES mode conditions of MS analysis were set as China). Standards of chlorogenic acid, caffeic acid, vanillin, follows: capillary voltage, 2.0 kV; source temperature, 150 C; ferulic acid, senkyunolide I, senkyunolide H, coniferyl desolvation temperature, 500 C; cone gas flow, 20 L/h; and ferulate, Z-ligustilide, butylphthalide, senkyunolide A, desolvation gas flow, 1000 L/h. ,e ES mode conditions Journal of Analytical Methods in Chemistry 3 were as follows: capillary voltage 2.0 kV; source temperature 2.7.1. ICP-MS Method. ,e mixed standard mother liquor ° ° 72 C; desolvation temperature 350 C; cone gas flow 1 L/h; of Na, Mg, K, Ca, Mn, Fe, Cu, and Zn was taken and diluted and desolvation gas flow 650 L/h. ,e cone voltage and to 5, 10, 20, 50, and 100 μg/mL with 10% HNO . Meanwhile, collision energy were set to match the MRM of each 10% HNO was used as blank. A standard solution was compound [24]. ,e dwell time was automatically set by prepared according to the level of the measured elements in Mass Lynx software. ,e summary of MS/MS detection the sample. A series of mass concentration standard solu- parameters is given in Table 1. tions of eight inorganic elements were determined. Ge, 115 209 In, and Bi internal standard solutions were added, and standard blank solution was prepared at the same time. With 2.4.2. Preparation of Sample. Each dried material was the standard mass concentration as abscissa (X) and the ratio pulverized to 65 mesh. Approximately 0.5 g of pulverized of peak signal value to reference peak response value of powder was accurately weighted, then extracted with 25 mL internal standard elements as longitudinal coordinate (Y), methanol by ultrasound extraction (300 W of efficiency, the standard curve was drawn, and the regression equation, 45 kHz of frequency) for 30 min, cooled to room temper- correlation coefficient, and linear range of each element ature, and supplemented weightlessness. ,e extraction standard were obtained. solution passed through a filter (0.22 μm mesh size). ,e limit of detection (LOD) and limit of quantitation (LOQ) were determined by a series of diluted standard solutions until the signal-to-noise (S/N) ratio was approx- 2.5. Determination of Inorganic Elements by ICP-MS imately 3 and 10, respectively. ,e precision of the method 2.5.1. Sample Pretreatment. All glass instruments and pol- was determined by the analysis of six consecutive injections ytetrafluoroethylene (PTFE) digestion tank were soaked using the same sample solution. Repeatability of the method about 6 h with 10% (V/V) nitric acid before the experiment. was evaluated by analyzing six samples from the same source Each dried material was pulverized to 50 mesh. Approxi- using the developed method. ,e stability was evaluated by mately 0.2 g of pulverized powder was accurately weighted storing the sample solutions at 25 C, then analyzed at 0, 2, 4, and placed in the PTFE digestion tank, and then 8 mL of 6, 8, and 12 h, respectively. To evaluate accuracy, a recovery nitric acid was added in the fume hood, and the samples test was conducted by standard protocol and calculated by were sealed and stayed overnight. ,e next day, all samples the formula [(total detected amount − original amount)/ were placed in a microwave digestion apparatus and pro- spiked amount] × 100%. Variations are expressed in terms of cessed according to the set digestion procedure. ,e di- the relative standard deviation (RSD) of the measurement in gestion conditions are shown in Table 2. After digestion, the all tests. samples were cooled to room temperature, the digestion ,e quantitative determination of 13 organic constitu- tank was removed, the acid was taken out of the fume hood, ents of DG and its different parts was performed under the and deionized water was transferred to a constant volume of optimal condition by UHPLC-MS/MS and that of 8 inor- 50 mL. Simultaneously, 8 mL concentrated nitric acid were ganic elements was performed by ICP-MS. ,e results of used as blank. ,e supernatant was collected and passed samples were shown as mean (mg/g)± SD (%). through a filter (0.22 μm mesh size). 2.8. Statistical Analysis. ,e result of analysis was performed 2.6. Solutions Preparation. ,e 21 reference compounds using OPLS-DA, HCA, and LSD of one-way ANOVA. were respectively prepared by completely dissolving in OPLS-DA and HCA were carried out by SIMCA-P 14.0 methanol, and their concentrations were as follows: software (Umetrics AB, Umea, Sweden). ,e sample vari- chlorogenic acid, 0.0217 mg/mL; caffeic acid, 0.0077 mg/mL; ation could be assessed by OPLS-DA, the parameters of the vanillin, 0.0156 mg/mL; ferulic acid, 0.0678 mg/mL; sen- 2 2 modeling (R and Q values) explain the quality of the fitting kyunolide I, 0.0226 mg/mL; senkyunolide H, 0.0075 mg/mL; model. In HCA, a dendrogram was obtained to characterize senkyunolide A, 0.0846 mg/mL; coniferyl ferulate, the classification result of the samples by Ward’s linkage as 0.1744 mg/mL; Z-ligustilide, 0.0954 mg/mL; butylideneph- cluster method. LSD of one-way ANOVA was carried out by thalide, 0.0142 mg/mL; neocnidilide, 0.0316 mg/mL; levis- SPSS 19.0 software (Palo Alto, CA, USA) and the differences tilide A, 0.0159 mg/mL. Na, Mg, K, Ca, Mn, Fe, Cu, and Zn were considered statistically significant when P< 0.05 and were 100 μg/mL. All the stock solutions were stored at 4 C were considered extremely significant when P< 0.01. before analysis. 3. Results and Discussion 2.7. Method Validation and Sample Determination. UHPLC-MS/MS method: ,is stock solution was further 3.1. Optimization of MS Conditions. In order to obtain an diluted to a series of different concentration solutions with accurate and sensitive quantitative method by UHPLC-MS/ methanol for the establishment of the calibration curves. MS, individual solutions of all standard compounds were ,ese mixture standard solutions were injected in triplicate, determined with the electrospray ionization (ESI) source by and calibration curves were constructed by plotting the peak a full-scan mass spectrometry (MS) method and in both area (Y-axis) versus the concentration (X-axis) of each positive and negative modes to optimize the parameters of analyte. cone voltage (CV) and collision energy (CE) with the highest 4 Journal of Analytical Methods in Chemistry Table 1: UHPLC-MS/MS parameters for MRM of compounds of sample. + − Molecular t [M + H] [M − H] MS/MS fragments Cone voltage Collision energy No. Compound formula (min) (m/z) (m/z) ions (V) (eV) 1 Chlorogenic acid C H O 1.89 — 353 191 31 30 16 18 9 2 Caffeic acid C H O 1.91 — 179 135, 134 32 34 9 8 4 3 Vanillin C H O 2.56 153 — 105, 93 31 28 8 8 3 4 Ferulic acid C H O 2.71 195 — 117, 89 32 26 10 10 4 5 Senkyunolide I C H O 4.64 225 — 189, 119 23 22 12 16 4 6 Senkyunolide H C H O 4.92 225 — 189, 119 23 21 12 16 4 7 Coniferyl ferulate C H O 6.98 — 355 163, 134 23 26 20 20 6 8 Senkyunolide A C H O 7.39 193 — 91 21 23 12 16 2 9 Butylphthalide C H O 7.41 191 — 145 29 14 12 14 2 10 Z-ligustilide C H O 9.68 191 — 115, 91 23 31 12 14 2 11 butylidenephthalide C H O 9.70 189 — 89 19 21 12 12 2 12 neocnidilide C H O 10.57 195 — 91 27 33 12 18 2 13 Levistilide A C H O 11.87 357 — 191 30 23 24 28 4 ferulate, Z-ligustilide, butylphthalide, senkyunolide A, Table 2: Microwave operating conditions for the digestion of samples. butylidenephthalide, neocnidilide, and levistilide A were unable, the interday results of precision were beyond 5%. Time (min) Temperature (℃) Power (W) ,e result is shown in Table 4. 0 Ordinary temperature 0 10 130 1550 15 130 1550 3.3.2. ICP-MS Method. ,e linearity of each element was 25 165 1550 2 good (R were above 0.9992) and within the range of 35 165 1550 0–100 μg/mL. Also, limit of detection (LOD, S/N � 3) and 40 180 1550 limit of quantification (LOQ, S/N � 10) were calculated. ,e accuracy, repeatability, stability (24 h), and recovery were evaluated based on the contents of eight inorganic sensitivity. Meanwhile, multiple reaction monitoring elements, with six samples in parallel, and were (MRM) from MS/MS spectrum was chosen when the most expressed as RSD (%) within 5%. ,e results are shown in abundant, specific, and stable fragment ions appeared. ,e Table 4. detailed information of retention time (t ), MS information, CV, and CE for each analyte was listed in Table 1 and Figure 1. 3.4. HCA. To compare the difference among DG and its different parts, HCA was performed. A total of 32 samples were selected for analysis, while the contents of 21 com- 3.2. Optimization of ICP-MS Conditions. ,e working pa- pounds (Table 5) were selected as variables. In the den- rameters of ICP-MS were set by automatic tuning, and the drogram of HCA (Figure 2), all samples were mainly divided working parameters of the instrument were optimized based into four categories. Firstly, T and others were respectively on sensitivity, background, stability, and other indicators. belonged to one category, indicating that the difference was ,e measurement conditions are shown in Table 3. ,e significant between T and others, respectively. Secondly, W measuring method used was the standard curve method, and 73 115 were clustered into one category, B and H were clustered the reading method was peak strength. Using Ge, In, into other category, indicating that the difference was some and Bi as internal standards to monitor the change of the significant between W and B, H, respectively. ,en, B and H signal can effectively overcome the drift of the instrument were clustered into category, respectively, indicating that the signal and correct any matrix effects. difference was significant between B and H. It indicated that the differences were significant among DG and its different 3.3. Validation of Methodology parts. 3.3.1. UHPLC-MS/MS Method. Calibration curves were 3.5. OPLS-DA. To further compare the difference among developed from the chromatographic peak area relative to the weights of each compound, respectively. Also, limit of DG and its different parts, OPLS-DA was performed. A detection (LOD, S/N � 3) and limit of quantification (LOQ, total of 32 samples were selected for analysis, while the S/N � 10) were calculated. ,e results showed that the R contents of 21 compounds (Table 5) were selected as value of the calibration curves of all components were above variables. In the OPLS-DA, the first three principal com- 2 2 0.9997. ,e precision, repeatability, stability (12 h), and ponents were selected, R X (cum) was 0.612, R Y (cum) was average recovery (low, medium, high) were evaluated by the 0.922, and Q (cum) was 0.85, and we generated score contents of 13 constituents, with six samples in parallel, and scatter plot, permutation, and variable importance plot they were expressed as RSD (%) within 5%. Because coniferyl (Figure 3). In the score scatter plot (Figure 3(a)), all samples Journal of Analytical Methods in Chemistry 5 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 (a) (b) Figure 1: MRM chromatogram of 13 compounds investigated in the mix standards (a) and sample (b) of DG. (1) Chlorogenic acid, (2) caffeic acid, (3) vanillin, (4) ferulic acid, (5) senkyunolide I, (6) senkyunolide H, (7) coniferyl ferulate, (8) senkyunolide A, (9) butylphthalide, (10) butylidenephthalide, (11) Z-ligustilide, (12) neocnidilide, and (13) levistilide A. Table 3: Optimum ICP-MS operating conditions for the analysis of samples. Instrument parameter Condition Plasma radio frequency power 1550 W Plasma gas 15 L/min Auxiliary gas flow rate 1 L/min Spray flow rate 1 L/min Compensation/dilution gas 1 L/min Spray chamber temperature 2 C Peristaltic pump speed 0.3 rps Integral time 1 s delay time 1 s Repetition times 3 Isotopes measured Na, Mg, K, Ca, Mn, Fe, Cu, Zn 73 115 209 internal standards Ge, In, Bi were divided into four parts, indicating that the differences 3.6. Analysis of 11 Difference Markers among DG and Its were significant among the different parts of DG and DG. Different Parts. Z-ligustilide, T> B> W> H; coniferyl fer- 2 2 ulate and senkyunolide A, W> B> H> T; Mg, In the permutation (Figure 3(b)), R was 0.303, Q was −0.465, and the values of left are lower than the right, T> W> H> B; Zn, T> H> B> W; Ca, T> H> W> B; Mn, indicating that the model was accurate and predictive. In T> B> H> W; Fe, W> T> H> B; Na, H> T> B> W; K, the variable importance plot (VIP) (Figure 3(c)), the value T> B> W> H; Cu, B> T> H> W (Figure 4). A total of 32 of VIP in decreasing order was as follows: Mg (1.32) � Ca samples were selected for analysis, while the contents of 11 (1.32)> Z-ligustilide (1.30)> Na (1.28)> Mn (1.26)> Fe QDMs were selected as variables and LSD of one-way (1.25) � K (1.25)> Zn (1.19)> Cu (1.14)> coniferyl ferulate ANOVA was performed (Table 6). It indicated that there (1.13)> senkyunolide A (1.06)> levistilide A (0.94)> caffeic were some differences in the 11 difference markers among acid (0.88)> neocnidilide (0.73)> Vanillin (0.69)> ferulic DG and its different parts, especially Z-ligustilide, coniferyl acid (0.64)> senkyunolide H (0.60)> butylphthalide (0.53) ferulate, Mg, and Zn; the differences were significant. > senkyunolide I (0.52)> chlorogenic acid (0.49) TCM is usually prepared by boiling herbs in water, and it is impossible to obtain all the chemical components of herbs. > butylidenephthalide (0.41). When VIP> 1, 11 constitu- ents (Ca, Z-ligustilide, Mg, Mn, Fe, Na, K, Cu, Zn, coniferyl ,e emerging potential methods of pretreatment and ex- ferulate, Senkyunolide A) were acquired, indicating that traction of active components with ammonia and hydrogen these constituents were difference markers among DG and peroxide [25–27] and presoaking [28–30] may bring un- its different parts. expected insights into the material basis of TCM. For DG, 6 Journal of Analytical Methods in Chemistry Table 4: Analytical parameters of the quantitation method. Precision Recovery (RSD, %) Range LOD LQD Stability Reproducibility No. Compound Linear R Low Medium High (μg/ml) (ng/ml) (ng/ml) (RSD, %) (RSD, %) Intra- Inter- Average RSD Average RSD Average RSD day day (%) (%) (%) (%) (%) (%) 1 Chlorogenic acid Y � 6001.7X + 0.0229 0.9999 0.339–21.7 1.90 5.76 0.32 0.10 0.66 0.71 97.32 1.12 98.60 1.12 98.33 1.02 2 Caffeic acid Y � 23817X − 0.008 0.9999 0.12–7.7 0.71 2.15 0.25 0.21 0.39 0.45 99.28 1.09 99.28 0.75 101.23 0.56 3 Vanillin Y � 24882X − 1.2387 1.000 0.244–15.6 4.23 12.81 0.71 0.19 0.58 0.81 102.64 0.98 100.1 1.08 100.76 0.33 4 Ferulic acid Y � 5998X − 2.345 0.9998 0.53–67.8 3.2 9.68 0.48 0.22 0.42 0.77 98.29 1.36 101.21 0.89 101.28 0.49 5 Senkyunolide I Y � 31125X + 0.9982 1.000 0.177–22.6 4.27 12.58 0.49 0.35 0.61 0.79 100.02 1.02 99.84 1.54 100.99 0.77 6 Senkyunolide H Y � 30991X + 1.0871 0.9999 0.116–7.5 2.17 6.63 0.51 0.87 0.82 1.09 101.25 0.98 102.78 1.42 99.87 0.82 7 Coniferyl ferulate Y � 5109X + 2.9871 0.9997 2.725–174.4 2.32 6.98 0.32 10.32 1.98 1.77 97.33 2.39 101.55 2.99 96.25 3.98 8 Senkyunolide A Y � 4768.1X + 1.216 0.9999 0.661–84.6 0.43 12.3 0.88 6.87 1.67 1.87 98.32 2.01 98.86 2.08 102.34 2.11 9 Butylphthalide Y � 13452X + 0.3876 0.9998 0.174–11.2 1.07 3.22 0.58 7.21 0.89 1.26 98.29 1.87 99.22 1.76 98.76 2.88 10 Butylidenephthalide Y � 15552x + 0.987 0.9998 0.322–41.2 1.06 3.22 1.21 9.34 0.98 1.88 97.49 2.34 97.33 1.08 99.23 3.72 11 Z-ligustilide Y � 10008X − 1.8766 0.9998 0.745–95.4 1.40 5.14 0.88 10.38 2.36 1.87 96.11 1.59 96.07 3.09 96.11 3.77 12 Neocnidilide Y � 16987x − 0.0087 0.9998 0.247–31.6 1.56 4.72 0.59 9.28 0.77 1.12 98.34 2.34 98.28 1.49 98.76 2.34 13 Levistilide A Y � 15992X + 0.4871 0.9998 0.248–15.9 0.71 2.12 0.76 8.77 0.99 1.65 95.82 1.89 99.65 1.09 99.12 2.97 14 Na Y � 1.3332X + 0.1325 0.9996 0∼100 12.3 38.1 0.98 1.30 2.33 1.46 98.45 1.02 97.32 3.01 101.22 1.33 15 Mg Y � 0.0607X + 0.0010 0.9992 0∼100 6.60 20.1 1.2 0.99 2.1 2.08 99.35 1.13 102.08 1.63 100.49 1.09 16 K Y � 0.0046X + 2.2888e − 4 0.9999 0∼100 24.1 74.7 0.45 0.76 1.58 1.47 102.43 2.32 98.23 1.99 102.33 1.29 17 Ca Y � 2.0325X + 0.0154 0.9998 0∼100 0.90 2.70 0.98 0.38 0.88 1.88 101.21 1.79 102.89 2.71 99.45 0.98 18 Mn Y � 4.5207X + 0.0802 1.000 0∼100 14.2 42.7 1.26 1.33 1.22 2.05 100.92 1.22 96.33 1.68 97.35 1.76 19 Fe Y � 14.5814X + 0.0172 0.9996 0∼100 0.40 1.20 0.94 0.96 1.76 2.31 101.24 0.98 98.25 2.38 98.78 1.32 20 Cu Y � 10.7285X + 0.0369 0.9999 0∼100 0.60 1.80 0.57 0.77 1.39 1.87 99.73 1.01 101.29 3.09 101.22 0.87 21 Zn Y � 0.8232X + 0.0520 0.9998 0∼100 4.30 13.1 1.02 1.03 2.48 1.59 97.65 1.04 103.19 3.18 100.21 0.79 Journal of Analytical Methods in Chemistry 7 Table 5: ,e contents of 21 effective components (mg/g) of DG and its different parts (mean ± SD). Sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 W-1 0.11± 0.14 0.02± 0.03 0.01± 0.02 0.10± 0.07 0.09± 0.06 0.02± 0.03 3.73± 0.98 1.61± 0.22 0.03± 0.02 0.34± 0.13 3.28± 0.56 0.12± 0.07 0.06± 0.09 8.05± 1.23 10.02± 0.98 7.29± 0.78 1.52± 0.88 0.07± 0.01 0.76± 0.08 0.10± 0.08 0.35± 0.25 W-2 0.08± 0.07 0.02± 0.02 0.01± 0.01 0.13± 0.08 0.11± 0.08 0.01± 0.02 3.55± 1.76 2.02± 0.28 0.03± 0.01 0.23± 0.03 2.92± 0.64 0.34± 0.23 0.07± 0.03 7.05± 0.91 11.08± 1.32 5.96± 0.88 0.93± 0.91 0.06± 0.09 0.70± 0.17 0.05± 0.02 0.23± 0.12 W-3 0.12± 0.22 0.01± 0.01 0.02± 0.01 0.14± 0.09 0.08± 0.07 0.01± 0.01 4.13± 1.21 2.21± 0.09 0.02± 0.01 0.07± 0.11 2.78± 0.04 0.13± 0.08 0.07± 0.06 6.23± 0.49 11.87± 1.43 6.40± 1.02 0.94± 1.21 0.06± 0.01 0.71± 0.21 0.03± 0.01 0.37± 0.15 W-4 0.07± 0.09 0.02± 0.03 0.02± 0.03 0.07± 0.08 0.09± 0.11 0.02± 0.01 3.97± 1.09 1.33± 0.78 0.02± 0.01 0.13± 0.09 3.01± 0.99 0.22± 0.14 0.08± 0.04 7.17± 1.09 11.61± 2.01 6.76± 0.69 1.28± 1.09 0.05± 0.03 0.76± 0.37 0.03± 0.02 0.27± 0.08 W-5 0.10± 0.07 0.02± 0.03 0.01± 0.02 0.10± 0.07 0.11± 0.81 0.02± 0.01 4.04± 0.89 0.92± 0.12 0.03± 0.02 0.24± 0.02 3.32± 1.01 0.30± 0.08 0.06± 0.02 8.12± 0.81 11.78± 2.83 7.40± 1.32 0.95± 0.34 0.04± 0.01 0.66± 0.18 0.05± 0.03 0.25± 0.02 W-6 0.11± 0.08 0.01± 0.02 0.01± 0.01 0.09± 0.13 0.10± 1.22 0.03± 0.01 3.69± 0.89 1.47± 0.91 0.03± 0.02 0.14± 0.07 2.21± 0.23 0.12± 0.09 0.08± 0.03 8.95± 0.75 10.31± 1.94 6.43± 0.81 1.14± 0.21 0.05± 0.08 0.68± 0.32 0.02± 0.03 0.29± 0.09 W-7 0.11± 0.13 0.02± 0.01 0.02± 0.03 0.12± 0.08 0.09± 0.87 0.01± 0.01 3.74± 0.43 1.63± 0.76 0.01± 0.01 0.16± 0.08 3.33± 0.21 0.02± 0.01 0.04± 0.01 8.18± 1.02 9.96± 3.02 6.74± 1.31 1.39± 0.29 0.07± 0.02 0.61± 0.34 0.06± 0.02 0.22± 0.13 W-8 0.09± 0.06 0.02± 0.03 0.02± 0.01 0.10± 0.06 0.09± 0.98 0.02± 0.02 3.82± 0.89 1.57± 0.68 0.03± 0.01 0.10± 0.09 3.08± 0.82 0.10± 0.09 0.06± 0.02 7.93± 1.13 11.08± 0.98 5.05± 0.99 1.44± 0.72 0.06± 0.01 0.56± 0.18 0.01± 0.02 0.25± 0.03 T-1 0.11± 0.09 0.03± 0.02 0.01± 0.02 0.11± 0.13 0.11± 0.07 0.02± 0.03 2.76± 0.81 0.78± 0.21 0.02± 0.03 0.16± 0.12 5.84± 0.31 0.09± 0.04 0.04± 0.02 9.12± 0.92 14.02± 2.77 12.81± 0.48 2.78± 0.62 0.15± 0.09 0.69± 0.17 0.11± 0.03 1.15± 0.31 T-2 0.10± 0.13 0.01± 0.03 0.01± 0.01 0.09± 0.14 0.08± 0.05 0.01± 0.01 2.35± 0.32 0.01± 0.01 0.02± 0.01 0.17± 0.09 4.98± 0.49 0.03± 0.01 0.05± 0.07 9.61± 0.33 14.30± 1.73 12.70± 0.89 2.70± 0.33 0.17± 0.03 0.63± 0.12 0.08± 0.02 1.18± 0.29 T-3 0.08± 0.22 0.03± 0.02 0.02± 0.03 0.09± 0.07 0.07± 0.06 0.02± 0.01 2.23± 0.49 0.05± 0.03 0.03± 0.02 0.10± 0.02 5.12± 0.39 0.10± 0.02 0.02± 0.01 9.88± 1.32 13.12± 1.09 12.09± 0.78 3.02± 0.81 0.13± 0.08 0.39± 0.18 0.07± 0.04 1.12± 0.32 T-4 0.11± 0.12 0.03± 0.01 0.02± 0.01 0.08± 0.07 0.10± 0.12 0.01± 0.01 2.25± 0.22 0.14± 0.71 0.02± 0.01 0.23± 0.09 5.23± 0.89 0.25± 0.12 0.02± 0.01 10.05± 0.81 12.91± 2.31 12.26± 1.21 2.48± 0.82 0.21± 0.01 0.71± 0.09 0.09± 0.03 1.14± 0.45 T-5 0.12± 0.14 0.02± 0.03 0.03± 0.07 0.08± 0.12 0.09± 0.07 0.01± 0.02 2.73± 0.11 0.58± 0.09 0.04± 0.02 0.15± 0.07 5.77± 1.05 0.19± 0.07 0.04± 0.02 9.32± 1.03 10.96± 1.97 11.40± 1.38 2.52± 1.03 0.16± 0.03 0.66± 0.22 0.09± 0.02 1.11± 0.23 T-6 0.14± 0.09 0.02± 0.01 0.03± 0.02 0.08± 0.05 0.10± 0.11 0.02± 0.01 2.83± 0.78 0.47± 0.31 0.02± 0.01 0.11± 0.09 5.91± 0.89 0.20± 0.01 0.04± 0.01 9.52± 0.81 12.07± 1.84 12.30± 1.04 2.72± 1.09 0.15± 0.09 0.61± 0.33 0.11± 0.01 1.17± 0.44 T-7 0.11± 0.05 0.03± 0.05 0.02± 0.01 0.10± 0.07 0.12± 0.13 0.02± 0.02 2.23± 0.79 0.24± 0.11 0.03± 0.01 0.25± 0.12 5.49± 0.32 0.24± 0.11 0.03± 0.01 10.06± 1.04 12.41± 1.93 12.87± 0.72 2.70± 0.91 0.12± 0.09 0.43± 0.21 0.10± 0.03 1.20± 0.32 T-8 0.11± 0.04 0.03± 0.01 0.02± 0.02 0.11± 0.03 0.11± 0.09 0.02± 0.01 2.48± 0.56 0.08± 0.06 0.03± 0.02 0.14± 0.17 5.88± 1.12 0.18± 0.08 0.04± 0.02 10.18± 0.73 12.72± 1.72 11.89± 0.43 2.83± 0.88 0.15± 0.03 0.48± 0.12 0.11± 0.02 1.15± 0.21 B-1 0.11± 0.13 0.03± 0.05 0.03± 0.01 0.10± 0.09 0.10± 0.02 0.02± 0.01 3.33± 1.78 0.94± 0.02 0.02± 0.01 0.09± 0.03 4.24± 1.29 0.23± 0.12 0.07± 0.01 9.62± 0.83 8.97± 0.87 6.75± 1.09 1.44± 1.42 0.16± 0.01 0.46± 0.32 0.10± 0.03 0.37± 0.19 B-2 0.08± 0.09 0.03± 0.04 0.02± 0.01 0.07± 0.05 0.09± 0.04 0.02± 0.02 3.14± 1.09 0.89± 0.79 0.03± 0.02 0.15± 0.02 4.19± 0.79 0.05± 0.02 0.06± 0.03 8.96± 0.52 7.22± 1.22 6.63± 1.21 1.28± 1.32 0.12± 0.03 0.48± 0.08 0.09± 0.08 0.35± 0.07 B-3 0.07± 0.59 0.02± 0.01 0.02± 0.02 0.08± 0.02 0.10± 0.01 0.01± 0.02 3.11± 1.21 0.78± 0.33 0.02± 0.01 0.23± 0.09 4.45± 0.88 0.29± 0.09 0.04± 0.03 8.92± 0.38 8.08± 0.92 7.31± 0.82 1.15± 0.76 0.12± 0.02 0.42± 0.05 0.11± 0.14 0.35± 0.04 B-4 0.10± 0.05 0.02± 0.01 0.02± 0.01 0.09± 0.07 0.10± 0.09 0.02± 0.02 3.31± 0.97 1.01± 0.09 0.01± 0.01 0.21± 0.04 3.78± 1.04 0.13± 0.08 0.05± 0.02 8.76± 1.03 8.16± 1.02 6.87± 1.32 0.84± 0.63 0.14± 0.05 0.38± 0.11 0.10± 0.07 0.33± 0.12 B-5 0.09± 0.03 0.03± 0.02 0.02± 0.01 0.10± 0.02 0.08± 0.04 0.02± 0.01 3.57± 1.13 1.11± 0.21 0.02± 0.01 0.14± 0.09 3.29± 0.59 0.18± 0.08 0.06± 0.03 8.52± 0.71 8.85± 1.08 6.53± 0.83 1.06± 0.85 0.16± 0.03 0.45± 0.21 0.10± 0.12 0.30± 0.18 B-6 0.11± 0.13 0.01± 0.02 0.01± 0.01 0.10± 0.13 0.07± 0.09 0.03± 0.01 3.03± 0.75 0.69± 0.18 0.02± 0.01 0.17± 0.03 3.67± 0.49 0.10± 0.05 0.09± 0.01 9.16± 0.22 9.02± 1.57 6.30± 0.77 0.94± 0.49 0.15± 0.11 0.43± 0.19 0.09± 0.08 0.37± 0.09 B-7 0.10± 0.08 0.03± 0.02 0.02± 0.01 0.11± 0.19 0.11± 0.08 0.02± 0.01 3.16± 0.32 1.77± 0.29 0.03± 0.02 0.11± 0.09 4.13± 0.92 — 0.06± 0.01 9.29± 0.74 9.08± 2.31 5.80± 1.92 0.89± 0.21 0.17± 0.12 0.46± 0.11 0.11± 0.09 0.32± 0.19 B-8 0.11± 0.07 0.03± 0.01 0.02± 0.02 0.10± 0.03 0.10± 0.09 0.02± 0.01 2.93± 0.87 0.95± 0.39 0.02± 0.01 0.13± 0.08 4.33± 1.01 0.12± 0.07 0.07± 0.02 9.39± 0.48 9.18± 2.45 5.86± 0.98 0.93± 1.05 0.16± 0.09 0.48± 0.33 0.11± 0.03 0.37± 0.33 H-1 0.11± 0.09 0.02± 0.01 0.02± 0.01 0.07± 0.09 0.10± 0.03 0.02± 0.01 3.04± 0.39 1.17± 0.47 0.03± 0.01 0.14± 0.03 2.08± 0.31 0.13± 0.01 0.06± 0.02 10.21± 0.72 10.63± 1.39 5.91± 1.22 2.93± 0.37 0.06± 0.03 0.57± 0.42 0.08± 0.07 0.61± 0.16 H-2 0.11± 0.12 0.02± 0.03 0.01± 0.01 0.08± 0.11 0.09± 0.04 0.01± 0.01 3.01± 0.78 0.92± 0.19 0.03± 0.02 0.09± 0.02 2.11± 0.49 0.09± 0.03 0.05± 0.01 10.22± 0.28 10.83± 1.29 5.74± 0.93 2.79± 0.44 0.03± 0.08 0.61± 0.48 0.07± 0.03 0.61± 0.08 H-3 0.12± 0.10 0.01± 0.02 0.02± 0.01 0.09± 0.06 0.09± 0.06 0.01± 0.02 2.81± 0.39 0.88± 0.31 0.02± 0.01 0.07± 0.09 1.87± 0.71 0.02± 0.01 0.07± 0.03 9.71± 0.33 10.43± 1.03 5.51± 1.42 2.83± 0.67 0.05± 0.06 0.50± 0.39 0.07± 0.11 0.59± 0.18 H-4 0.10± 0.07 0.02± 0.01 0.02± 0.01 0.10± 0.11 0.08± 0.03 0.02± 0.01 2.59± 0.88 1.19± 0.33 0.03± 0.02 0.11± 0.09 1.38± 1.04 0.16± 0.07 0.06± 0.02 9.72± 0.21 10.23± 1.78 5.54± 1.81 2.59± 0.34 0.08± 0.13 0.53± 0.31 0.05± 0.03 0.61± 0.19 H-5 0.09± 0.03 0.03± 0.01 0.03± 0.02 0.10± 0.12 0.08± 0.06 0.02± 0.03 2.50± 0.89 0.91± 0.29 0.03± 0.01 0.24± 0.02 1.69± 0.34 0.20± 0.07 0.05± 0.01 9.38± 0.26 9.63± 0.77 5.65± 0.62 2.28± 0.13 0.06± 0.11 0.54± 0.21 0.06± 0.02 0.51± 0.11 H-6 0.09± 0.06 0.02± 0.01 0.02± 0.01 0.11± 0.02 0.10± 0.05 0.03± 0.02 2.80± 0.57 0.78± 0.39 0.01± 1.01 0.14± 0.08 1.88± 0.82 0.09± 0.08 0.04± 0.03 9.22± 0.49 9.07± 0.89 5.20± 1.71 2.38± 0.39 0.06± 0.09 0.55± 0.12 0.06± 0.04 0.61± 0.21 H-7 0.10± 0.21 0.01± 0.01 0.02± 0.03 0.09± 0.08 0.11± 0.03 0.02± 0.01 3.34± 0.29 0.89± 0.18 0.03± 0.02 0.30± 0.03 1.77± 0.82 0.02± 0.01 0.06± 0.02 10.29± 0.83 9.52± 0.71 5.08± 1.09 2.47± 0.33 0.07± 0.06 0.43± 0.17 0.03± 0.02 0.53± 0.10 H-8 0.11± 0.09 0.02± 0.03 0.02± 0.01 0.10± 0.08 0.10± 0.01 0.02± 0.01 3.01± 0.82 0.68± 0.38 0.03± 0.01 0.23± 0.09 2.21± 0.88 0.08± 0.03 0.06± 0.02 10.83± 0.87 10.06± 0.93 4.51± 1.72 2.70± 0.53 0.06± 0.04 0.52± 0.32 0.04± 0.02 0.55± 0.14 8 Journal of Analytical Methods in Chemistry Calculated with ward and sorted by size Group 1 Group 3 Group 2 Group 4 Figure 2: ,e result of dendrogram by HCA (group 1, T-1∼T-8; group 2, W-1∼W-8; group 3, H-1∼H-8; group 4, B-1∼B-8). W-3 W-2 T-2 W-4 W-5 T-1 H-2 W-1 W-6 1 T-4 T-5 W-8 H-8 H-3 T-6 T-7 W-7 H-1 H-4 H-7 T-8 T-3 H-5 H-6 –1 B-3 –2 B-7 B-5 B-2 B-6 B-1 B-8 –3 B-4 –4 –5 –8 –6 –4 –2 0246 t [1] 2 2 R X [1] = 0.316 R X [2] = 0.122 Ellipse: hotelling’s T2 (95%) W B T H (a) 0.8 0.6 0.4 0.2 –0.2 –0.4 –0.6 –0.8 –1 –0.2 0 0.2 0.4 0.6 0.8 1 20 permutations and 3 components R2 Q2 (b) Figure 3: Continued. t t t [2] [2] [2] t [1 2 3] T-1 T-5 T-2 T-4 T-3 T-7 T-6 T-8 W-2 W-3 W-4 W-1 W-5 W-7 W-6 W-8 B-3 B-2 B-1 B-8 B-4 B-7 B-5 B-6 H-2 H-8 H-1 H-3 H-6 H-7 H-4 H-5 Journal of Analytical Methods in Chemistry 9 1.5 0.5 –0.5 –1 17 11 15 18 19 14 16 20 21 7 8 13 2 12 439165 10 Var ID (primary) (c) Figure 3: ,e results of statistical analysis by OPLS-DA: (a) score scatter plot; (b) permutation; (c) VIP plot. 16 3.5 2.5 1.5 0.5 0 0 WT B H WT B H DG and its different parts DG and its different parts Coniferyl ferulate Mg Senkyunolide A Fe Z-ligustilide K Ca Cu Na Mn Zn (a) (b) Figure 4: ,e comparison of difference markers among DG and different parts. Table 6: ,e P results of LSD of one-way ANOVA. Constituents W T B H W — 0.000 0.000 0.000 T — — 0.000 0.002 Coniferyl ferulate B — — — 0.013 H — — — — W — 0.000 0.000 0.000 T — — 0.000 0.000 Z-ligustilide B — — — 0.000 H — — — — VIP [4] VIP [4] VIP [4] –1 Contents (mg·g ) –1 Contents (ma·g ) 10 Journal of Analytical Methods in Chemistry Table 6: Continued. Constituents W T B H W — 0.000 0.001 0.000 T — — 0.000 0.000 Senkyunolide A B — — — 0.563 H — — — — W — 0.000 0.000 0.000 T — — 0.031 0.422 Na B — — — 0.005 H — — — — W — 0.000 0.000 0.031 T — — 0.000 0.000 Mg B — — — 0.001 H — — — — W — 0.000 0.993 0.001 T — — 0.000 0.000 B — — — 0.000 H — — — — W — 0.000 0.228 0.000 T — — 0.000 0.372 Ca B — — — 0.000 H — — — — W — 0.000 0.000 0.896 T — — 0.434 0.000 Mn B — — — 0.000 H — — — — W — 0.011 0.000 0.001 T — — 0.002 0.269 Fe B — — — 0.034 H — — — — W — 0.000 0.000 0.149 T — — 0.506 0.000 Cu B — — — 0.000 H — — — — W — 0.000 0.002 0.000 T — — 0.000 0.000 Zn B — — — 0.000 H — — — — Z-ligustilide, coniferyl ferulate, and senkyunolide A, they Data Availability were liposoluble components, their solubility was low in ,e data used to support the findings of this study are decoction, especially coniferyl ferulate; it was easily hy- available from the corresponding author upon request. drolyzed to ferulic acid [31]. In addition, TCM contains water, and the determination of water content may be performed before extraction [32], which contributes to Conflicts of Interest accurate determination of active components in future research. ,e authors declare no conflicts of interest. References 4. Conclusions [1] Chinese Pharmacopoeia Commission, Chinese Pharmaco- ,e UHPLC-MS/MS method and ICP-MS method are poeia, Vol. 1, Chemical Industry Press, Beijing, China, 2015. accurate and reliable methods for the quantification of 21 [2] Y.-L. Yang, F. Cui, F. Hu et al., “Investigation on chro- bioactive components (13 organic components and 8 matogram-pharmacodynamics relationship of Angelica inorganic elements) in DG and its different parts. ,e sinensis on effect of replenishing blood,” China Journal of differences were significant among DG and its different Chinese Materia Medica, vol. 38, no. 22, pp. 3923–3927, 2013. parts. ,e difference markers were 11 bioactive con- [3] F. Yang, Z. W. Lin, T. Y. 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Quantification Analysis of 13 Organic Components and 8 Inorganic Elements in Angelica Sinensis Radix and Its Different Parts Combined with Chemical Recognition Pattern

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Hindawi Journal of Analytical Methods in Chemistry Volume 2020, Article ID 8836184, 11 pages https://doi.org/10.1155/2020/8836184 Research Article Quantification Analysis of 13 Organic Components and 8 Inorganic Elements in Angelica Sinensis Radix and Its Different Parts Combined with Chemical Recognition Pattern 1 2 3 4 1 5 Xi Li, Yixin Yao , Xiaoxiao Wang, Chang An, Shanshan Gao, Fangtao Xiang, and Yangli Dong Sichuan Institute for Food and Drug Control, Chengdu 611730, China Kangmei Pharmaceutical Co., Ltd., Shenzhen 518000, China Deyang Food and Drug Safety Inspection and Testing Center, Deyang 618000, China School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China Affiliated Hospital, Leshan Normal University, Leshan 614004, China Correspondence should be addressed to Yixin Yao; 14759155387@163.com Received 16 April 2020; Revised 4 August 2020; Accepted 19 August 2020; Published 31 August 2020 Academic Editor: Giuseppe Ruberto Copyright © 2020 Xi Li et al. ,is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Angelica Sinensis Radix (Danggui, DG) is one of the most commonly prescribed traditional Chinese medicines. ,e organic components include phthalides and phenolic acids. Meanwhile, inorganic elements play an important role in clinical effect. DG and its different parts have different effects. ,ere is no relevant report on the analysis of organic compounds and inorganic elements among them. ,erefore, ultra-high-performance liquid chromatography coupled with triple quadrupole mass spec- trometry was developed for the simultaneous determination of 13 organic components (8 phthalides and 5 phenolic acids), and 8 inorganic elements were determined by inductively coupled plasma mass spectrometry. ,e contents of 32 samples were analyzed by orthogonal partial least squares discrimination analysis, hierarchical cluster analysis, and least-significant difference of one-way analysis of variance. ,e results showed that the differences were significant among DG and its different parts. 11 difference markers (Ca, Z-ligustilide, Mg, Mn, Fe, Na, K, Cu, Zn, coniferyl ferulate, and senkyunolide A) were obtained by variable importance for the project. ,ese difference markers were some different among DG and its different parts, especially Z-ligustilide, coniferyl ferulate, Mg, Zn, the differences were significant. ,is study can provide a reference for DG research. components mainly consist of phthalides and phenolic acids. 1. Introduction Phthalides include senkyunolide I, senkyunolide H, Z-lig- Angelica Sinensis Radix (Danggui, DG), the dried root of ustilide, senkyunolide A, etc. Phenolic acids comprise ferulic Angelica sinensis (Oliv) Diels. (Umbelliferae), is one of the acid, chlorogenic acid, caffeic acid, and coniferyl ferulate [5–7]. In recent years, numerous studies have found that the most commonly prescribed traditional Chinese medicines (TCM). DG is commonly used to enrich blood and regulate efficacy of TCM is not only related to the organic compo- menstruation and employed in the treatment of blood de- nents but also closely related to inorganic elements. In the ficiency and chlorosis, vertigo and palpitation, irregular participation and regulation of metabolism, inorganic ele- menstruation, amenorrhea and dysmenorrhea, asthenia cold ments also represent an important factor for the exertion of abdominalgia, intestinal dryness, and constipation [1]. pharmacological effects [8, 9]. Mn-superoxide dismutase Modern studies have shown that DG can increase red blood (Mn-SOD) has a strong correlation with epithelial ovarian cells and hemoglobin, improve hemorheology and acute tumors [10]. ,e low ratio of Cu to Zn is prone to hyper- myocardial infarction, etc [2–4]. ,e organic bioactive lipidemia and coronary heart disease, and Cu deficiency is an 2 Journal of Analytical Methods in Chemistry important risk factor of coronary heart disease [11, 12]. In butylidenephthalide, neocnidilide, and levistilide A 2+ ischemic diseases, the excessive increase of [Ca ] in (purities≥ 98% by HPLC) were purchased from Chengdu myocardium will lead to calcium overload and cell death. Pufei De Biotech Co., Ltd. (Chengdu, China). Calibration 2+ −1 Ca in cardiomyocytes is mainly involved in the excitation solutions of 1,000 mg·L of Na, Mg, K, Ca, Mn, Fe, Cu, and 2+ contraction coupling of myocardium. Meanwhile, Ca Zn were all purchased from Agilent Technologies Inc. homeostasis is regulated by a variety of proteins, including (USA). Internal standard elements, consisting of + 2+ 2+ −1 73 115 209 Na /Ca exchanger, etc [13, 14]. ,e content of serum Mg 1,000 mg·L of Ge, In, and Bi, were obtained from 2+ and Ca is low in patients with cerebral infarction [15, 16]. National Institute of Metrology (China). All experimental Fe enriches blood and is related with heart failure [17]. solutions were prepared with ultrapure water −1 Modern research indicates that the efficacy of DG is strongly (18.2 MΩ·cm ), which was produced by a purification related with uterus disease and cardiovascular and cere- system (Milli-Q Gradient, Millipore, USA). brovascular diseases [18]. It indicates that these inorganic elements are also the bioactive components of DG. 2.2. Apparatus. BSA224S Precision electronic balance was ,ere are different parts of DG: head (H), body (B), and purchased from Beijing Sartorius Scientific Instrument Co., tail (T). Whole DG (W) and its different parts have different Ltd. (Beijing, China). KQ-500VDE double frequency digital effects in TCM [19]. Recent research shows that the contents ultrasonic cleaning instrument was purchased from Kun- of bioactive components and pharmacological actions are shan Ultrasonic Instrument Co., Ltd. (Kunshan, China). different in DG and its different parts [20–23]. However, Chromatographic analysis was performed on a Waters there is no study of DG and its different parts based on Acquity UHPLC system (Waters, Corp., Milford, MA, USA), organic constituents and inorganic elements at the same consisting of a binary pump solvent management system, an time. online degasser, and an autosampler. Mass spectrometry Ultra-high-performance liquid chromatography coupled detection was performed using a Xevo Triple Quadrupole with triple quadrupole mass spectrometry (UHPLC–MS/ MS (Waters Corp., Milford, MA, USA) equipped with an MS) has the characteristics of high accuracy, high sensitivity electrospray ionization source (ESI). ,e ESI-MS spectra and rapid analysis and is suitable for the quantitative de- were acquired by using multiple reaction monitoring termination of minor compounds with complex matrix and (MRM). Inorganic elements analysis was performed on serious interference. It had been widely used in the analysis Agilent 7800 ICP-MS system (Agilent Technologies Inc., research of TCM [5, 6]. Inductively coupled plasma mass USA). CEM MARS6 microwave digestion apparatus was spectrometry (ICP-MS) is a rapid development of element purchased from BERGHOF Co., Ltd. (CEM MARS6, Ber- analysis technology in recent years, with the advantages of ghof Co., Germany). rapid, accurate, and simultaneous determination of multi- element and it is widely used in the inorganic elements of TCM [8, 9]. 2.3. Samples Collection. DG samples (8 samples, 2-year- ,erefore, the UHPLC-MS/MS method was developed olds) were collected from Minxian, Gansu province, and to simultaneously determine 13 organic components further were identified as dried radix of Angelica sinensis (chlorogenic acid, caffeic acid, vanillin, ferulic acid, sen- (Oliv.) Diels. by chief pharmacist Liu Maogui, director of kyunolide I, senkyunolide H, coniferyl ferulate, Z-ligustilide, quality management department, Kangmei Pharmaceutical butylphthalide, senkyunolide A, butylidenephthalide, neo- Co., Ltd. DG samples were divided into H, B, and T. All cnidilide, and levistilide A) in different parts of DG. 8 in- samples were deposited in the traditional Chinese Medicine organic elements, including Na, Mg, K, Ca, Mn, Fe, Cu, and Laboratory of Puning Production Base of Kangmei Zn, were simultaneously quantified by inductively coupled Pharmaceutical. plasma mass spectrometry (ICP-MS). ,e results were an- alyzed by hierarchical cluster analysis (HCA), orthogonal partial least squares discrimination analysis (OPLS-DA), 2.4. Determination of Organic Components by and least-significant difference (LSD) of one-way analysis of UHPLC-MS/MS variance (ANOVA). It provides useful information for DG research. 2.4.1. Condition of UHPLC-MS/MS. ,e column was an Agilent Eclipse Plus C18 column (1.8 μm, 50 × 2.1 mm, Agilent), and the column temperature was kept at 35 C. ,e 2. Materials and Methods −1 flow rate was set at 0.3 mL·min . ,e injection volume was 2.1. Materials and Reagents. Acetonitrile (HPLC grade) was 2 μL. 0.1% formic acid (V/V) was selected as mobile phase A, obtained from Fisher Corporation (Waltham, MA, USA). and acetonitrile was selected as mobile phase B. ,e linear Glacial acetic acid was analytical grade and acquired from gradient elution of A was performed as follows: 5%A at Guangzhou Chemical Reagent Factory (Guangzhou, 0–2 min, 5%–25% A at 2–5 min, 24%–45% A at 5-6 min, China). Nitric acid 67% was MOS grade and purchased 45%–70% A at 6–12 min, and 70%–100% A at 12–15 min. from Tianjin Kemiou Chemical Reagent Co., Ltd. (Tianjin, ,e ES mode conditions of MS analysis were set as China). Standards of chlorogenic acid, caffeic acid, vanillin, follows: capillary voltage, 2.0 kV; source temperature, 150 C; ferulic acid, senkyunolide I, senkyunolide H, coniferyl desolvation temperature, 500 C; cone gas flow, 20 L/h; and ferulate, Z-ligustilide, butylphthalide, senkyunolide A, desolvation gas flow, 1000 L/h. ,e ES mode conditions Journal of Analytical Methods in Chemistry 3 were as follows: capillary voltage 2.0 kV; source temperature 2.7.1. ICP-MS Method. ,e mixed standard mother liquor ° ° 72 C; desolvation temperature 350 C; cone gas flow 1 L/h; of Na, Mg, K, Ca, Mn, Fe, Cu, and Zn was taken and diluted and desolvation gas flow 650 L/h. ,e cone voltage and to 5, 10, 20, 50, and 100 μg/mL with 10% HNO . Meanwhile, collision energy were set to match the MRM of each 10% HNO was used as blank. A standard solution was compound [24]. ,e dwell time was automatically set by prepared according to the level of the measured elements in Mass Lynx software. ,e summary of MS/MS detection the sample. A series of mass concentration standard solu- parameters is given in Table 1. tions of eight inorganic elements were determined. Ge, 115 209 In, and Bi internal standard solutions were added, and standard blank solution was prepared at the same time. With 2.4.2. Preparation of Sample. Each dried material was the standard mass concentration as abscissa (X) and the ratio pulverized to 65 mesh. Approximately 0.5 g of pulverized of peak signal value to reference peak response value of powder was accurately weighted, then extracted with 25 mL internal standard elements as longitudinal coordinate (Y), methanol by ultrasound extraction (300 W of efficiency, the standard curve was drawn, and the regression equation, 45 kHz of frequency) for 30 min, cooled to room temper- correlation coefficient, and linear range of each element ature, and supplemented weightlessness. ,e extraction standard were obtained. solution passed through a filter (0.22 μm mesh size). ,e limit of detection (LOD) and limit of quantitation (LOQ) were determined by a series of diluted standard solutions until the signal-to-noise (S/N) ratio was approx- 2.5. Determination of Inorganic Elements by ICP-MS imately 3 and 10, respectively. ,e precision of the method 2.5.1. Sample Pretreatment. All glass instruments and pol- was determined by the analysis of six consecutive injections ytetrafluoroethylene (PTFE) digestion tank were soaked using the same sample solution. Repeatability of the method about 6 h with 10% (V/V) nitric acid before the experiment. was evaluated by analyzing six samples from the same source Each dried material was pulverized to 50 mesh. Approxi- using the developed method. ,e stability was evaluated by mately 0.2 g of pulverized powder was accurately weighted storing the sample solutions at 25 C, then analyzed at 0, 2, 4, and placed in the PTFE digestion tank, and then 8 mL of 6, 8, and 12 h, respectively. To evaluate accuracy, a recovery nitric acid was added in the fume hood, and the samples test was conducted by standard protocol and calculated by were sealed and stayed overnight. ,e next day, all samples the formula [(total detected amount − original amount)/ were placed in a microwave digestion apparatus and pro- spiked amount] × 100%. Variations are expressed in terms of cessed according to the set digestion procedure. ,e di- the relative standard deviation (RSD) of the measurement in gestion conditions are shown in Table 2. After digestion, the all tests. samples were cooled to room temperature, the digestion ,e quantitative determination of 13 organic constitu- tank was removed, the acid was taken out of the fume hood, ents of DG and its different parts was performed under the and deionized water was transferred to a constant volume of optimal condition by UHPLC-MS/MS and that of 8 inor- 50 mL. Simultaneously, 8 mL concentrated nitric acid were ganic elements was performed by ICP-MS. ,e results of used as blank. ,e supernatant was collected and passed samples were shown as mean (mg/g)± SD (%). through a filter (0.22 μm mesh size). 2.8. Statistical Analysis. ,e result of analysis was performed 2.6. Solutions Preparation. ,e 21 reference compounds using OPLS-DA, HCA, and LSD of one-way ANOVA. were respectively prepared by completely dissolving in OPLS-DA and HCA were carried out by SIMCA-P 14.0 methanol, and their concentrations were as follows: software (Umetrics AB, Umea, Sweden). ,e sample vari- chlorogenic acid, 0.0217 mg/mL; caffeic acid, 0.0077 mg/mL; ation could be assessed by OPLS-DA, the parameters of the vanillin, 0.0156 mg/mL; ferulic acid, 0.0678 mg/mL; sen- 2 2 modeling (R and Q values) explain the quality of the fitting kyunolide I, 0.0226 mg/mL; senkyunolide H, 0.0075 mg/mL; model. In HCA, a dendrogram was obtained to characterize senkyunolide A, 0.0846 mg/mL; coniferyl ferulate, the classification result of the samples by Ward’s linkage as 0.1744 mg/mL; Z-ligustilide, 0.0954 mg/mL; butylideneph- cluster method. LSD of one-way ANOVA was carried out by thalide, 0.0142 mg/mL; neocnidilide, 0.0316 mg/mL; levis- SPSS 19.0 software (Palo Alto, CA, USA) and the differences tilide A, 0.0159 mg/mL. Na, Mg, K, Ca, Mn, Fe, Cu, and Zn were considered statistically significant when P< 0.05 and were 100 μg/mL. All the stock solutions were stored at 4 C were considered extremely significant when P< 0.01. before analysis. 3. Results and Discussion 2.7. Method Validation and Sample Determination. UHPLC-MS/MS method: ,is stock solution was further 3.1. Optimization of MS Conditions. In order to obtain an diluted to a series of different concentration solutions with accurate and sensitive quantitative method by UHPLC-MS/ methanol for the establishment of the calibration curves. MS, individual solutions of all standard compounds were ,ese mixture standard solutions were injected in triplicate, determined with the electrospray ionization (ESI) source by and calibration curves were constructed by plotting the peak a full-scan mass spectrometry (MS) method and in both area (Y-axis) versus the concentration (X-axis) of each positive and negative modes to optimize the parameters of analyte. cone voltage (CV) and collision energy (CE) with the highest 4 Journal of Analytical Methods in Chemistry Table 1: UHPLC-MS/MS parameters for MRM of compounds of sample. + − Molecular t [M + H] [M − H] MS/MS fragments Cone voltage Collision energy No. Compound formula (min) (m/z) (m/z) ions (V) (eV) 1 Chlorogenic acid C H O 1.89 — 353 191 31 30 16 18 9 2 Caffeic acid C H O 1.91 — 179 135, 134 32 34 9 8 4 3 Vanillin C H O 2.56 153 — 105, 93 31 28 8 8 3 4 Ferulic acid C H O 2.71 195 — 117, 89 32 26 10 10 4 5 Senkyunolide I C H O 4.64 225 — 189, 119 23 22 12 16 4 6 Senkyunolide H C H O 4.92 225 — 189, 119 23 21 12 16 4 7 Coniferyl ferulate C H O 6.98 — 355 163, 134 23 26 20 20 6 8 Senkyunolide A C H O 7.39 193 — 91 21 23 12 16 2 9 Butylphthalide C H O 7.41 191 — 145 29 14 12 14 2 10 Z-ligustilide C H O 9.68 191 — 115, 91 23 31 12 14 2 11 butylidenephthalide C H O 9.70 189 — 89 19 21 12 12 2 12 neocnidilide C H O 10.57 195 — 91 27 33 12 18 2 13 Levistilide A C H O 11.87 357 — 191 30 23 24 28 4 ferulate, Z-ligustilide, butylphthalide, senkyunolide A, Table 2: Microwave operating conditions for the digestion of samples. butylidenephthalide, neocnidilide, and levistilide A were unable, the interday results of precision were beyond 5%. Time (min) Temperature (℃) Power (W) ,e result is shown in Table 4. 0 Ordinary temperature 0 10 130 1550 15 130 1550 3.3.2. ICP-MS Method. ,e linearity of each element was 25 165 1550 2 good (R were above 0.9992) and within the range of 35 165 1550 0–100 μg/mL. Also, limit of detection (LOD, S/N � 3) and 40 180 1550 limit of quantification (LOQ, S/N � 10) were calculated. ,e accuracy, repeatability, stability (24 h), and recovery were evaluated based on the contents of eight inorganic sensitivity. Meanwhile, multiple reaction monitoring elements, with six samples in parallel, and were (MRM) from MS/MS spectrum was chosen when the most expressed as RSD (%) within 5%. ,e results are shown in abundant, specific, and stable fragment ions appeared. ,e Table 4. detailed information of retention time (t ), MS information, CV, and CE for each analyte was listed in Table 1 and Figure 1. 3.4. HCA. To compare the difference among DG and its different parts, HCA was performed. A total of 32 samples were selected for analysis, while the contents of 21 com- 3.2. Optimization of ICP-MS Conditions. ,e working pa- pounds (Table 5) were selected as variables. In the den- rameters of ICP-MS were set by automatic tuning, and the drogram of HCA (Figure 2), all samples were mainly divided working parameters of the instrument were optimized based into four categories. Firstly, T and others were respectively on sensitivity, background, stability, and other indicators. belonged to one category, indicating that the difference was ,e measurement conditions are shown in Table 3. ,e significant between T and others, respectively. Secondly, W measuring method used was the standard curve method, and 73 115 were clustered into one category, B and H were clustered the reading method was peak strength. Using Ge, In, into other category, indicating that the difference was some and Bi as internal standards to monitor the change of the significant between W and B, H, respectively. ,en, B and H signal can effectively overcome the drift of the instrument were clustered into category, respectively, indicating that the signal and correct any matrix effects. difference was significant between B and H. It indicated that the differences were significant among DG and its different 3.3. Validation of Methodology parts. 3.3.1. UHPLC-MS/MS Method. Calibration curves were 3.5. OPLS-DA. To further compare the difference among developed from the chromatographic peak area relative to the weights of each compound, respectively. Also, limit of DG and its different parts, OPLS-DA was performed. A detection (LOD, S/N � 3) and limit of quantification (LOQ, total of 32 samples were selected for analysis, while the S/N � 10) were calculated. ,e results showed that the R contents of 21 compounds (Table 5) were selected as value of the calibration curves of all components were above variables. In the OPLS-DA, the first three principal com- 2 2 0.9997. ,e precision, repeatability, stability (12 h), and ponents were selected, R X (cum) was 0.612, R Y (cum) was average recovery (low, medium, high) were evaluated by the 0.922, and Q (cum) was 0.85, and we generated score contents of 13 constituents, with six samples in parallel, and scatter plot, permutation, and variable importance plot they were expressed as RSD (%) within 5%. Because coniferyl (Figure 3). In the score scatter plot (Figure 3(a)), all samples Journal of Analytical Methods in Chemistry 5 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 (a) (b) Figure 1: MRM chromatogram of 13 compounds investigated in the mix standards (a) and sample (b) of DG. (1) Chlorogenic acid, (2) caffeic acid, (3) vanillin, (4) ferulic acid, (5) senkyunolide I, (6) senkyunolide H, (7) coniferyl ferulate, (8) senkyunolide A, (9) butylphthalide, (10) butylidenephthalide, (11) Z-ligustilide, (12) neocnidilide, and (13) levistilide A. Table 3: Optimum ICP-MS operating conditions for the analysis of samples. Instrument parameter Condition Plasma radio frequency power 1550 W Plasma gas 15 L/min Auxiliary gas flow rate 1 L/min Spray flow rate 1 L/min Compensation/dilution gas 1 L/min Spray chamber temperature 2 C Peristaltic pump speed 0.3 rps Integral time 1 s delay time 1 s Repetition times 3 Isotopes measured Na, Mg, K, Ca, Mn, Fe, Cu, Zn 73 115 209 internal standards Ge, In, Bi were divided into four parts, indicating that the differences 3.6. Analysis of 11 Difference Markers among DG and Its were significant among the different parts of DG and DG. Different Parts. Z-ligustilide, T> B> W> H; coniferyl fer- 2 2 ulate and senkyunolide A, W> B> H> T; Mg, In the permutation (Figure 3(b)), R was 0.303, Q was −0.465, and the values of left are lower than the right, T> W> H> B; Zn, T> H> B> W; Ca, T> H> W> B; Mn, indicating that the model was accurate and predictive. In T> B> H> W; Fe, W> T> H> B; Na, H> T> B> W; K, the variable importance plot (VIP) (Figure 3(c)), the value T> B> W> H; Cu, B> T> H> W (Figure 4). A total of 32 of VIP in decreasing order was as follows: Mg (1.32) � Ca samples were selected for analysis, while the contents of 11 (1.32)> Z-ligustilide (1.30)> Na (1.28)> Mn (1.26)> Fe QDMs were selected as variables and LSD of one-way (1.25) � K (1.25)> Zn (1.19)> Cu (1.14)> coniferyl ferulate ANOVA was performed (Table 6). It indicated that there (1.13)> senkyunolide A (1.06)> levistilide A (0.94)> caffeic were some differences in the 11 difference markers among acid (0.88)> neocnidilide (0.73)> Vanillin (0.69)> ferulic DG and its different parts, especially Z-ligustilide, coniferyl acid (0.64)> senkyunolide H (0.60)> butylphthalide (0.53) ferulate, Mg, and Zn; the differences were significant. > senkyunolide I (0.52)> chlorogenic acid (0.49) TCM is usually prepared by boiling herbs in water, and it is impossible to obtain all the chemical components of herbs. > butylidenephthalide (0.41). When VIP> 1, 11 constitu- ents (Ca, Z-ligustilide, Mg, Mn, Fe, Na, K, Cu, Zn, coniferyl ,e emerging potential methods of pretreatment and ex- ferulate, Senkyunolide A) were acquired, indicating that traction of active components with ammonia and hydrogen these constituents were difference markers among DG and peroxide [25–27] and presoaking [28–30] may bring un- its different parts. expected insights into the material basis of TCM. For DG, 6 Journal of Analytical Methods in Chemistry Table 4: Analytical parameters of the quantitation method. Precision Recovery (RSD, %) Range LOD LQD Stability Reproducibility No. Compound Linear R Low Medium High (μg/ml) (ng/ml) (ng/ml) (RSD, %) (RSD, %) Intra- Inter- Average RSD Average RSD Average RSD day day (%) (%) (%) (%) (%) (%) 1 Chlorogenic acid Y � 6001.7X + 0.0229 0.9999 0.339–21.7 1.90 5.76 0.32 0.10 0.66 0.71 97.32 1.12 98.60 1.12 98.33 1.02 2 Caffeic acid Y � 23817X − 0.008 0.9999 0.12–7.7 0.71 2.15 0.25 0.21 0.39 0.45 99.28 1.09 99.28 0.75 101.23 0.56 3 Vanillin Y � 24882X − 1.2387 1.000 0.244–15.6 4.23 12.81 0.71 0.19 0.58 0.81 102.64 0.98 100.1 1.08 100.76 0.33 4 Ferulic acid Y � 5998X − 2.345 0.9998 0.53–67.8 3.2 9.68 0.48 0.22 0.42 0.77 98.29 1.36 101.21 0.89 101.28 0.49 5 Senkyunolide I Y � 31125X + 0.9982 1.000 0.177–22.6 4.27 12.58 0.49 0.35 0.61 0.79 100.02 1.02 99.84 1.54 100.99 0.77 6 Senkyunolide H Y � 30991X + 1.0871 0.9999 0.116–7.5 2.17 6.63 0.51 0.87 0.82 1.09 101.25 0.98 102.78 1.42 99.87 0.82 7 Coniferyl ferulate Y � 5109X + 2.9871 0.9997 2.725–174.4 2.32 6.98 0.32 10.32 1.98 1.77 97.33 2.39 101.55 2.99 96.25 3.98 8 Senkyunolide A Y � 4768.1X + 1.216 0.9999 0.661–84.6 0.43 12.3 0.88 6.87 1.67 1.87 98.32 2.01 98.86 2.08 102.34 2.11 9 Butylphthalide Y � 13452X + 0.3876 0.9998 0.174–11.2 1.07 3.22 0.58 7.21 0.89 1.26 98.29 1.87 99.22 1.76 98.76 2.88 10 Butylidenephthalide Y � 15552x + 0.987 0.9998 0.322–41.2 1.06 3.22 1.21 9.34 0.98 1.88 97.49 2.34 97.33 1.08 99.23 3.72 11 Z-ligustilide Y � 10008X − 1.8766 0.9998 0.745–95.4 1.40 5.14 0.88 10.38 2.36 1.87 96.11 1.59 96.07 3.09 96.11 3.77 12 Neocnidilide Y � 16987x − 0.0087 0.9998 0.247–31.6 1.56 4.72 0.59 9.28 0.77 1.12 98.34 2.34 98.28 1.49 98.76 2.34 13 Levistilide A Y � 15992X + 0.4871 0.9998 0.248–15.9 0.71 2.12 0.76 8.77 0.99 1.65 95.82 1.89 99.65 1.09 99.12 2.97 14 Na Y � 1.3332X + 0.1325 0.9996 0∼100 12.3 38.1 0.98 1.30 2.33 1.46 98.45 1.02 97.32 3.01 101.22 1.33 15 Mg Y � 0.0607X + 0.0010 0.9992 0∼100 6.60 20.1 1.2 0.99 2.1 2.08 99.35 1.13 102.08 1.63 100.49 1.09 16 K Y � 0.0046X + 2.2888e − 4 0.9999 0∼100 24.1 74.7 0.45 0.76 1.58 1.47 102.43 2.32 98.23 1.99 102.33 1.29 17 Ca Y � 2.0325X + 0.0154 0.9998 0∼100 0.90 2.70 0.98 0.38 0.88 1.88 101.21 1.79 102.89 2.71 99.45 0.98 18 Mn Y � 4.5207X + 0.0802 1.000 0∼100 14.2 42.7 1.26 1.33 1.22 2.05 100.92 1.22 96.33 1.68 97.35 1.76 19 Fe Y � 14.5814X + 0.0172 0.9996 0∼100 0.40 1.20 0.94 0.96 1.76 2.31 101.24 0.98 98.25 2.38 98.78 1.32 20 Cu Y � 10.7285X + 0.0369 0.9999 0∼100 0.60 1.80 0.57 0.77 1.39 1.87 99.73 1.01 101.29 3.09 101.22 0.87 21 Zn Y � 0.8232X + 0.0520 0.9998 0∼100 4.30 13.1 1.02 1.03 2.48 1.59 97.65 1.04 103.19 3.18 100.21 0.79 Journal of Analytical Methods in Chemistry 7 Table 5: ,e contents of 21 effective components (mg/g) of DG and its different parts (mean ± SD). Sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 W-1 0.11± 0.14 0.02± 0.03 0.01± 0.02 0.10± 0.07 0.09± 0.06 0.02± 0.03 3.73± 0.98 1.61± 0.22 0.03± 0.02 0.34± 0.13 3.28± 0.56 0.12± 0.07 0.06± 0.09 8.05± 1.23 10.02± 0.98 7.29± 0.78 1.52± 0.88 0.07± 0.01 0.76± 0.08 0.10± 0.08 0.35± 0.25 W-2 0.08± 0.07 0.02± 0.02 0.01± 0.01 0.13± 0.08 0.11± 0.08 0.01± 0.02 3.55± 1.76 2.02± 0.28 0.03± 0.01 0.23± 0.03 2.92± 0.64 0.34± 0.23 0.07± 0.03 7.05± 0.91 11.08± 1.32 5.96± 0.88 0.93± 0.91 0.06± 0.09 0.70± 0.17 0.05± 0.02 0.23± 0.12 W-3 0.12± 0.22 0.01± 0.01 0.02± 0.01 0.14± 0.09 0.08± 0.07 0.01± 0.01 4.13± 1.21 2.21± 0.09 0.02± 0.01 0.07± 0.11 2.78± 0.04 0.13± 0.08 0.07± 0.06 6.23± 0.49 11.87± 1.43 6.40± 1.02 0.94± 1.21 0.06± 0.01 0.71± 0.21 0.03± 0.01 0.37± 0.15 W-4 0.07± 0.09 0.02± 0.03 0.02± 0.03 0.07± 0.08 0.09± 0.11 0.02± 0.01 3.97± 1.09 1.33± 0.78 0.02± 0.01 0.13± 0.09 3.01± 0.99 0.22± 0.14 0.08± 0.04 7.17± 1.09 11.61± 2.01 6.76± 0.69 1.28± 1.09 0.05± 0.03 0.76± 0.37 0.03± 0.02 0.27± 0.08 W-5 0.10± 0.07 0.02± 0.03 0.01± 0.02 0.10± 0.07 0.11± 0.81 0.02± 0.01 4.04± 0.89 0.92± 0.12 0.03± 0.02 0.24± 0.02 3.32± 1.01 0.30± 0.08 0.06± 0.02 8.12± 0.81 11.78± 2.83 7.40± 1.32 0.95± 0.34 0.04± 0.01 0.66± 0.18 0.05± 0.03 0.25± 0.02 W-6 0.11± 0.08 0.01± 0.02 0.01± 0.01 0.09± 0.13 0.10± 1.22 0.03± 0.01 3.69± 0.89 1.47± 0.91 0.03± 0.02 0.14± 0.07 2.21± 0.23 0.12± 0.09 0.08± 0.03 8.95± 0.75 10.31± 1.94 6.43± 0.81 1.14± 0.21 0.05± 0.08 0.68± 0.32 0.02± 0.03 0.29± 0.09 W-7 0.11± 0.13 0.02± 0.01 0.02± 0.03 0.12± 0.08 0.09± 0.87 0.01± 0.01 3.74± 0.43 1.63± 0.76 0.01± 0.01 0.16± 0.08 3.33± 0.21 0.02± 0.01 0.04± 0.01 8.18± 1.02 9.96± 3.02 6.74± 1.31 1.39± 0.29 0.07± 0.02 0.61± 0.34 0.06± 0.02 0.22± 0.13 W-8 0.09± 0.06 0.02± 0.03 0.02± 0.01 0.10± 0.06 0.09± 0.98 0.02± 0.02 3.82± 0.89 1.57± 0.68 0.03± 0.01 0.10± 0.09 3.08± 0.82 0.10± 0.09 0.06± 0.02 7.93± 1.13 11.08± 0.98 5.05± 0.99 1.44± 0.72 0.06± 0.01 0.56± 0.18 0.01± 0.02 0.25± 0.03 T-1 0.11± 0.09 0.03± 0.02 0.01± 0.02 0.11± 0.13 0.11± 0.07 0.02± 0.03 2.76± 0.81 0.78± 0.21 0.02± 0.03 0.16± 0.12 5.84± 0.31 0.09± 0.04 0.04± 0.02 9.12± 0.92 14.02± 2.77 12.81± 0.48 2.78± 0.62 0.15± 0.09 0.69± 0.17 0.11± 0.03 1.15± 0.31 T-2 0.10± 0.13 0.01± 0.03 0.01± 0.01 0.09± 0.14 0.08± 0.05 0.01± 0.01 2.35± 0.32 0.01± 0.01 0.02± 0.01 0.17± 0.09 4.98± 0.49 0.03± 0.01 0.05± 0.07 9.61± 0.33 14.30± 1.73 12.70± 0.89 2.70± 0.33 0.17± 0.03 0.63± 0.12 0.08± 0.02 1.18± 0.29 T-3 0.08± 0.22 0.03± 0.02 0.02± 0.03 0.09± 0.07 0.07± 0.06 0.02± 0.01 2.23± 0.49 0.05± 0.03 0.03± 0.02 0.10± 0.02 5.12± 0.39 0.10± 0.02 0.02± 0.01 9.88± 1.32 13.12± 1.09 12.09± 0.78 3.02± 0.81 0.13± 0.08 0.39± 0.18 0.07± 0.04 1.12± 0.32 T-4 0.11± 0.12 0.03± 0.01 0.02± 0.01 0.08± 0.07 0.10± 0.12 0.01± 0.01 2.25± 0.22 0.14± 0.71 0.02± 0.01 0.23± 0.09 5.23± 0.89 0.25± 0.12 0.02± 0.01 10.05± 0.81 12.91± 2.31 12.26± 1.21 2.48± 0.82 0.21± 0.01 0.71± 0.09 0.09± 0.03 1.14± 0.45 T-5 0.12± 0.14 0.02± 0.03 0.03± 0.07 0.08± 0.12 0.09± 0.07 0.01± 0.02 2.73± 0.11 0.58± 0.09 0.04± 0.02 0.15± 0.07 5.77± 1.05 0.19± 0.07 0.04± 0.02 9.32± 1.03 10.96± 1.97 11.40± 1.38 2.52± 1.03 0.16± 0.03 0.66± 0.22 0.09± 0.02 1.11± 0.23 T-6 0.14± 0.09 0.02± 0.01 0.03± 0.02 0.08± 0.05 0.10± 0.11 0.02± 0.01 2.83± 0.78 0.47± 0.31 0.02± 0.01 0.11± 0.09 5.91± 0.89 0.20± 0.01 0.04± 0.01 9.52± 0.81 12.07± 1.84 12.30± 1.04 2.72± 1.09 0.15± 0.09 0.61± 0.33 0.11± 0.01 1.17± 0.44 T-7 0.11± 0.05 0.03± 0.05 0.02± 0.01 0.10± 0.07 0.12± 0.13 0.02± 0.02 2.23± 0.79 0.24± 0.11 0.03± 0.01 0.25± 0.12 5.49± 0.32 0.24± 0.11 0.03± 0.01 10.06± 1.04 12.41± 1.93 12.87± 0.72 2.70± 0.91 0.12± 0.09 0.43± 0.21 0.10± 0.03 1.20± 0.32 T-8 0.11± 0.04 0.03± 0.01 0.02± 0.02 0.11± 0.03 0.11± 0.09 0.02± 0.01 2.48± 0.56 0.08± 0.06 0.03± 0.02 0.14± 0.17 5.88± 1.12 0.18± 0.08 0.04± 0.02 10.18± 0.73 12.72± 1.72 11.89± 0.43 2.83± 0.88 0.15± 0.03 0.48± 0.12 0.11± 0.02 1.15± 0.21 B-1 0.11± 0.13 0.03± 0.05 0.03± 0.01 0.10± 0.09 0.10± 0.02 0.02± 0.01 3.33± 1.78 0.94± 0.02 0.02± 0.01 0.09± 0.03 4.24± 1.29 0.23± 0.12 0.07± 0.01 9.62± 0.83 8.97± 0.87 6.75± 1.09 1.44± 1.42 0.16± 0.01 0.46± 0.32 0.10± 0.03 0.37± 0.19 B-2 0.08± 0.09 0.03± 0.04 0.02± 0.01 0.07± 0.05 0.09± 0.04 0.02± 0.02 3.14± 1.09 0.89± 0.79 0.03± 0.02 0.15± 0.02 4.19± 0.79 0.05± 0.02 0.06± 0.03 8.96± 0.52 7.22± 1.22 6.63± 1.21 1.28± 1.32 0.12± 0.03 0.48± 0.08 0.09± 0.08 0.35± 0.07 B-3 0.07± 0.59 0.02± 0.01 0.02± 0.02 0.08± 0.02 0.10± 0.01 0.01± 0.02 3.11± 1.21 0.78± 0.33 0.02± 0.01 0.23± 0.09 4.45± 0.88 0.29± 0.09 0.04± 0.03 8.92± 0.38 8.08± 0.92 7.31± 0.82 1.15± 0.76 0.12± 0.02 0.42± 0.05 0.11± 0.14 0.35± 0.04 B-4 0.10± 0.05 0.02± 0.01 0.02± 0.01 0.09± 0.07 0.10± 0.09 0.02± 0.02 3.31± 0.97 1.01± 0.09 0.01± 0.01 0.21± 0.04 3.78± 1.04 0.13± 0.08 0.05± 0.02 8.76± 1.03 8.16± 1.02 6.87± 1.32 0.84± 0.63 0.14± 0.05 0.38± 0.11 0.10± 0.07 0.33± 0.12 B-5 0.09± 0.03 0.03± 0.02 0.02± 0.01 0.10± 0.02 0.08± 0.04 0.02± 0.01 3.57± 1.13 1.11± 0.21 0.02± 0.01 0.14± 0.09 3.29± 0.59 0.18± 0.08 0.06± 0.03 8.52± 0.71 8.85± 1.08 6.53± 0.83 1.06± 0.85 0.16± 0.03 0.45± 0.21 0.10± 0.12 0.30± 0.18 B-6 0.11± 0.13 0.01± 0.02 0.01± 0.01 0.10± 0.13 0.07± 0.09 0.03± 0.01 3.03± 0.75 0.69± 0.18 0.02± 0.01 0.17± 0.03 3.67± 0.49 0.10± 0.05 0.09± 0.01 9.16± 0.22 9.02± 1.57 6.30± 0.77 0.94± 0.49 0.15± 0.11 0.43± 0.19 0.09± 0.08 0.37± 0.09 B-7 0.10± 0.08 0.03± 0.02 0.02± 0.01 0.11± 0.19 0.11± 0.08 0.02± 0.01 3.16± 0.32 1.77± 0.29 0.03± 0.02 0.11± 0.09 4.13± 0.92 — 0.06± 0.01 9.29± 0.74 9.08± 2.31 5.80± 1.92 0.89± 0.21 0.17± 0.12 0.46± 0.11 0.11± 0.09 0.32± 0.19 B-8 0.11± 0.07 0.03± 0.01 0.02± 0.02 0.10± 0.03 0.10± 0.09 0.02± 0.01 2.93± 0.87 0.95± 0.39 0.02± 0.01 0.13± 0.08 4.33± 1.01 0.12± 0.07 0.07± 0.02 9.39± 0.48 9.18± 2.45 5.86± 0.98 0.93± 1.05 0.16± 0.09 0.48± 0.33 0.11± 0.03 0.37± 0.33 H-1 0.11± 0.09 0.02± 0.01 0.02± 0.01 0.07± 0.09 0.10± 0.03 0.02± 0.01 3.04± 0.39 1.17± 0.47 0.03± 0.01 0.14± 0.03 2.08± 0.31 0.13± 0.01 0.06± 0.02 10.21± 0.72 10.63± 1.39 5.91± 1.22 2.93± 0.37 0.06± 0.03 0.57± 0.42 0.08± 0.07 0.61± 0.16 H-2 0.11± 0.12 0.02± 0.03 0.01± 0.01 0.08± 0.11 0.09± 0.04 0.01± 0.01 3.01± 0.78 0.92± 0.19 0.03± 0.02 0.09± 0.02 2.11± 0.49 0.09± 0.03 0.05± 0.01 10.22± 0.28 10.83± 1.29 5.74± 0.93 2.79± 0.44 0.03± 0.08 0.61± 0.48 0.07± 0.03 0.61± 0.08 H-3 0.12± 0.10 0.01± 0.02 0.02± 0.01 0.09± 0.06 0.09± 0.06 0.01± 0.02 2.81± 0.39 0.88± 0.31 0.02± 0.01 0.07± 0.09 1.87± 0.71 0.02± 0.01 0.07± 0.03 9.71± 0.33 10.43± 1.03 5.51± 1.42 2.83± 0.67 0.05± 0.06 0.50± 0.39 0.07± 0.11 0.59± 0.18 H-4 0.10± 0.07 0.02± 0.01 0.02± 0.01 0.10± 0.11 0.08± 0.03 0.02± 0.01 2.59± 0.88 1.19± 0.33 0.03± 0.02 0.11± 0.09 1.38± 1.04 0.16± 0.07 0.06± 0.02 9.72± 0.21 10.23± 1.78 5.54± 1.81 2.59± 0.34 0.08± 0.13 0.53± 0.31 0.05± 0.03 0.61± 0.19 H-5 0.09± 0.03 0.03± 0.01 0.03± 0.02 0.10± 0.12 0.08± 0.06 0.02± 0.03 2.50± 0.89 0.91± 0.29 0.03± 0.01 0.24± 0.02 1.69± 0.34 0.20± 0.07 0.05± 0.01 9.38± 0.26 9.63± 0.77 5.65± 0.62 2.28± 0.13 0.06± 0.11 0.54± 0.21 0.06± 0.02 0.51± 0.11 H-6 0.09± 0.06 0.02± 0.01 0.02± 0.01 0.11± 0.02 0.10± 0.05 0.03± 0.02 2.80± 0.57 0.78± 0.39 0.01± 1.01 0.14± 0.08 1.88± 0.82 0.09± 0.08 0.04± 0.03 9.22± 0.49 9.07± 0.89 5.20± 1.71 2.38± 0.39 0.06± 0.09 0.55± 0.12 0.06± 0.04 0.61± 0.21 H-7 0.10± 0.21 0.01± 0.01 0.02± 0.03 0.09± 0.08 0.11± 0.03 0.02± 0.01 3.34± 0.29 0.89± 0.18 0.03± 0.02 0.30± 0.03 1.77± 0.82 0.02± 0.01 0.06± 0.02 10.29± 0.83 9.52± 0.71 5.08± 1.09 2.47± 0.33 0.07± 0.06 0.43± 0.17 0.03± 0.02 0.53± 0.10 H-8 0.11± 0.09 0.02± 0.03 0.02± 0.01 0.10± 0.08 0.10± 0.01 0.02± 0.01 3.01± 0.82 0.68± 0.38 0.03± 0.01 0.23± 0.09 2.21± 0.88 0.08± 0.03 0.06± 0.02 10.83± 0.87 10.06± 0.93 4.51± 1.72 2.70± 0.53 0.06± 0.04 0.52± 0.32 0.04± 0.02 0.55± 0.14 8 Journal of Analytical Methods in Chemistry Calculated with ward and sorted by size Group 1 Group 3 Group 2 Group 4 Figure 2: ,e result of dendrogram by HCA (group 1, T-1∼T-8; group 2, W-1∼W-8; group 3, H-1∼H-8; group 4, B-1∼B-8). W-3 W-2 T-2 W-4 W-5 T-1 H-2 W-1 W-6 1 T-4 T-5 W-8 H-8 H-3 T-6 T-7 W-7 H-1 H-4 H-7 T-8 T-3 H-5 H-6 –1 B-3 –2 B-7 B-5 B-2 B-6 B-1 B-8 –3 B-4 –4 –5 –8 –6 –4 –2 0246 t [1] 2 2 R X [1] = 0.316 R X [2] = 0.122 Ellipse: hotelling’s T2 (95%) W B T H (a) 0.8 0.6 0.4 0.2 –0.2 –0.4 –0.6 –0.8 –1 –0.2 0 0.2 0.4 0.6 0.8 1 20 permutations and 3 components R2 Q2 (b) Figure 3: Continued. t t t [2] [2] [2] t [1 2 3] T-1 T-5 T-2 T-4 T-3 T-7 T-6 T-8 W-2 W-3 W-4 W-1 W-5 W-7 W-6 W-8 B-3 B-2 B-1 B-8 B-4 B-7 B-5 B-6 H-2 H-8 H-1 H-3 H-6 H-7 H-4 H-5 Journal of Analytical Methods in Chemistry 9 1.5 0.5 –0.5 –1 17 11 15 18 19 14 16 20 21 7 8 13 2 12 439165 10 Var ID (primary) (c) Figure 3: ,e results of statistical analysis by OPLS-DA: (a) score scatter plot; (b) permutation; (c) VIP plot. 16 3.5 2.5 1.5 0.5 0 0 WT B H WT B H DG and its different parts DG and its different parts Coniferyl ferulate Mg Senkyunolide A Fe Z-ligustilide K Ca Cu Na Mn Zn (a) (b) Figure 4: ,e comparison of difference markers among DG and different parts. Table 6: ,e P results of LSD of one-way ANOVA. Constituents W T B H W — 0.000 0.000 0.000 T — — 0.000 0.002 Coniferyl ferulate B — — — 0.013 H — — — — W — 0.000 0.000 0.000 T — — 0.000 0.000 Z-ligustilide B — — — 0.000 H — — — — VIP [4] VIP [4] VIP [4] –1 Contents (mg·g ) –1 Contents (ma·g ) 10 Journal of Analytical Methods in Chemistry Table 6: Continued. Constituents W T B H W — 0.000 0.001 0.000 T — — 0.000 0.000 Senkyunolide A B — — — 0.563 H — — — — W — 0.000 0.000 0.000 T — — 0.031 0.422 Na B — — — 0.005 H — — — — W — 0.000 0.000 0.031 T — — 0.000 0.000 Mg B — — — 0.001 H — — — — W — 0.000 0.993 0.001 T — — 0.000 0.000 B — — — 0.000 H — — — — W — 0.000 0.228 0.000 T — — 0.000 0.372 Ca B — — — 0.000 H — — — — W — 0.000 0.000 0.896 T — — 0.434 0.000 Mn B — — — 0.000 H — — — — W — 0.011 0.000 0.001 T — — 0.002 0.269 Fe B — — — 0.034 H — — — — W — 0.000 0.000 0.149 T — — 0.506 0.000 Cu B — — — 0.000 H — — — — W — 0.000 0.002 0.000 T — — 0.000 0.000 Zn B — — — 0.000 H — — — — Z-ligustilide, coniferyl ferulate, and senkyunolide A, they Data Availability were liposoluble components, their solubility was low in ,e data used to support the findings of this study are decoction, especially coniferyl ferulate; it was easily hy- available from the corresponding author upon request. drolyzed to ferulic acid [31]. 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