Validation and Standardization of the Content of Three DCQAs (Di-caffeoylquinic acid) in Korean Ligularia fischeri by Region of Origin

Validation and Standardization of the Content of Three DCQAs (Di-caffeoylquinic acid) in Korean Ligularia fischeri by Region of Origin | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Validation and Standardization of the Content of Three DCQAs (Di-caffeoylquinic acid) in Korean Ligularia fischeri by Region of Origin Hun Hwan KIM, Se Hyo Jeong, Pritam Bhangwan Bhosale, Tae Yang Kim, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6576668/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Ligularia fischeri is a perennial plant in the Asteraceae family, native to Japan, China, Eastern Siberia, and Korea. In general, it is said to be good for anti-aging, bronchial diseases, anti-cancer, and constipation. In this study, we obtained five regions (Hamyang, Hoengseong, Jeongseon, Nonsan, and Yangsan) of the Korean cultivated Ligularia fischeri and analyzed its compounds by HPLC-MS/MS and chromatograms with standards to confirm the absence of interfering substances and confirmed that it contains three types of DCQA (Di-caffeoylquinic acid): 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA. The linearity, precision, limit of quantification (LOQ) and limit of detection (LOD), and recovery were then measured by quantitative analysis to confirm the content of the three DCQAs. The results of the analysis of three types of DCQA content in Ligularia fischeri obtained from five regions (Hamyang, Hoengseong, Jeongseon, Nonsan, and Yangsan) using three different solvent concentrations (100% DW, 30% EtOH, and 50% EtOH) are as follows (5 g of raw material/50 mL of extraction solvent). In 100% distilled water, 3,4-DCQA was highest in Nonsan (9.29 mg/g), 3,5-DCQA was highest in Hoengseong (5.32 mg/g), and 4,5-DCQA was highest in Nonsan (3.38 mg/g). In 30% ethanol, 3,4-DCQA was highest in Nonsan (19.15 mg/g), 3,5-DCQA was highest in Hoengseong (9.98 mg/g), and 4,5-DCQA was highest in Nonsan (11.79 mg/g). In 50% ethanol, 3,4-DCQA was highest in Nonsan (21.52 mg/g), 3,5-DCQA was highest in Hoengseong (17.06 mg/g), and 4,5-DCQA was highest in Nonsan (11.25 mg/g). Biological sciences/Biochemistry Biological sciences/Developmental biology Biological sciences/Drug discovery Health sciences/Biomarkers Ligularia fischeri 3 4-DCQA 3 5-DCQA 4 5-DCQA Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Ligularia fischeri is a plant of the Asteraceae family native to the high mountain regions of Northeast Asia, particularly Korea, China, and Japan. Its roots and leaves have been widely used traditionally for medicinal and edible purposes [ 1 ]. Recent studies have revealed that Ligularia fischeri contains various bioactive compounds and is attracting attention for its physiological effects, such as antioxidant, anti-inflammatory, and anti-cancer properties. [ 2 ]. The main bioactive compounds of Ligularia fischeri include polyphenolic compounds, flavonoids, terpenoids, and sesquiterpenoids, which have been also reported to contain various bioactive effects such as antioxidant, anti-inflammatory, and antibacterial activities [ 3 – 5 ]. In particular, polyphenols, flavonoids, and chlorogenic acid are known to have positive effects on the prevention of chronic diseases [ 6 ]. Additionally, some studies suggest that Ligularia fischeri extract may exhibit physiological functions such as inhibiting cancer cell proliferation, lowering blood sugar levels, protecting the liver, and treating rheumatoid arthritis [ 7 – 10 ]. However, scientific research on Ligularia fischeri is still in its early stages, and standardized methods are required to ensure reliable and reproducible experimental results, particularly in analytical procedures. A search on PubMed using the keywords “ Ligularia fischeri” and “DCQA” found a total of three papers. Among these, only one paper actually addressed the separation and identification of the compounds. Specifically, Shang et al. (2010) analyzed Ligularia fischeri collected from Daegwallyeong, Gangwon-do, South Korea, and identified three types of DCQA (dicaffeoylquinic acids), with 4,5-DCQA showing the highest content. The three types of DCQA identified were 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA, with 4,5-DCQA having the highest content [ 11 ]. Kuroda et al. (2016) analyzed samples of Ligularia fischeri from two species in Sichuan Province and four species in Chongqing, totaling six species, and found differences in the distribution and composition of benzo[a]pyranoquinones and eremophilan compounds among the samples [ 12 ]. Additionally, according to a study conducted in Korea, leaf extracts of Ligularia fischeri collected in summer (June) had higher polyphenol and flavonoid content and stronger antioxidant and antibacterial activity compared to samples collected in winter (December) [ 13 ]. Thus, to date, there has been limited research on regional differences in the content of DCQA in Korea. Therefore, in this study, five regions (Hamyang, Hoengseong, Jeongseon, Nonsan, and Yangsan) where Ligularia fischeri is cultivated in Korea and analyzed the compounds using HPLC-MS/MS and chromatograms, confirming the absence of interfering compounds. As a result, seven main peaks were identified, including three DCQAs (dicaffeoylquinic acid): 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA. After that, the content differences of three types of DCQAs (3,4-DCQA, 3,5-DCQA, and 4,5-DCQA) were confirmed using standard compounds, and linearity, precision, limit of quantification (LOD), limit of detection (LOQ), and recovery rate were measured for quantitative analysis. This study aims to scientifically confirm the potential value of Ligularia fischeri as a functional food ingredient, as well as regional and solvent-specific differences in content and validation methods, thereby providing foundational data for future industrial applications. 2. Materials and Methods 2.1. Chemicals and Reagents The chemical standards of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC-MS/MS grade solvents (acetonitrile and triple deionized water) and methanol were obtained from Duksan Pure Chemical Co. Ltd. (Dongdaemun-gu, Seoul, Korea). 2.2. Preparation of Plant Materials Ligularia fischeri was collected from Hamyang (Code No. 00951A) and Yangsan (Code No. 00952A), Gyeongsangnam-do; Hoengseong (Code No. 00953A) and Jeongseon (Code No. 00954A), Gangwon-do; and Nonsan (Code No. 00955A), Chungcheongnam-do. After obtaining permission from each local farm to collect and provided by Kick the Hurdle Inc. The samples were stored in the Animal Bio Resources Bank, Korea, a nationally designated research materials bank, and each source was assigned a code number (Fig. 1 ). The leaves and stems of the plants provided were washed with water, finely chopped, and dried in a drying oven at 56°C for 24 hours. They were then placed in sealed polyethylene bags containing silica gel and stored at -20°C until use. 2.3. Preparation of Sample and Standard Solutions The preparation of plants and standards solution for LC-MS was performed using a modified technique [ 14 ]. Ligularia fischeri (5g) was extracted for 72 hours at 60°C in a water bath using three solvents: 100% DW, 30% EtOH, and 50% EtOH, each in 50 mL. The extract was centrifuged at 3000 rpm for 10 minutes. After centrifugation, the supernatant was filtered through filter paper (Whatman, Qualitative, Circles, 110mm Dia, Cat No. 1001 − 110). A rotary evaporator (N-1110, Eyela, Tokyo, Japan) was rotated at 100 rpm and used under reduced pressure at 45°C to completely remove any residual ethanol. Subsequently, the sample was freeze-dried to obtain a powder. Prepared Ligularia fischeri sample powder and standard of 3,4-DCQA, 3,5-DCQA, 4,5-DCQA were prepared at a concentration of 1 mg/mL in methanol. All samples were filtered through a 0.45 µm PVDF syringe filter prior to analysis and prepared in the same method as above prior to injection for HPLC-MS/MS analysis. 2.4. HPLC-MS/MS Instrumentation and Analysis HPLC and LC-MS/MS was performed on a Shimadzu Nexera Lite LC-40D HPLC system (Shimadzu, Corp., Kyoto, Japan) and Ultra Quadrupole Time of flight LC/MS/MS System (X500R) operated in positive ion mode (spray voltage set at − 4.5 kV). The solvent used was DW and Acetonitrile containing 0.1% formic acid, a gradient system was used at a flow rate of 0.5 mL/min for analysis, and a Prontosil C18 column (length, 250 mm; inner diameter, 4.6 mm; particle size, 5 µm; Phenomenex Co., Ltd., California, USA, Biochoff Chromatography) was used. The solvent conditions used in the mobile phases were 0–10 min at 10–15% B, 10–20 min at 20% B, 20–30 min at 25%, 30–40 min at 40%, 40–50 min at 70%, 50–60 min at 95%, and 60–70 min at 95%. The analysis was performed at a wavelength of 284 nm, and temperature of 35°C. The mass spectrometry conditions for qualitative analysis of peaks identified by HPLC were performed in positive mode using electrospray ionization (ESI) and multiscan between m/z 100–2000, with a desolvation temperature set at 500°C, spray voltage at 5500 V, ion source gas at 50 psi, curtain gas at 30 psi, and declustering potential (DP) at 80 V; MS/MS spectra were set with a collision energy of 35 ± 15V. The obtained ion chromatogram data were generated using SCIEX OS software (3.0.0). 2.5. Quantification and Validation of the Analytical Method The analytical method validation was conducted in accordance with the ICH and U.S. Food and Drug Administration bioanalytical method validation guidelines [ 15 , 16 ]. The quantification of compounds detected in Ligularia fischeri extract was performed at 284 nm, and the concentrations of the three DCQAs were calculated using the following formula: (Calibration curve results (ug/mL) × Final volume (mL) × dilution factor × standard solution purity) / (sample weight (g) × 1000 (ug/mg)). Specificity, linearity, detection limit (LOD), quantification limit (LOQ), precision, and recovery rate were measured to evaluate the performance of this method. 2.5.1. Specificity The specificity of the chromatogram and PDA spectrum patterns of Ligularia fischeri extract was confirmed as follows. It was confirmed by comparison with standard solutions based on specificity derived from the selective quantification of various compounds in complex mixtures. 2.5.2. Linearity and Range Standard solutions of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA at concentrations of 100, 250, 500, 750, and 1000 µg/mL (n = 5) were prepared and analyzed to confirm the linearity of the calibration curve. The standard solution analysis was repeated five times for each concentration. The eluted peaks were subjected to linear regression (n = 5) and measured as the ratio of peak area to analyte concentration. The linearity of the association was assessed using the correlation coefficients (R 2 ) that were computed from the calibration curves. 2.5.3. LOD and LOQ The minimum quantity of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA that may be found in the Ligularia fischeri extract sample is known as the LOD. The LOQ is the minimum quantity that can be accurately and quantitatively suitable for precision. The standard deviations of the y-intercepts and slopes of the calibration curves were used to determine the LOD and LOQ values. The calibration curve through standard deviation and slope was used to verify linearity. 2.5.4. Precision and Recovery The precision of the validation method was determined using standard solutions of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA by inter-day and intra-day at five different concentrations (100, 250, 500, 750, 1000 µg/mL). Intra-day was five times a day to analyze precision, and tests were run five times a day for three days in a row to analyze inter-day precision. To determine precision, the relative standard deviation (RSD) was calculated. The accuracy of the suggested approach was verified by a recovery analysis. Recovery was assessed after measuring the Ligularia fischeri extract with five concentrations of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA. Each analysis was repeated five times. Recovery was calculated based on the sample peak area value, standard peak area value, and peak area value. 3. Results 3.1. Separation and characteristic analysis of phenolic compounds in Ligularia fischeri extract The qualitative and quantitative analysis of compounds contained in Ligularia fischeri by region (Hamyang, Hoengseong, Jeongseon, Nonsan, and Yangsan) and extraction solvent (100% DW, 30% EtOH, and 50% EtOH) was performed using HPLC-MS/MS. A total of seven peaks were identified by HPLC retention time and UV-vis spectra (Fig. 1 ). The seven compounds obtained at a wavelength of 284 nm were identified as chlorogenic acid [ 17 ], Caffeic acid [ 18 ], Hyperin [ 19 ], 3,4-Dicaffeoylquinic acid [ 20 ], 3,5- Dicaffeoylquinic acid, 4,5- Dicaffeoylquinic acid [ 21 ], and an unknown peak. Table 1 provides the analysis results of mass spectrometry data based on existing literature. This is followed by the predicted fragmentation of compounds obtained through LC-MS/MS (Fig. 2 ). Table 1 The HPLC-MS/MS data of phenolic compounds from Ligularia fischeri extract with different origins. Peak No. Rt(min) Formula Compound UV max [M + H] + MS/MS Reference 1 16.95 C 16 H 18 O 9 Chlorogenic acid 325, 250 355 181 (C 9 H 8 O 4 ) [M + H-C 7 H 10 O 5 ] + 163 (C 9 H 6 O 3 ) [M + H-C 7 H 10 O 5 -H 2 O] + 135 (C 8 H 6 O 2 ) [M + H-C 7 H 12 O 6 -CO] + [ 17 ] 2 21.16 C 9 H 8 O 4 Caffeic acid 330 181 163 (C 9 H 6 O 3 ) [M + H-H 2 O] + 145 (C 9 H 5 O 2 − ) [M + H-H 2 O- H 2 O] 135 (C 8 H 6 O 2 ) [M + H-COOH] + 117 (C 8 H 5 O − ) [M + H-COOH-H 2 O] [ 18 ] 3 31.62 C 21 H 20 O 12 Hyperin 355, 255 465 303 (C 15 H 10 O 7 ) [M + H-C 6 H 10 O 5 ] + 153 (C 8 H 8 O 3 ) [M + H-C 6 H 10 O 5 -RDA] 109 (C 7 H 8 O) [M + H- C 6 H 10 O 5 -RDA-H 2 O] [ 19 ] 4 33.18 C 25 H 24 O 12 3,4-Dicaffeoylquinic acid 325, 290 517 499 (C 25 H 22 O 11 ) [M + H-H 2 O] + 337 (C 16 H 16 O 8 ) [M + H-H 2 O-C 9 H 6 O 3 ] + 193 (C 7 H 12 O 6 ) [M + H-C 18 H 12 O 6 ] + 175 (C 7 H 10 O 5 ) [M + H-C 9 H 8 O 4 -C 9 H 6 O 3 ] + [ 20 ] 5 35.01 C 25 H 24 O 12 3,5- Dicaffeoylquinic acid 330, 290 517 355 (C 16 H 18 O 9 ) [M + H-C 9 H 6 O 3 ] + 193 (C 7 H 12 O 6 ) [M + H-C 9 H 6 O 3 -C 9 H 6 O 3 ] + 181 (C 9 H 8 O 4 ) [M + H-C 9 H 6 O 3 -C 7 H 10 O 5 ] + 137 (C 8 H 8 O 2 ) [M + H-C 16 H 16 O 8 -O] + [ 21 ] 6 38.31 C 25 H 24 O 12 4,5- Dicaffeoylquinic acid 330, 290 517 355 (C 16 H 18 O 9 ) [M + H-C 9 H 6 O 3 ] 193 (C 7 H 12 O 6 ) [M + H- C 9 H 6 O 3 -C 9 H 6 O 3 ] 175 (C 7 H 10 O 5 ) [M + H-C 18 H 18 O 6 -H 2 O] [ 21 ] 7 42.84 - Unknown - - - - 3.2. Optimization of HPLC–MS/MS Condition Standard solutions of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA (100 µg/mL) were used to optimize the HPLC-MS/MS conditions. A Pronto SIL column (250 × 4.6 mm, 5 µm, 120-5-C18 SH, Bischoff Chromatography, Leonberg, Germany) was used to identify detectable phenolic compounds, and the system was set up using a gradient method with a column temperature of 35°C and a mobile phase consisting of water and acetonitrile, each containing 0.01% formic acid. Based on a literature review, the maximum absorbance of chlorogenic acid was found to be around 290 nm, so the UV wavelength range of 200–400 nm was set to detect its derivatives, 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA [ 22 ]. For the identification of compounds in the sample, positive ESI conditions were applied because ion fragments showed higher safety than negative mode. Subsequently, when the sample was prepared using methanol, it exhibited sufficiently excellent peak shapes. 3.3 Method of Validation 3.3.1 Specificity Through the chromatograms of the standard solution and sample solution, we confirmed that the peaks in the Ligularia fischeri extract were clearly separated and that there was no interference from other compounds. Specificity was confirmed by verifying that the retention time (RT) patterns between the standard solution and sample solution matched and that when the standard solutions were mixed, each standard solution was detected at different times (Fig. 3 ). 3.3.2. Linearity, Range and LOD, and LOQ Linearity verification was performed five times at each concentration, and the linearity of 3,4-DCQA (R 2 ≥ 0.9998), 3,5-DCQA (R² ≥ 0.9998), and 4,5-DCQA (R² ≥ 0.9997) were confirmed. Subsequently, we obtained the linear expressions for 3,4-DCQA (y = 4390.5x − 34206), 3,5-DCQA (y = 5093.5x − 106812), and 4,5-DCQA (y = 5549.9x − 143191). The Limit of Detection (LOD) and Limit of Quantitation (LOQ) were determined, with the LOD and LOQ for 3,4-DCQA being 0.795 mg/L and 2.386 mg/L, respectively. The LOD and LOQ for 3,5-DCQA were 0.637 mg/L and 1.910 mg/L, respectively. The LOD and LOQ for 4,5-DCQA were 0.589 mg/L and 1.766 mg/L, respectively (Table 2 ). Table 2 Calibration curve data for the quantification of 3 type of DCQAs. Compound Slopes of Calibration Correlation Coefficient (R 2 ) LOD (mg/L) LOQ (mg/L) 3,4-DCQA 4390.5 0.9998 0.795 2.386 3,5-DCQA 5093.5 0.9998 0.637 1.910 4,5-DCQA 5549.9 0.9997 0.589 1.766 LOD: Limit of Detection; LOQ: Limit of Quantitation, ( ո = 5) 3.3.3 Precision Table 3 shows the intraday precision (repeatability) values of the standard solutions of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA, which were measured under the same conditions within a single day. The tests were conducted five times within a concentration range of 100–1000 µg/mL, and the relative standard deviation (RSD) values for each standard solution concentration were within 0.25%. Table 4 shows the interday precision (intermediate precision) values of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA in Ligularia fischeri extract obtained from five regions, using the same equipment under the same conditions but on different days. This confirmed the reliability and reproducibility of the HPLC-PDA analysis method. Table 3 Intra-day precision for 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA in Ligularia fischeri extract using the HPLC-PDA method. Compound Nominal Concentration (ug/mL) Intraday Observed Concentration Precision RSD (%) 3,4-DCQA 100 98.0465 ± 0.2410 0.2458 250 255.2384 ± 0.6584 0.2579 500 498.5773 ± 0.8622 0.1729 750 744.1814 ± 0.8564 0.1151 1000 1003.9732 ± 1.0276 0.1024 3,5-DCQA 100 99.5182 ± 0.1927 0.1936 250 255.2977 ± 0.4544 0.1780 500 494.9713 ± 0.6239 0.1260 750 745.8983 ± 0.7657 0.1027 1000 1003.0994 ± 0.9077 0.0905 4,5-DCQA 100 103.1656 ± 0.1782 0.1727 250 254.2724 ± 0.3253 0.1280 500 489.4829 ± 0.5913 0.1208 750 748.1448 ± 0.7020 0.0938 1000 1004.3856 ± 0.8119 0.0808 Data were expressed as the mean SD ( n = 5). Table 4 Inter-day precision for 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA in Ligularia fischeri extract using the HPLC-PDA method. Compound Origin Solvent ratio (%) Interday Observed Concentration Precision RSD (%) 3,4-DCQA Hamyang 100 400.9328 ± 0.7392 0.1844 70 694.7095 ± 1.0489 0.1510 50 628.7130 ± 0.6949 0.1105 Hoengseong 100 799.1811 ± 0.4865 0.0609 70 1675.8443 ± 1.2639 0.0754 50 1876.1564 ± 1.1760 0.0627 Jeongseon 100 782.4230 ± 0.5818 0.5818 70 894.3677 ± 0.6668 0.0746 50 952.0126 ± 1.0759 0.1130 Nonsan 100 929.3770 ± 0.9992 0.1075 70 1914.7634 ± 1.2271 0.0641 50 2152.2849 ± 2.0497 0.0952 Yangsan 100 194.0019 ± 0.7225 0.7225 70 300.7707 ± 0.5716 0.1900 50 381.5616 ± 0.4148 0.1087 3,5-DCQA Hamyang 100 347.1160 ± 0.2238 0.0645 70 637.3278 ± 1.1169 0.1752 50 1147.2962 ± 0.9989 0.0871 Hoengseong 100 532.2904 ± 0.9027 0.1696 70 995.4297 ± 0.5177 0.0520 50 1706.0361 ± 1.1197 0.0656 Jeongseon 100 320.7918 ± 0.4125 0.1286 70 550.6090 ± 0.4755 0.0864 50 1070.2532 ± 0.5016 0.0469 Nonsan 100 495.3014 ± 0.5545 0.1120 70 987.9929 ± 0.7045 0.5158 50 1470.6069 ± 1.0601 0.0721 Yangsan 100 171.6034 ± 0.4994 0.2910 70 373.1339 ± 0.5158 0.1382 50 843.3912 ± 0.7800 0.0925 4,5-DCQA Hamyang 100 180.3627 ± 0.6340 0.3515 70 749.1779 ± 0.4227 0.0564 50 1044.9344 ± 0.9620 0.0921 Hoengseong 100 304.4347 ± 0.4813 0.1581 70 950.7943 ± 0.3793 0.0399 50 988.9631 ± 0.9003 0.0910 Jeongseon 100 213.6833 ± 0.4025 0.1884 70 705.7721 ± 0.7480 0.1060 50 927.4137 ± 0.7327 0.0790 Nonsan 100 338.2645 ± 0.4671 0.1381 70 1179.4910 ± 0.8460 0.0717 50 1125.3682 ± 0.6967 0.0619 Yangsan 100 86.7630 ± 0.4404 0.5076 70 435.0737 ± 0.3509 0.0807 50 466.1191 ± 0.6737 0.1445 Solvent; DW (with Ethanol), ( ո = 5) 3.3.4. Recovery The recovery study was conducted using HPLC-PDA analysis with a mixture of Ligularia fischeri extract and standard solutions of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA. The overall recovery rates for 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA were 96.9675–99.1368%, with RSD values all below 1% (Table 5 ). Table 5 Recovery study of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA in Ligularia fischeri extract Compound Concentration Recovery (%) SD RSD (%) Extract Standard Extract with Standard 3,4-DCQA 1079.5395 ± 1.2749 508.7541 ± 4.1856 1540.1124 ± 6.4529 96.9675 0.6123 0.631472 3,5-DCQA 745.7905 ± 3.0325 514.3746 ± 2.4918 1228.5486 ± 9.4095 99.1368 0.7146 0.720803 4,5-DCQA 576.4081 ± 5.8836 516.3915 ± 6.3605 1126.8748 ± 11.7163 97.4385 0.8892 0.912598 3.3.5 Quantitative Analysis of the 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA in Ligularia fischeri extract by solvent ratio and origin. Analysis of the content of three types of DCQA by the concentration of extraction solvent in Ligularia fischeri . from five regions showed that in 100% distilled water, 3,4-DCQA was highest in Nonsan (929.3770 ± 0.9992 ug/mL), Hoengseong (799.1811 ± 0.4865 ug/mL), and Jeongseon (782.4230 ± 0.5818 ug/mL); 3,5-DCQA was higher in the order of Hoengseong (532.2904 ± 0.9027 ug/mL) Nonsan (495.3014 ± 0.5545 ug/mL) and Hamyang (347.1160 ± 0.2238 ug/mL); and 4,5-DCQA was higher in the order of Nonsan (338.2645 ± 0.4671 ug/mL), Hoengseong (304.4347 ± 0.4813 ug/mL), and Jeongseon (213.6833 ± 0.4025 ug/mL). In 30% ethanol, 3,4-DCQA was Nonsan (1914.7634 ± 1.2271 ug/mL), Hoengseong (1675.8443 ± 1.2639 ug/mL), and Jeongseon (894.3677 ± 0.6668 ug/mL); 3,5-DCQA was Hoengseong (995.4297 ± 0.5177 ug/mL), Nonsan (987.9929 ± 0.7045 ug/mL), and Hamyang (637.3278 ± 1.1169 ug/mL); and 4,5-DCQA was Nonsan (1179.4910 ± 0.8460 ug/mL), Hoengseong (950.7943 ± 0.3793 ug/mL), and Hamyang (749.1779 ± 0.4227 ug/mL). In 50% ethanol, 3,4-DCQA was Nonsan (2152.2849 ± 2.0497 ug/mL), Hoengseong (1876.1564 ± 1.1760 ug/mL), and Jeongseon (952.0126 ± 1.0759 ug/mL); 3,5-DCQA was Hoengseong (1706.0361 ± 1.1197 ug/mL), Nonsan (1470 ± 6069 ± 1.0601 ug/mL), and Hamyang(1147.2962 ± 0.9989 ug/mL); and 4,5-DCQA was Nonsan (1125.3682 ± 0.6967 ug/mL), Hamyang(1044.9344 ± 0.9620 ug/mL), and Hoengseong (988.9631 ± 0.9003 ug/mL) (Table 6 ). Table 6 Changes in content of the 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA according to extraction by solvent ratio and region. Compound Name Solvent ratio (%) Concentration (ug/mL) Hamyang Hoengseong Jeongseon Nonsan Yangsan 3,4-DCQA 100 400.9328 ± 0.7392 E 799.1811 ± 0.4865 A 782.4230 ± 0.5818 C 929.3770 ± 0.9992 B 194.0019 ± 0.7225 D 70 694.7095 ± 1.0489 E 1675.8443 ± 1.2639 A 894.3677 ± 0.6668 C 1914.7634 ± 1.2271 B 300.7707 ± 0.5716 D 50 628.7130 ± 0.6949 E 1876.1564 ± 1.1760 A 952.0126 ± 1.0759 C 2152.2849 ± 2.0497 B 381.5616 ± 0.4148 D 3,5-DCQA 100 347.1160 ± 0.2238 E 532.2904 ± 0.9027 C 320.7918 ± 0.4125 A 495.3014 ± 0.5545 D 171.6034 ± 0.4994 B 70 637.3278 ± 1.1169 E 995.4297 ± 0.5177 C 550.6090 ± 0.4755 A 987.9929 ± 0.7045 D 373.1339 ± 0.5158 B 50 1147.2962 ± 0.9989 E 1706.0361 ± 1.1197 C 1070.2532 ± 0.5016 A 1470 ± 6069 ± 1.0601 D 843.3912 ± 0.7800 B 4,5-DCQA 100 180.3627 ± 0.6340 E 304.4347 ± 0.4813 A 213.6833 ± 0.4025 C 338.2645 ± 0.4671 B 86.7630 ± 0.4404 D 70 749.1779 ± 0.4227 E 950.7943 ± 0.3793 C 705.7721 ± 0.7480 A 1179.4910 ± 0.8460 B 435.0737 ± 0.3509 D 50 1044.9344 ± 0.9620 E 988.9631 ± 0.9003 C 927.4137 ± 0.7327 B 1125.3682 ± 0.6967 A 466.1191 ± 0.6737 D Solvent; DW (with Ethanol), ( ո = 5) All values are mean ± SD (n = 3). A-E Means with different superscripts in the same row were significantly different at p < 0.05 using Duncan's multiple range test. Also tested based on superscript A in the same row. 4. Discussion Ligularia fischeri contains dicaffeoylquinic acids (DCQAs), one of the main bioactive compounds, which are promising substances that can contribute to health promotion through their antioxidant and anti-inflammatory effects [ 23 , 24 ]. DCQA is a compound in which a caffeine acid residue is bound to a quinic acid structure and varies in form depending on its position, such as 3,4-, 3,5-, and 4,5-DCQA. Recent studies have shown that 3,5-DCQA inhibits NO and suppresses the expression of inflammatory mediators such as INOS, COX2, and TNF-α. Additionally, 4,5-DCQA has been found to regulate an anti-inflammatory pathway mediated by the TRPV1 receptor and inhibit COX2 expression [ 23 , 25 ]. Especially, in the case of Ligularia fischeri , 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA are evenly contained and exist at a certain concentration [ 26 ]. Therefore, there is a lot of research being conducted on the development of various functional foods and pharmaceuticals using DCQAs. The study of analyzing and verifying three types of DCQA contained in Ligularia fischeri extract as marker compounds is already conducted. [ 27 ]. In addition, studies on the antioxidant and physiological activities of Ligularia fischeri have been conducted based on its extraction method, but these studies are limited to simple extracts [ 28 ]. However, in this study, we compared the concentrations of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA, which act as marker compounds of Ligularia fischeri extract. Additionally, we compared and analyzed the concentrations of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA in Ligularia fischeri extract using three different solvents and performed methodological analytical validity verification. The results of the content analysis in Table 6 show that the concentration of 3,4-DCQA was highest at 2152.2849 ± 2.0497 ug/mL in the 50% ethanol extract from the Nonsan region, while the concentration of 3,5-DCQA was highest at 1706.0361 ± 1.1197 ug/mL in the 50% ethanol extract from the Hoengseong region, while the highest concentration of 4,5-DCQA was found in the 30% ethanol extract from the Nonsan region at 1179.4910 ± 0.8460 ug/mL. This study examined the importance of validation procedures for ensuring data validity and standardization processes for enhancing data comparability, as well as practical application methods for these procedures. Through this, we confirmed that not only can data quality be improved, but the reproducibility and generalizability of research results can also be enhanced. 5. Conclusions We screened six phenolic compounds contained in Ligularia fischeri and applied an HPLC-MS/MS system to perform quantitative analysis of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA. A validation system was established to evaluate specificity, linearity, detection limits, quantification limits, precision, and recovery rates, and differences in content levels were confirmed based on extraction solvents and regional variations. This study contributed to the standardization of the three DCQA compounds as marker components of Ligularia fischeri extracts , providing valuable insights for analyzing compounds in related plants. Additionally, these findings are expected to facilitate the development of various health functional products and pharmaceuticals with potential applications in antioxidant, anti-inflammatory, blood sugar regulation, liver protection, and bone health improvement. Declarations Author Contributions Conceptualization, H.H.K. and S.H.J.; methodology, H.H.K.; writing—original draft preparation, S.H.J. and H.H.K.; writing—review and editing, P.B.B., Y.G.M., and K.H.H,; investigation, S.H.J.; validation H.H.K. and S.H.J.; project administration, T.Y.K., J.W.P. and G.I.K..; supervision, G.S.K. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Informed Consent Statement Not applicable. Data Availability Statement The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Acknowledgments This study was supported by the National Research Foundation of Korea, funded by the Ministry of Science and ICT (grant no. RS-2023-00243376, and RS-2024-00411709) and the Kick the Hurdle Co., Ltd. Conflicts of Interest The authors declare no conflicts of interest. References Park, H. S., Choi, H. Y. & Kim, G. H. Preventive effect of Ligularia fischerion inhibition of nitric oxide in lipopolysaccharide-stimulated RAW 264.7 macrophages depending on cooking method. Biol. Res. 47 , 69. 10.1186/0717-6287-47-69 (2014). Kim, T. H., Truong, V. L. & Jeong, W. S. Phytochemical Composition and Antioxidant and Anti-Inflammatory Activities of Ligularia fischeri Turcz: A Comparison between Leaf and Root Extracts. Plants (Basel Switzerland) . 11 10.3390/plants11213005 (2022). Liu, X. et al. A New Sesquiterpene from Ligularia fischeri. Chem. Nat. Compd. 52 , 642–646. 10.1007/s10600-016-1729-x (2016). Park, Y. J. et al. Identification of drought-responsive phenolic compounds and their biosynthetic regulation under drought stress in Ligularia fischeri. Volume 14–2023 , (2023). 10.3389/fpls.2023.1140509 Liu, X., Li, J., Li, J., Liu, Q. & Xun, M. A New Flavonoid Glycoside from Ligularia fischeri. Chem. Nat. Compd. 55 , 638–641. 10.1007/s10600-019-02767-8 (2019). Shanmugam, G. Polyphenols: potent protectors against chronic diseases. Nat. Prod. Res. 1–3. 10.1080/14786419.2024.2386402 (2024). Cho, Y. R., Kim, J. K., Kim, J. H., Oh, J. S. & Seo, D. W. Ligularia fischeri regulates lung cancer cell proliferation and migration through down-regulation of epidermal growth factor receptor and integrin β1 expression. Genes Genomics . 35 , 741–746. 10.1007/s13258-013-0124-2 (2013). Baek, H. J. et al. Antihyperglycemic and Antilipidemic Effects of the Ethanol Extract Mixture of Ligularia fischeri and Momordica charantia in Type II Diabetes-Mimicking Mice. Evidence-based complementary and alternative medicine: eCAM 2018 , 3468040, (2018). 10.1155/2018/3468040 Yoo, J. H. et al. Hepatoprotective effect of Handaeri-gomchi (Ligularia fischeri var. spiciformis Nakai) extract against chronic alcohol-induced liver damage in rats. Food Sci. Biotechnol. 20 , 1655–1661. 10.1007/s10068-011-0228-x (2011). Choi, E. M. & Suh, K. S. Ligularia fischeri leaf extract suppresses proinflammatory mediators in SW982 human synovial cells. Phytother Res. 23 , 1575–1580. 10.1002/ptr.2823 (2009). Shang, Y. F. et al. Isolation and Identification of Antioxidant Compounds from Ligularia fischeri. 75 , C530–C535, (2010). https://doi.org/10.1111/j.1750-3841.2010.01714.x Kuroda, C. et al. Chemical Lineages of Ligularia fischeri. Nat. Prod. Commun. 11 , 139–143 (2016). Rekha, K., Sivasubramanian, C. & Thiruvengadam, M. Evaluation of polyphenol composition and biological activities of two samples from summer and winter seasons of Ligularia fischeri var. Spiciformis Nakai %J Acta Biologica Hungarica Acta Biologica Hungarica . 66 , 179–191. https://doi.org/10.1556/018.66.2015.2.5 (2015). Yang, L., Li, C. L., Cheng, Y. Y. & Tsai, T. H. Development of a Validated UPLC-MS/MS Method for Analyzing Major Ginseng Saponins from Various Ginseng Species. 24 , 4065. (2019). Tiwari, A., Bose, D., Mishra, P., Jain, A. & Jain, S. K. Determination of Oxaliplatin and Curcumin in Combination via Micellar HPLC and Its Method Validation. J. AOAC Int. 105 , 999–1007. 10.1093/jaoacint/qsac042 (2022). Liu, G. et al. Development and validation of an HPLC-MS/MS method to determine clopidogrel in human plasma. Acta Pharm. Sinica B . 6 , 55–63. 10.1016/j.apsb.2015.11.001 (2016). Willems, J. L. et al. Analysis of a series of chlorogenic acid isomers using differential ion mobility and tandem mass spectrometry. Anal. Chim. Acta . 933 , 164–174. 10.1016/j.aca.2016.05.041 (2016). Santos, J. L. et al. Evaluation of chemical constituents and antioxidant activity of coconut water (Cocus nucifera L.) and caffeic acid in cell culture. Anais da Acad. Brasileira de Ciencias . 85 , 1235–1247. 10.1590/0001-37652013105312 (2013). Cao, S. et al. Chemical Constituent Analysis of Ranunculus sceleratus L. Using Ultra-High-Performance Liquid Chromatography Coupled with Quadrupole-Orbitrap High-Resolution Mass Spectrometry. 27 , 3299. (2022). Sun, L. et al. Qualitative analysis and quality control of Traditional Chinese Medicine preparation Tanreqing injection by LC-TOF/MS and HPLC-DAD-ELSD. Anal. Methods . 5 , 6431–6440. 10.1039/C3AY40681D (2013). Peres, R. G., Tonin, F. G., Tavares, M. F. & Rodriguez-Amaya, D. B. HPLC-DAD-ESI/MS identification and quantification of phenolic compounds in Ilex paraguariensis beverages and on-line evaluation of individual antioxidant activity. Molecules 18 , 3859–3871. 10.3390/molecules18043859 (2013). Misto, M. et al. Identification of Chlorogenic Acid, Caffeine, Melanoidin, Sucrose, and Protein Content of Local Indonesia Arabica Coffee Base on Its Cupping and Variety Variation. BIO Web of Conferences 101 , 01003, (2024). 10.1051/bioconf/202410101003 Hong, S., Joo, T. & Jhoo, J. W. Antioxidant and anti-inflammatory activities of 3,5-dicaffeoylquinic acid isolated from Ligularia fischeri leaves. Food Sci. Biotechnol. 24 , 257–263. 10.1007/s10068-015-0034-y (2015). Sang-Min, K., Suk-Woo, K. & Byung-Hun, U. Extraction Conditions of Radical Scavenging Caffeoylquinic Acids from Gomchui (Ligularia fischeri) Tea. J. Korean Soc. Food Sci. Nutr. 39 , 399–405 (2010). Mijangos-Ramos, I. F. et al. Bioactive dicaffeoylquinic acid derivatives from the root extract of Calea urticifolia. Revista Brasileira de Farmacognosia . 28 , 339–343. https://doi.org/10.1016/j.bjp.2018.01.010 (2018). Shang, Y. F. et al. Isolation and identification of antioxidant compounds from Ligularia fischeri. J. Food Sci. 75 , C530–535. 10.1111/j.1750-3841.2010.01714.x (2010). Jin Gwan, K. et al. Joa Sub, O. Method for Validation of Caffeoylquinic Acid Derivatives in Ligularia fischeri Leaf Extract as Functional Ingredients. J. Korean Soc. Food Sci. Nutr. 45 , 61–67 (2016). Woo, Y. J., Shin, S. R. & Hong, J. Y. Study on antioxidant and physiological activities of extract from Ligularia fischeri by extraction methods. Korean J. Food Preservation . 24 , 1113–1121. 10.11002/kjfp.2017.24.8.1113 (2017). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 21 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 26 May, 2025 Reviews received at journal 25 May, 2025 Reviews received at journal 21 May, 2025 Reviews received at journal 20 May, 2025 Reviewers agreed at journal 14 May, 2025 Reviewers agreed at journal 14 May, 2025 Reviewers agreed at journal 13 May, 2025 Reviewers invited by journal 09 May, 2025 Editor assigned by journal 09 May, 2025 Editor invited by journal 09 May, 2025 Submission checks completed at journal 08 May, 2025 First submitted to journal 02 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6576668","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":456173066,"identity":"ce5c5215-86ee-4286-b167-a0633c8967e6","order_by":0,"name":"Hun Hwan KIM","email":"","orcid":"","institution":"Biological Resources Research Group, Gyeongnam Department of Environment Toxicology and Chemistry, Korea Institute of Toxicology","correspondingAuthor":false,"prefix":"","firstName":"Hun","middleName":"Hwan","lastName":"KIM","suffix":""},{"id":456173068,"identity":"e248faba-46d4-431d-bd80-7d39d154f1b3","order_by":1,"name":"Se Hyo 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08:23:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6576668/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6576668/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-10636-4","type":"published","date":"2025-08-21T16:29:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82747221,"identity":"fa5128c6-613d-44d9-b34b-82d0fb975ce3","added_by":"auto","created_at":"2025-05-14 19:07:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":344815,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLeaf shapes of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLigularia fischeri\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in each cultivation area. (A) Yangsan, (B) Nonsan, (C) Jeongseon, (D) Hoengseong and (E) Hamyang.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6576668/v1/8a3b963e4e7081459ffdd4d2.png"},{"id":82747220,"identity":"baea0007-0276-4705-805a-751faf53760d","added_by":"auto","created_at":"2025-05-14 19:07:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":95116,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromatograms of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLigularia fischeri\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e extract based on origin and extraction solvent.\u003c/strong\u003e Orange represents 100% DW, blue represents 30% EtOH, and green represents 50% EtOH peaks. Each region is labeled as (A) Hamyang, (B) Hoengseong, (C) Jeongseon, (D) Nonsan, and (E) Yangsan. The compounds detected at 284 nm are Chlorogenic acid (1), Caffeic acid (2), Hyperin (3), 3,4-Dicaffeoyl quinicacid (4), 3,5-Dicaffeoylquinic acid (5), 4,5-Dicaffeoylquinic acid (6), and unknown (7).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6576668/v1/85593cee0d7da944411161e5.png"},{"id":82747688,"identity":"c9cf4dda-ec93-43b4-8ead-25fe72eac661","added_by":"auto","created_at":"2025-05-14 19:23:11","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":150046,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFragmentation scheme of identified compounds in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLigularia fischeri \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eextract.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6576668/v1/67d23b28c05c27a742a6dc20.jpg"},{"id":82747536,"identity":"db8deb89-58e1-4bbe-8662-494c4a4f8ccc","added_by":"auto","created_at":"2025-05-14 19:15:11","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":62336,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromatograms of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLigularia fischeri\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e extracts and three DCQAs as potential functional compounds.\u003c/strong\u003e (A) The black peak is from \u003cem\u003eLigularia fischeri\u003c/em\u003e extract, and the blue peak is from a mixture of three standard DCQAs. (B) The mixture of three standard DCQAs is shown as a pink peak, and 3,4-Dicaffeoyl quinic acid is shown as a black peak. (C) The pink peak represents the mixture of the three standard DCQAs, and the black peak represents 3,5-dicaffeoyl quinic acid. (D) The pink peak represents the mixture of the three standard DCQAs, and the black peak represents 4,5-dicaffeoyl quinic acid.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6576668/v1/36c569dbbccec7b247093d25.jpg"},{"id":89847219,"identity":"0d4b7991-bdee-4ca5-9176-a1b98c7cdd98","added_by":"auto","created_at":"2025-08-25 16:42:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2337660,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6576668/v1/89309bb7-b074-4288-a9a5-50441aeaeb7a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Validation and Standardization of the Content of Three DCQAs (Di-caffeoylquinic acid) in Korean Ligularia fischeri by Region of Origin","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eLigularia fischeri\u003c/em\u003e is a plant of the Asteraceae family native to the high mountain regions of Northeast Asia, particularly Korea, China, and Japan. Its roots and leaves have been widely used traditionally for medicinal and edible purposes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Recent studies have revealed that \u003cem\u003eLigularia fischeri contains\u003c/em\u003e various bioactive compounds and is attracting attention for its physiological effects, such as antioxidant, anti-inflammatory, and anti-cancer properties. [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe main bioactive compounds of \u003cem\u003eLigularia fischeri\u003c/em\u003e include polyphenolic compounds, flavonoids, terpenoids, and sesquiterpenoids, which have been also reported to contain various bioactive effects such as antioxidant, anti-inflammatory, and antibacterial activities [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In particular, polyphenols, flavonoids, and chlorogenic acid are known to have positive effects on the prevention of chronic diseases [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Additionally, some studies suggest that \u003cem\u003eLigularia fischeri\u003c/em\u003e extract may exhibit physiological functions such as inhibiting cancer cell proliferation, lowering blood sugar levels, protecting the liver, and treating rheumatoid arthritis [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, scientific research on \u003cem\u003eLigularia fischeri\u003c/em\u003e is still in its early stages, and standardized methods are required to ensure reliable and reproducible experimental results, particularly in analytical procedures.\u003c/p\u003e \u003cp\u003eA search on PubMed using the keywords \u0026ldquo;\u003cem\u003eLigularia\u003c/em\u003e fischeri\u0026rdquo; and \u0026ldquo;DCQA\u0026rdquo; found a total of three papers. Among these, only one paper actually addressed the separation and identification of the compounds. Specifically, Shang et al. (2010) analyzed \u003cem\u003eLigularia fischeri\u003c/em\u003e collected from Daegwallyeong, Gangwon-do, South Korea, and identified three types of DCQA (dicaffeoylquinic acids), with 4,5-DCQA showing the highest content. The three types of DCQA identified were 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA, with 4,5-DCQA having the highest content [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Kuroda et al. (2016) analyzed samples of \u003cem\u003eLigularia fischeri\u003c/em\u003e from two species in Sichuan Province and four species in Chongqing, totaling six species, and found differences in the distribution and composition of benzo[a]pyranoquinones and eremophilan compounds among the samples [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Additionally, according to a study conducted in Korea, leaf extracts of \u003cem\u003eLigularia fischeri\u003c/em\u003e collected in summer (June) had higher polyphenol and flavonoid content and stronger antioxidant and antibacterial activity compared to samples collected in winter (December) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Thus, to date, there has been limited research on regional differences in the content of DCQA in Korea.\u003c/p\u003e \u003cp\u003eTherefore, in this study, five regions (Hamyang, Hoengseong, Jeongseon, Nonsan, and Yangsan) where \u003cem\u003eLigularia fischeri\u003c/em\u003e is cultivated in Korea and analyzed the compounds using HPLC-MS/MS and chromatograms, confirming the absence of interfering compounds. As a result, seven main peaks were identified, including three DCQAs (dicaffeoylquinic acid): 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA. After that, the content differences of three types of DCQAs (3,4-DCQA, 3,5-DCQA, and 4,5-DCQA) were confirmed using standard compounds, and linearity, precision, limit of quantification (LOD), limit of detection (LOQ), and recovery rate were measured for quantitative analysis. This study aims to scientifically confirm the potential value of \u003cem\u003eLigularia fischeri\u003c/em\u003e as a functional food ingredient, as well as regional and solvent-specific differences in content and validation methods, thereby providing foundational data for future industrial applications.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals and Reagents\u003c/h2\u003e \u003cp\u003eThe chemical standards of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC-MS/MS grade solvents (acetonitrile and triple deionized water) and methanol were obtained from Duksan Pure Chemical Co. Ltd. (Dongdaemun-gu, Seoul, Korea).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of Plant Materials\u003c/h2\u003e \u003cp\u003e\u003cem\u003eLigularia fischeri\u003c/em\u003e was collected from Hamyang (Code No. 00951A) and Yangsan (Code No. 00952A), Gyeongsangnam-do; Hoengseong (Code No. 00953A) and Jeongseon (Code No. 00954A), Gangwon-do; and Nonsan (Code No. 00955A), Chungcheongnam-do. After obtaining permission from each local farm to collect and provided by Kick the Hurdle Inc. The samples were stored in the Animal Bio Resources Bank, Korea, a nationally designated research materials bank, and each source was assigned a code number (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The leaves and stems of the plants provided were washed with water, finely chopped, and dried in a drying oven at 56\u0026deg;C for 24 hours. They were then placed in sealed polyethylene bags containing silica gel and stored at -20\u0026deg;C until use.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation of Sample and Standard Solutions\u003c/h2\u003e \u003cp\u003eThe preparation of plants and standards solution for LC-MS was performed using a modified technique [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. \u003cem\u003eLigularia fischeri\u003c/em\u003e (5g) was extracted for 72 hours at 60\u0026deg;C in a water bath using three solvents: 100% DW, 30% EtOH, and 50% EtOH, each in 50 mL. The extract was centrifuged at 3000 rpm for 10 minutes. After centrifugation, the supernatant was filtered through filter paper (Whatman, Qualitative, Circles, 110mm Dia, Cat No. 1001\u0026thinsp;\u0026minus;\u0026thinsp;110). A rotary evaporator (N-1110, Eyela, Tokyo, Japan) was rotated at 100 rpm and used under reduced pressure at 45\u0026deg;C to completely remove any residual ethanol. Subsequently, the sample was freeze-dried to obtain a powder. Prepared \u003cem\u003eLigularia fischeri\u003c/em\u003e sample powder and standard of 3,4-DCQA, 3,5-DCQA, 4,5-DCQA were prepared at a concentration of 1 mg/mL in methanol. All samples were filtered through a 0.45 \u0026micro;m PVDF syringe filter prior to analysis and prepared in the same method as above prior to injection for HPLC-MS/MS analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. HPLC-MS/MS Instrumentation and Analysis\u003c/h2\u003e \u003cp\u003eHPLC and LC-MS/MS was performed on a Shimadzu Nexera Lite LC-40D HPLC system (Shimadzu, Corp., Kyoto, Japan) and Ultra Quadrupole Time of flight LC/MS/MS System (X500R) operated in positive ion mode (spray voltage set at \u0026minus;\u0026thinsp;4.5 kV). The solvent used was DW and Acetonitrile containing 0.1% formic acid, a gradient system was used at a flow rate of 0.5 mL/min for analysis, and a Prontosil C18 column (length, 250 mm; inner diameter, 4.6 mm; particle size, 5 \u0026micro;m; Phenomenex Co., Ltd., California, USA, Biochoff Chromatography) was used. The solvent conditions used in the mobile phases were 0\u0026ndash;10 min at 10\u0026ndash;15% B, 10\u0026ndash;20 min at 20% B, 20\u0026ndash;30 min at 25%, 30\u0026ndash;40 min at 40%, 40\u0026ndash;50 min at 70%, 50\u0026ndash;60 min at 95%, and 60\u0026ndash;70 min at 95%. The analysis was performed at a wavelength of 284 nm, and temperature of 35\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe mass spectrometry conditions for qualitative analysis of peaks identified by HPLC were performed in positive mode using electrospray ionization (ESI) and multiscan between m/z 100\u0026ndash;2000, with a desolvation temperature set at 500\u0026deg;C, spray voltage at 5500 V, ion source gas at 50 psi, curtain gas at 30 psi, and declustering potential (DP) at 80 V; MS/MS spectra were set with a collision energy of 35\u0026thinsp;\u0026plusmn;\u0026thinsp;15V. The obtained ion chromatogram data were generated using SCIEX OS software (3.0.0).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Quantification and Validation of the Analytical Method\u003c/h2\u003e \u003cp\u003eThe analytical method validation was conducted in accordance with the ICH and U.S. Food and Drug Administration bioanalytical method validation guidelines [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The quantification of compounds detected in \u003cem\u003eLigularia fischeri\u003c/em\u003e extract was performed at 284 nm, and the concentrations of the three DCQAs were calculated using the following formula: (Calibration curve results (ug/mL) \u0026times; Final volume (mL) \u0026times; dilution factor \u0026times; standard solution purity) / (sample weight (g) \u0026times; 1000 (ug/mg)). Specificity, linearity, detection limit (LOD), quantification limit (LOQ), precision, and recovery rate were measured to evaluate the performance of this method.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. Specificity\u003c/h2\u003e \u003cp\u003eThe specificity of the chromatogram and PDA spectrum patterns of \u003cem\u003eLigularia fischeri\u003c/em\u003e extract was confirmed as follows. It was confirmed by comparison with standard solutions based on specificity derived from the selective quantification of various compounds in complex mixtures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2. Linearity and Range\u003c/h2\u003e \u003cp\u003eStandard solutions of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA at concentrations of 100, 250, 500, 750, and 1000 \u0026micro;g/mL (n\u0026thinsp;=\u0026thinsp;5) were prepared and analyzed to confirm the linearity of the calibration curve. The standard solution analysis was repeated five times for each concentration. The eluted peaks were subjected to linear regression (n\u0026thinsp;=\u0026thinsp;5) and measured as the ratio of peak area to analyte concentration. The linearity of the association was assessed using the correlation coefficients (R\u003csup\u003e2\u003c/sup\u003e) that were computed from the calibration curves.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3. LOD and LOQ\u003c/h2\u003e \u003cp\u003eThe minimum quantity of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA that may be found in the \u003cem\u003eLigularia fischeri\u003c/em\u003e extract sample is known as the LOD. The LOQ is the minimum quantity that can be accurately and quantitatively suitable for precision. The standard deviations of the y-intercepts and slopes of the calibration curves were used to determine the LOD and LOQ values. The calibration curve through standard deviation and slope was used to verify linearity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.5.4. Precision and Recovery\u003c/h2\u003e \u003cp\u003eThe precision of the validation method was determined using standard solutions of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA by inter-day and intra-day at five different concentrations (100, 250, 500, 750, 1000 \u0026micro;g/mL). Intra-day was five times a day to analyze precision, and tests were run five times a day for three days in a row to analyze inter-day precision. To determine precision, the relative standard deviation (RSD) was calculated. The accuracy of the suggested approach was verified by a recovery analysis. Recovery was assessed after measuring the \u003cem\u003eLigularia fischeri\u003c/em\u003e extract with five concentrations of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA. Each analysis was repeated five times. Recovery was calculated based on the sample peak area value, standard peak area value, and peak area value.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Separation and characteristic analysis of phenolic compounds in \u003cem\u003eLigularia fischeri\u003c/em\u003e extract\u003c/h2\u003e \u003cp\u003eThe qualitative and quantitative analysis of compounds contained in \u003cem\u003eLigularia fischeri\u003c/em\u003e by region (Hamyang, Hoengseong, Jeongseon, Nonsan, and Yangsan) and extraction solvent (100% DW, 30% EtOH, and 50% EtOH) was performed using HPLC-MS/MS. A total of seven peaks were identified by HPLC retention time and UV-vis spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The seven compounds obtained at a wavelength of 284 nm were identified as chlorogenic acid [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], Caffeic acid [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], Hyperin [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], 3,4-Dicaffeoylquinic acid [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], 3,5- Dicaffeoylquinic acid, 4,5- Dicaffeoylquinic acid [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and an unknown peak. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e provides the analysis results of mass spectrometry data based on existing literature. This is followed by the predicted fragmentation of compounds obtained through LC-MS/MS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe HPLC-MS/MS data of phenolic compounds from \u003cem\u003eLigularia fischeri\u003c/em\u003e extract with different origins.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePeak\u003c/p\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRt(min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFormula\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUV max\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMS/MS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChlorogenic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e325, 250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e355\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e181 (C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e) [M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e163 (C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e) [M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e-H\u003csub\u003e2\u003c/sub\u003eO]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e135 (C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) [M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e-CO]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e21.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCaffeic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e330\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e181\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e163 (C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e) [M\u0026thinsp;+\u0026thinsp;H-H\u003csub\u003e2\u003c/sub\u003eO]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e145 (C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) [M\u0026thinsp;+\u0026thinsp;H-H\u003csub\u003e2\u003c/sub\u003eO- H\u003csub\u003e2\u003c/sub\u003eO]\u003c/p\u003e \u003cp\u003e135 (C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) [M\u0026thinsp;+\u0026thinsp;H-COOH]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e117 (C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eO\u003csup\u003e\u0026minus;\u003c/sup\u003e) [M\u0026thinsp;+\u0026thinsp;H-COOH-H\u003csub\u003e2\u003c/sub\u003eO]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e31.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHyperin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e355, 255\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e465\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e303 (C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e)\u003c/p\u003e \u003cp\u003e[M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e153 (C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e)\u003c/p\u003e \u003cp\u003e[M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e-RDA]\u003c/p\u003e \u003cp\u003e109 (C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO)\u003c/p\u003e \u003cp\u003e[M\u0026thinsp;+\u0026thinsp;H- C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e-RDA-H\u003csub\u003e2\u003c/sub\u003eO]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e33.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3,4-Dicaffeoylquinic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e325, 290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e517\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e499 (C\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e) [M\u0026thinsp;+\u0026thinsp;H-H\u003csub\u003e2\u003c/sub\u003eO]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e337 (C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e) [M\u0026thinsp;+\u0026thinsp;H-H\u003csub\u003e2\u003c/sub\u003eO-C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e193 (C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e) [M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e175 (C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e) [M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3,5- Dicaffeoylquinic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e330, 290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e517\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e355 (C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e) [M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e193 (C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e) [M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e-C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e181 (C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e) [M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e-C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e137 (C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) [M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e-O]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e38.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4,5- Dicaffeoylquinic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e330, 290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e517\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e355 (C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e)\u003c/p\u003e \u003cp\u003e[M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e]\u003c/p\u003e \u003cp\u003e193 (C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e)\u003c/p\u003e \u003cp\u003e[M\u0026thinsp;+\u0026thinsp;H- C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e-C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e]\u003c/p\u003e \u003cp\u003e175 (C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e)\u003c/p\u003e \u003cp\u003e[M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e-H\u003csub\u003e2\u003c/sub\u003eO]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Optimization of HPLC\u0026ndash;MS/MS Condition\u003c/h2\u003e \u003cp\u003eStandard solutions of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA (100 \u0026micro;g/mL) were used to optimize the HPLC-MS/MS conditions. A Pronto SIL column (250 \u0026times; 4.6 mm, 5 \u0026micro;m, 120-5-C18 SH, Bischoff Chromatography, Leonberg, Germany) was used to identify detectable phenolic compounds, and the system was set up using a gradient method with a column temperature of 35\u0026deg;C and a mobile phase consisting of water and acetonitrile, each containing 0.01% formic acid. Based on a literature review, the maximum absorbance of chlorogenic acid was found to be around 290 nm, so the UV wavelength range of 200\u0026ndash;400 nm was set to detect its derivatives, 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor the identification of compounds in the sample, positive ESI conditions were applied because ion fragments showed higher safety than negative mode. Subsequently, when the sample was prepared using methanol, it exhibited sufficiently excellent peak shapes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Method of Validation\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Specificity\u003c/h2\u003e \u003cp\u003eThrough the chromatograms of the standard solution and sample solution, we confirmed that the peaks in the \u003cem\u003eLigularia fischeri\u003c/em\u003e extract were clearly separated and that there was no interference from other compounds. Specificity was confirmed by verifying that the retention time (RT) patterns between the standard solution and sample solution matched and that when the standard solutions were mixed, each standard solution was detected at different times (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2. Linearity, Range and LOD, and LOQ\u003c/h2\u003e \u003cp\u003eLinearity verification was performed five times at each concentration, and the linearity of 3,4-DCQA (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.9998), 3,5-DCQA (R\u0026sup2; \u0026ge; 0.9998), and 4,5-DCQA (R\u0026sup2; \u0026ge; 0.9997) were confirmed. Subsequently, we obtained the linear expressions for 3,4-DCQA (y\u0026thinsp;=\u0026thinsp;4390.5x \u0026minus;\u0026thinsp;34206), 3,5-DCQA (y\u0026thinsp;=\u0026thinsp;5093.5x \u0026minus;\u0026thinsp;106812), and 4,5-DCQA (y\u0026thinsp;=\u0026thinsp;5549.9x \u0026minus;\u0026thinsp;143191). The Limit of Detection (LOD) and Limit of Quantitation (LOQ) were determined, with the LOD and LOQ for 3,4-DCQA being 0.795 mg/L and 2.386 mg/L, respectively. The LOD and LOQ for 3,5-DCQA were 0.637 mg/L and 1.910 mg/L, respectively. The LOD and LOQ for 4,5-DCQA were 0.589 mg/L and 1.766 mg/L, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eCalibration curve data for the quantification of 3 type of DCQAs.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSlopes of Calibration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCorrelation Coefficient (R\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLOD\u003c/p\u003e \u003cp\u003e(mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLOQ\u003c/p\u003e \u003cp\u003e(mg/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3,4-DCQA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4390.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9998\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.795\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.386\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3,5-DCQA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5093.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9998\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.637\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.910\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4,5-DCQA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5549.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9997\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.589\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.766\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eLOD: Limit of Detection; LOQ: Limit of Quantitation, (\u003cem\u003eո\u003c/em\u003e = 5)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3 Precision\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the intraday precision (repeatability) values of the standard solutions of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA, which were measured under the same conditions within a single day. The tests were conducted five times within a concentration range of 100\u0026ndash;1000 \u0026micro;g/mL, and the relative standard deviation (RSD) values for each standard solution concentration were within 0.25%. Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the interday precision (intermediate precision) values of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA in \u003cem\u003eLigularia fischeri\u003c/em\u003e extract obtained from five regions, using the same equipment under the same conditions but on different days. This confirmed the reliability and reproducibility of the HPLC-PDA analysis method.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eIntra-day precision for 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA in\u003c/b\u003e \u003cb\u003eLigularia fischeri\u003c/b\u003e \u003cb\u003eextract using the HPLC-PDA method.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNominal Concentration\u003c/p\u003e \u003cp\u003e(ug/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eIntraday\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eObserved Concentration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePrecision RSD (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e3,4-DCQA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98.0465\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2458\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e255.2384\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6584\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2579\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e498.5773\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8622\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1729\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e750\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e744.1814\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8564\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1151\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1003.9732\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0276\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1024\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e3,5-DCQA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.5182\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1927\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1936\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e255.2977\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4544\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1780\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e494.9713\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6239\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1260\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e750\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e745.8983\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7657\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1027\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1003.0994\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9077\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0905\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e4,5-DCQA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e103.1656\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1782\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1727\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e254.2724\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3253\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1280\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e489.4829\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5913\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1208\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e750\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e748.1448\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0938\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1004.3856\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8119\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0808\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eData were expressed as the mean SD (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eInter-day precision for 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA in\u003c/b\u003e \u003cb\u003eLigularia fischeri\u003c/b\u003e \u003cb\u003eextract using the HPLC-PDA method.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOrigin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSolvent ratio (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eInterday\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eObserved Concentration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePrecision RSD (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"14\" rowspan=\"15\"\u003e \u003cp\u003e3,4-DCQA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eHamyang\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e400.9328\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7392\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1844\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e694.7095\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0489\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1510\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e628.7130\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1105\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eHoengseong\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e799.1811\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4865\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0609\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1675.8443\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2639\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0754\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1876.1564\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1760\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0627\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eJeongseon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e782.4230\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5818\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5818\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e894.3677\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6668\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0746\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e952.0126\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0759\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1130\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eNonsan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e929.3770\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9992\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1075\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1914.7634\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2271\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0641\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2152.2849\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0497\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0952\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eYangsan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e194.0019\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7225\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.7225\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e300.7707\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5716\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1900\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e381.5616\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4148\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1087\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"14\" rowspan=\"15\"\u003e \u003cp\u003e3,5-DCQA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eHamyang\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e347.1160\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2238\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0645\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e637.3278\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1752\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1147.2962\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9989\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0871\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eHoengseong\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e532.2904\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9027\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1696\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e995.4297\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5177\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0520\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1706.0361\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1197\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0656\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eJeongseon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e320.7918\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1286\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e550.6090\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4755\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0864\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1070.2532\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0469\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eNonsan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e495.3014\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5545\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1120\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e987.9929\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7045\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5158\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1470.6069\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0601\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0721\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eYangsan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e171.6034\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4994\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.2910\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e373.1339\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1382\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e843.3912\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0925\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"14\" rowspan=\"15\"\u003e \u003cp\u003e4,5-DCQA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eHamyang\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e180.3627\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6340\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.3515\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e749.1779\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4227\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0564\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1044.9344\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9620\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0921\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eHoengseong\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e304.4347\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4813\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1581\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e950.7943\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3793\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0399\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e988.9631\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0910\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eJeongseon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e213.6833\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1884\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e705.7721\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7480\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1060\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e927.4137\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7327\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0790\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eNonsan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e338.2645\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4671\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1381\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1179.4910\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8460\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0717\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1125.3682\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6967\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0619\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eYangsan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e86.7630\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5076\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e435.0737\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3509\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0807\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e466.1191\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6737\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1445\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eSolvent; DW (with Ethanol), (\u003cem\u003eո\u003c/em\u003e = 5)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.3.4. Recovery\u003c/h2\u003e \u003cp\u003eThe recovery study was conducted using HPLC-PDA analysis with a mixture of \u003cem\u003eLigularia fischeri\u003c/em\u003e extract and standard solutions of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA. The overall recovery rates for 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA were 96.9675\u0026ndash;99.1368%, with RSD values all below 1% (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eRecovery study of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA in\u003c/b\u003e \u003cb\u003eLigularia fischeri\u003c/b\u003e \u003cb\u003eextract\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRecovery (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRSD\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExtract\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStandard\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eExtract with Standard\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3,4-DCQA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1079.5395\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2749\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e508.7541\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1856\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1540.1124\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4529\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e96.9675\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.6123\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.631472\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3,5-DCQA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e745.7905\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0325\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e514.3746\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4918\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1228.5486\u0026thinsp;\u0026plusmn;\u0026thinsp;9.4095\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e99.1368\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.7146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.720803\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4,5-DCQA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e576.4081\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8836\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e516.3915\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3605\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1126.8748\u0026thinsp;\u0026plusmn;\u0026thinsp;11.7163\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e97.4385\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.8892\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.912598\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3.5\u003c/b\u003e \u003cb\u003eQuantitative Analysis of the\u003c/b\u003e \u003cb\u003e3,4-DCQA, 3,5-DCQA, and 4,5-DCQA\u003c/b\u003e \u003cb\u003ein Ligularia fischeri\u003c/b\u003e \u003cb\u003eextract by solvent ratio and origin.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAnalysis of the content of three types of DCQA by the concentration of extraction solvent in \u003cem\u003eLigularia fischeri\u003c/em\u003e. from five regions showed that in 100% distilled water, 3,4-DCQA was highest in Nonsan (929.3770\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9992 ug/mL), Hoengseong (799.1811\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4865 ug/mL), and Jeongseon (782.4230\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5818 ug/mL); 3,5-DCQA was higher in the order of Hoengseong (532.2904\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9027 ug/mL) Nonsan (495.3014\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5545 ug/mL) and Hamyang (347.1160\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2238 ug/mL); and 4,5-DCQA was higher in the order of Nonsan (338.2645\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4671 ug/mL), Hoengseong (304.4347\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4813 ug/mL), and Jeongseon (213.6833\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4025 ug/mL). In 30% ethanol, 3,4-DCQA was Nonsan (1914.7634\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2271 ug/mL), Hoengseong (1675.8443\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2639 ug/mL), and Jeongseon (894.3677\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6668 ug/mL); 3,5-DCQA was Hoengseong (995.4297\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5177 ug/mL), Nonsan (987.9929\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7045 ug/mL), and Hamyang (637.3278\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1169 ug/mL); and 4,5-DCQA was Nonsan (1179.4910\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8460 ug/mL), Hoengseong (950.7943\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3793 ug/mL), and Hamyang (749.1779\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4227 ug/mL). In 50% ethanol, 3,4-DCQA was Nonsan (2152.2849\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0497 ug/mL), Hoengseong (1876.1564\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1760 ug/mL), and Jeongseon (952.0126\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0759 ug/mL); 3,5-DCQA was Hoengseong (1706.0361\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1197 ug/mL), Nonsan (1470\u0026thinsp;\u0026plusmn;\u0026thinsp;6069\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0601 ug/mL), and Hamyang(1147.2962\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9989 ug/mL); and 4,5-DCQA was Nonsan (1125.3682\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6967 ug/mL), Hamyang(1044.9344\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9620 ug/mL), and Hoengseong (988.9631\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9003 ug/mL) (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eChanges in content of\u003c/b\u003e \u003cb\u003ethe\u003c/b\u003e \u003cb\u003e3,4-DCQA, 3,5-DCQA, and 4,5-DCQA according to extraction by solvent ratio and region.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompound Name\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSolvent ratio (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eConcentration (ug/mL)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHamyang\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHoengseong\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJeongseon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNonsan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYangsan\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e3,4-DCQA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e400.9328\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7392 \u003csup\u003eE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e799.1811\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4865 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e782.4230\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5818 \u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e929.3770\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9992 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e194.0019\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7225 \u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e694.7095\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0489 \u003csup\u003eE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1675.8443\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2639 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e894.3677\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6668 \u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1914.7634\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2271 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e300.7707\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5716 \u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e628.7130\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6949 \u003csup\u003eE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1876.1564\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1760 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e952.0126\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0759 \u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2152.2849\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0497 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e381.5616\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4148 \u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e3,5-DCQA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e347.1160\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2238 \u003csup\u003eE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e532.2904\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9027 \u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e320.7918\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4125 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e495.3014\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5545 \u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e171.6034\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4994 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e637.3278\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1169 \u003csup\u003eE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e995.4297\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5177 \u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e550.6090\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4755 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e987.9929\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7045 \u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e373.1339\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5158 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1147.2962\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9989 \u003csup\u003eE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1706.0361\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1197 \u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1070.2532\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5016 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1470\u0026thinsp;\u0026plusmn;\u0026thinsp;6069\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0601 \u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e843.3912\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7800 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e4,5-DCQA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e180.3627\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6340 \u003csup\u003eE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e304.4347\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4813 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e213.6833\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4025 \u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e338.2645\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4671 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e86.7630\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4404 \u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e749.1779\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4227 \u003csup\u003eE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e950.7943\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3793 \u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e705.7721\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7480 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1179.4910\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8460 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e435.0737\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3509 \u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1044.9344\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9620 \u003csup\u003eE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e988.9631\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9003 \u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e927.4137\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7327 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1125.3682\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6967 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e466.1191\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6737 \u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eSolvent; DW (with Ethanol), (\u003cem\u003eո\u003c/em\u003e = 5)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAll values are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3). A-E Means with different superscripts in the same row were significantly different at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 using Duncan's multiple range test. Also tested based on superscript A in the same row.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cem\u003eLigularia fischeri\u003c/em\u003e contains dicaffeoylquinic acids (DCQAs), one of the main bioactive compounds, which are promising substances that can contribute to health promotion through their antioxidant and anti-inflammatory effects [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. DCQA is a compound in which a caffeine acid residue is bound to a quinic acid structure and varies in form depending on its position, such as 3,4-, 3,5-, and 4,5-DCQA. Recent studies have shown that 3,5-DCQA inhibits NO and suppresses the expression of inflammatory mediators such as INOS, COX2, and TNF-α. Additionally, 4,5-DCQA has been found to regulate an anti-inflammatory pathway mediated by the TRPV1 receptor and inhibit COX2 expression [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Especially, in the case of \u003cem\u003eLigularia fischeri\u003c/em\u003e, 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA are evenly contained and exist at a certain concentration [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Therefore, there is a lot of research being conducted on the development of various functional foods and pharmaceuticals using DCQAs.\u003c/p\u003e \u003cp\u003eThe study of analyzing and verifying three types of DCQA contained in \u003cem\u003eLigularia fischeri\u003c/em\u003e extract as marker compounds is already conducted. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In addition, studies on the antioxidant and physiological activities of \u003cem\u003eLigularia\u003c/em\u003e fischeri have been conducted based on its extraction method, but these studies are limited to simple extracts [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, in this study, we compared the concentrations of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA, which act as marker compounds of \u003cem\u003eLigularia fischeri\u003c/em\u003e extract. Additionally, we compared and analyzed the concentrations of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA in \u003cem\u003eLigularia fischeri\u003c/em\u003e extract using three different solvents and performed methodological analytical validity verification.\u003c/p\u003e \u003cp\u003eThe results of the content analysis in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e show that the concentration of 3,4-DCQA was highest at 2152.2849\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0497 ug/mL in the 50% ethanol extract from the Nonsan region, while the concentration of 3,5-DCQA was highest at 1706.0361\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1197 ug/mL in the 50% ethanol extract from the Hoengseong region, while the highest concentration of 4,5-DCQA was found in the 30% ethanol extract from the Nonsan region at 1179.4910\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8460 ug/mL.\u003c/p\u003e \u003cp\u003eThis study examined the importance of validation procedures for ensuring data validity and standardization processes for enhancing data comparability, as well as practical application methods for these procedures. Through this, we confirmed that not only can data quality be improved, but the reproducibility and generalizability of research results can also be enhanced.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eWe screened six phenolic compounds contained in \u003cem\u003eLigularia fischeri\u003c/em\u003e and applied an HPLC-MS/MS system to perform quantitative analysis of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA. A validation system was established to evaluate specificity, linearity, detection limits, quantification limits, precision, and recovery rates, and differences in content levels were confirmed based on extraction solvents and regional variations. This study contributed to the standardization of the three DCQA compounds as marker components of \u003cem\u003eLigularia fischeri extracts\u003c/em\u003e, providing valuable insights for analyzing compounds in related plants. Additionally, these findings are expected to facilitate the development of various health functional products and pharmaceuticals with potential applications in antioxidant, anti-inflammatory, blood sugar regulation, liver protection, and bone health improvement.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, H.H.K. and S.H.J.; methodology, H.H.K.; writing\u0026mdash;original draft preparation, S.H.J. and H.H.K.; writing\u0026mdash;review and editing, P.B.B., Y.G.M., and K.H.H,; investigation, S.H.J.; validation H.H.K. and S.H.J.; project administration, T.Y.K., J.W.P. and G.I.K..; supervision, G.S.K. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Research Foundation of Korea, funded by the Ministry of Science and ICT (grant no. RS-2023-00243376, and RS-2024-00411709)\u0026nbsp;and the Kick the Hurdle Co., Ltd.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePark, H. S., Choi, H. Y. \u0026amp; Kim, G. H. Preventive effect of Ligularia fischerion inhibition of nitric oxide in lipopolysaccharide-stimulated RAW 264.7 macrophages depending on cooking method. \u003cem\u003eBiol. Res.\u003c/em\u003e \u003cb\u003e47\u003c/b\u003e, 69. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/0717-6287-47-69\u003c/span\u003e\u003cspan address=\"10.1186/0717-6287-47-69\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, T. H., Truong, V. L. \u0026amp; Jeong, W. S. 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Food Preservation\u003c/em\u003e. \u003cb\u003e24\u003c/b\u003e, 1113\u0026ndash;1121. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.11002/kjfp.2017.24.8.1113\u003c/span\u003e\u003cspan address=\"10.11002/kjfp.2017.24.8.1113\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ligularia fischeri, 3,4-DCQA, 3,5-DCQA, 4,5-DCQA","lastPublishedDoi":"10.21203/rs.3.rs-6576668/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6576668/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eLigularia fischeri\u003c/em\u003e is a perennial plant in the Asteraceae family, native to Japan, China, Eastern Siberia, and Korea. In general, it is said to be good for anti-aging, bronchial diseases, anti-cancer, and constipation. In this study, we obtained five regions (Hamyang, Hoengseong, Jeongseon, Nonsan, and Yangsan) of the Korean cultivated \u003cem\u003eLigularia fischeri\u003c/em\u003e and analyzed its compounds by HPLC-MS/MS and chromatograms with standards to confirm the absence of interfering substances and confirmed that it contains three types of DCQA (Di-caffeoylquinic acid): 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA. The linearity, precision, limit of quantification (LOQ) and limit of detection (LOD), and recovery were then measured by quantitative analysis to confirm the content of the three DCQAs. The results of the analysis of three types of DCQA content in \u003cem\u003eLigularia fischeri\u003c/em\u003e obtained from five regions (Hamyang, Hoengseong, Jeongseon, Nonsan, and Yangsan) using three different solvent concentrations (100% DW, 30% EtOH, and 50% EtOH) are as follows (5 g of raw material/50 mL of extraction solvent). In 100% distilled water, 3,4-DCQA was highest in Nonsan (9.29 mg/g), 3,5-DCQA was highest in Hoengseong (5.32 mg/g), and 4,5-DCQA was highest in Nonsan (3.38 mg/g). In 30% ethanol, 3,4-DCQA was highest in Nonsan (19.15 mg/g), 3,5-DCQA was highest in Hoengseong (9.98 mg/g), and 4,5-DCQA was highest in Nonsan (11.79 mg/g). In 50% ethanol, 3,4-DCQA was highest in Nonsan (21.52 mg/g), 3,5-DCQA was highest in Hoengseong (17.06 mg/g), and 4,5-DCQA was highest in Nonsan (11.25 mg/g).\u003c/p\u003e","manuscriptTitle":"Validation and Standardization of the Content of Three DCQAs (Di-caffeoylquinic acid) in Korean Ligularia fischeri by Region of Origin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-14 19:07:07","doi":"10.21203/rs.3.rs-6576668/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-26T08:14:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-25T12:17:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-21T11:31:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-20T13:52:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"289757977723242788891657170816676900890","date":"2025-05-14T06:30:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"92728841094153685428028823182324916984","date":"2025-05-14T04:57:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"234594552867407131477546099721856460460","date":"2025-05-13T21:12:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-09T06:23:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-09T06:22:33+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-09T04:28:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-08T05:16:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-02T08:14:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4d1deb8a-64f8-4683-83a2-72c2a6cc61cd","owner":[],"postedDate":"May 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48490170,"name":"Biological sciences/Biochemistry"},{"id":48490171,"name":"Biological sciences/Developmental biology"},{"id":48490172,"name":"Biological sciences/Drug discovery"},{"id":48490173,"name":"Health sciences/Biomarkers"}],"tags":[],"updatedAt":"2025-08-25T16:36:06+00:00","versionOfRecord":{"articleIdentity":"rs-6576668","link":"https://doi.org/10.1038/s41598-025-10636-4","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-08-21 16:29:30","publishedOnDateReadable":"August 21st, 2025"},"versionCreatedAt":"2025-05-14 19:07:07","video":"","vorDoi":"10.1038/s41598-025-10636-4","vorDoiUrl":"https://doi.org/10.1038/s41598-025-10636-4","workflowStages":[]},"version":"v1","identity":"rs-6576668","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6576668","identity":"rs-6576668","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}