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Hindawi Journal of Analytical Methods in Chemistry Volume 2020, Article ID 8831938, 11 pages https://doi.org/10.1155/2020/8831938 Research Article Simultaneous Determination of Five Components of Chaihu-Shugan-San in Beagle Plasma by HPLC-MS/MS and Its Application to a Pharmacokinetic Study after a Single Dose of Chaihu-Shugan-San 1 1 1 1 1 Yong-liang Zhu , Hui-jun Wang, Hao Xue, Yi Zhang, Qian-shi Cheng, 2 1 Ling-yun Chen , and Xiang-jun Qiu School of Basic Medicine of Henan University of Science and Technology, Luoyang, China School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, China Correspondence should be addressed to Ling-yun Chen; email@example.com and Xiang-jun Qiu; firstname.lastname@example.org Received 12 April 2020; Revised 20 June 2020; Accepted 13 July 2020; Published 19 August 2020 Academic Editor: Eulogio J. Llorent Martinez Copyright © 2020 Yong-liang Zhu et al. -is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Chaihu-shugan-san (CHSGS) has been widely used in China to treat depression and gastrointestinal diseases for thousands of years, but little is known about its pharmacokinetic properties. -e purpose of our study is to develop a reliable and sensitive high- performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method to detect ﬁve components in beagle plasma and study their pharmacokinetic after oral administration of CHSGS in beagles. An Agilent C18 column (2.1 × 150 mm, 3.5 μm) was used to separate the analytes, and the column temperature was maintained at 40 C. A gradient elution procedure was used with solvent A (acetonitrile) and solvent B (0.1% formic acid, aqueous) as mobile phases. -e elution procedure was 60% B—10% B (0–3 min) and 10% B—60% B (3.1–4 min). -e ﬂow rate was 0.3 mL/min, and the total measurement time was 4 min. Within the determined range, the standard calibration curves of the ﬁve analytes had a satisfactory linear relationship (r ≥ 0.9923). -e recovery rate (n � 6) of the ﬁve analytes was between 85.42% and 90.85%, and the matrix eﬀects (n � 6) were between 94.52% and 103.91%. -ese results show that the validated method could be successfully applied to study the phar- macokinetic in beagles after a single dose of CHSGS. antioxidant, and coordinated gastrointestinal motility [8, 9]. 1. Introduction To our knowledge, the ﬂavonoid proﬁle contained in CHSGS Chaihu-shugan-san (CHSGS), a classic prescription of tra- is mainly composed of naringin, neohesperidin, hesperidin, ditional Chinese medicine (TCM), is derived from the Book paeoniﬂorin, and liquiritin. Naringin and neohesperidin are of Jingyue and recorded in the China Pharmacopoeia (2015 two major active ingredients in Zhi-qiao, which are one of edition) . CHSGS is famous for its ability to relieve qi the antidepressant mechanisms of CHSGS . Hesperidin, stagnation and coordinate gastrointestinal function . the main active ingredient in Chen-pi, has been proved to With the development of research, CHSGS has been proven coordinate gastrointestinal movements . Paeoniﬂorin to have an excellent eﬀect on depression [3, 4] and functional and liquiritin, which are main active ingredients in Bai-shao dyspepsia [5, 6]. CHSGS is a complex prescription consisting and Zhi-gan-cao, respectively, are proved to have been used of seven components, which is shown in Table 1 . -e in the treatment of hyperprolactinemia-related disorders main chemical components of CHSGS are saponins, ﬂa- , in addition to their anti-inﬂammatory eﬀects . vonoids, phenolic acids, and terpenes. Recent studies have In recent years, a large number of studies have focused shown that CHSGS has antidepressant, anti-inﬂammatory, on reporting the mechanism of CHSGS [9, 13] and its 2 Journal of Analytical Methods in Chemistry clinical eﬃcacy . However, to our knowledge, there is no C18 column (2.1 × 150 mm, 3.5 μm) and the column report to elucidate the pharmacokinetic characteristics of temperature was 40 C. -e gradient elution solutions CHSGS in vivo. It is known that a pharmacokinetic study of consisted of mobile phase A (acetonitrile) and mobile TCM is important to evaluate the rationality and safety of phase B (0.1% formic acid aqueous solution). -e speciﬁc drug prescription . -e eﬃcacy of TCM often depends gradient elution procedure was as follows: 0–3 min (60% on the synergy between the active ingredients. -erefore, the B—10% B) and 3-4 min (10% B—60% B). -e total determination of active ingredients in biological samples is measurement time was 4 min, the ﬂow rate was 0.3 mL/ necessary for a pharmacokinetic study of TCM. Although min, and the injection volume was 10 μL. CHSGS has been widely used in clinical application for Mass spectrometry measurements were performed on thousands of years, little is known about its pharmacokinetic a Sciex API 4000 Qtrap MS system equipped with a Turbo properties. -erefore, due to the lack of scientiﬁc evidence Ionspray interface. Samples were analyzed in negative ion and research methods, there is no good understanding of the mode and monitored in multiple reaction monitoring pharmacokinetics of CHSGS in vivo. To improve the de- (MRM) mode. -e MS parameters of all analytes are shown velopment of CHSGS, comprehensive studies of CHSGS are in Table 3. -e data acquisition and control of the in- required and a validated bioanalytical method is necessary to strument were performed by Analyst 1.5 software. support some pharmacokinetic researches. In this experiment, an eﬃcient, simple, and sensitive HPLC-MS/MS method was established for the ﬁrst time to 2.4. Standard Solutions, Calibration Standards, and Quality Control (QC) Samples. Stock solutions of all analytes were simultaneously detect naringin, neohesperidin, hesperidin, paeoniﬂorin, and liquiritin. Additionally, this validated prepared in the same manner (both at a concentration of 1 mg/mL): accurately weighed 10 mg of the standard and method was ﬁrst successfully applied to a pharmacokinetic study of CHSGS in beagles. dissolved it in methanol to a constant volume of 10 mL. A working solution for calibration and quality control (QC) was prepared by diluting the stock solution with methanol. 2. Materials and Methods A calibration curve standard and QC samples (three 2.1. Materials. All analyte standards were purchased from concentration levels, low, medium and high) were prepared Chengdu Mansite Biotechnology Co., Ltd. (Chendu, China). by adding an appropriate amount of working solution to Naringenin, which was used as internal standard (IS), was the blank beagle plasma. Table 4 shows the speciﬁc con- bought from Shanghai Yuanye Biotechnology Co., Ltd. centrations of calibration standards and QC samples. -e (Shanghai, China). Table 2 shows the speciﬁc information of IS working solution 100 ng/mL was also prepared by di- ﬁve standards. Meanwhile, the structure of all standards is luting the stock solution. All solution samples were stored shown in Figure 1. -e deionized water used in the ex- at −20 C. periment was produced by Milli Q system (Millipore, Bedford, MA, USA). Both acetonitrile and methanol were HPLC grade and were purchased from Merck (Darmstadt, 2.5. Preparation of Samples. -e samples in this experiment Germany). were prepared by ethyl acetate extraction method. After the plasma samples were thawed at room temperature, 100 μL of the plasma sample, 20 μL of IS working solution 2.2. Chaihu-Shugan-San Preparation. According to the 100 ng/mL, and 1 mL of ethyl acetate were added into formula of the China Pharmacopoeia (2015 edition), Chai- a 2.0 mL Eppendorf tube. -e mixed solution was vortexed hu (18 g), Chuan-xiong (15 g), Zhi-qiao (15 g), Chen-pi for 1 min and then was centrifuged at 6,000 ×g for 5 min to (18 g), Bai-shao (15 g), Xiang-fu (15 g), and Zhi-gan-cao obtain a supernatant. After the supernatant was dried with (9 g) were weighed and air-dried. All materials were nitrogen, acetonitrile-water 40 : 60 was added to re- bought from Tong Ren Tang Technologies Co., Ltd. constitute. 10 μL of the ﬁnal solution was taken into the (Beijing, China) and had been identiﬁed by HPLC-MS/MS HPLC-MS/MS system to analyze. method for chemical compositions of plants. In this re- port, we followed the methods of Li et al.  to prepare CHSGS extracts. -e raw materials made according to the 2.6. Method Veriﬁcation. In this report, we followed the ratios presented in Table 1 were soaked in 1050 mL ul- methods of Zhu et al. . Method validation included trapure water (solid/solvent, 1/10) for 30 min, decocted speciﬁcity, linearity, precision, accuracy, recovery, and with a large ﬁre until boiling, and then kept boiling for stability. In this experiment, the HPLC-MS/MS method had 30 min. After the solution was cooled to room tempera- been validated according to the US Food and Drug Ad- ture, the solution was ﬁltered using a 0.25 μm microporous ministration (FDA) guidelines . membrane. After the solution was concentrated, lyophi- To verify the speciﬁcity of the experimental method, lized powder was prepared by freeze-drying. three groups of blood samples were analyzed by HPLC-MS/ MS: (a) six individual beagle blank plasma samples; (b) plasma samples added with all analytes and IS; and (c) real 2.3. Instrumentation and Conditions. In this report, we used plasma samples after giving a single dose of CHSGS. All HPLC-MS/MS (Shimadzu, Kyoto, Japan) system to ana- blood samples were obtained from beagles. -ree groups of lyze samples. -e chromatographic column was an Agilent Journal of Analytical Methods in Chemistry 3 Table 1: -e information of components in chaihu-shugan-san (CHSGS). Botanical name Herbal name Chinese name Acquire from Ratio (%) Bupleurum Bupleuri radix Chai-hu Root 17.14 Glycyrrhiza glabra L. Glycyrrhizae radix et rhizoma Zhi-gan-cao Root and rhizome 8.56 Paeonia lactiﬂora Pall. Paeoniae radix alba Bai-shao Root 14.29 Citrus reticulata Blanco Citri reticulatae pericarpium Chen-pi Pericarp 17.14 Ligusticum chuanxiong Hort. Chuanxiong rhizoma Chuan-xiong Rhizome 14.29 Citrus aurantium L. Aurantii fructus Zhi-qiao Fruit 14.29 Cyperus rotundus L. Cyperi rhizoma Xiang-fu Rhizome 14.29 OH OH HO HO OH HO O HO OH OO HO OH OH HO HO O O HO OH O OH OH HO OH O O OH HO HO O OH HO Naringin Neohesperidin Hesperidin (a) (b) (c) OH OH OH HO OH HO OH HO HO OH HO O O O HO OH Naringenin (IS) Liquiritin Paeoniflorin (d) (e) (f) Figure 1: -e structure of all analytes and IS. Table 2: -e speciﬁc information of all analytes and IS. Analytes Molecular formula Purity (%) Batch number Neohesperidin C H O ≥98.08 MUST-19040707 28 34 15 Hesperidin C H O ≥98.46 MUST-19070701 28 34 15 Naringin C H O ≥98.06 MUST-19050808 27 32 14 Paeoniﬂorin C H O ≥99.30 MUST-18032901 23 28 11 Liquiritin C H O ≥98.50 MUST-18032801 21 22 9 Naringenin (IS) C H O ≥98.00 L21O10Q100513 15 12 5 blood samples were tested for retention time and endoge- three consecutive days, each analyte in triplicate. -e linear nous interferences. relationship was established using a weighted (1/x ) least- To evaluate the linearity relationship of the method, squares linear regression. -e linearity of each standard a series of concentrations of all analytes were prepared for calibration curve was constructed by plotting the peak area 4 Journal of Analytical Methods in Chemistry Table 3: Main analytical parameters of all analytes. a b Analytes Parent ion (m/z) Product ion for quantiﬁcation (m/z) DP (V) CE (eV) Neohesperidin 609 301 −138 −65 Hesperidin 609 301 −115 −37 Naringin 579 271 −151 −49 Paeoniﬂorin 525 449 −66 −20 Liquiritin 417 254 −90 −28 a b DP, declustering potential; CE, collision energy. Declustering potential. Collision energy. Table 4: -e speciﬁc concentration of calibration curve standards and QC samples. Concentration of QC samples (ng/mL) Analyte Concentration range of calibration standards (ng/mL) Low Medium High Neohesperidin 0.5, 1, 2.5, 5, 10, 25, 50, 100 1 25 75 Hesperidin 0.5, 1, 2.5, 5, 10, 25, 50, 100 1 25 75 Naringin 1, 2.5, 5, 10, 25, 50, 100, 200 2.5 50 150 Paeoniﬂorin 1, 2.5, 5, 10, 25, 50, 100, 200 2.5 50 150 Liquiritin 1, 2.5, 5, 10, 25, 50, 100, 200 2.5 50 150 ratio of the all analytes to IS against the nominal concen- investigated by storing the same QC samples at −20 C in tration of all analytes in plasma. -e lower limit of quan- a refrigerator for 4 weeks. -e freeze and thaw stability were titation (LLOQ) was considered as the minimum value of the determined by analyzing the QC samples after three freeze- ° ° calibration curve. thaw cycles (−20 C∼25 C). -e RSD values were required to -e precision and accuracy of this experimental method be less than 15%, and the RE values were between −15% and were veriﬁed by repeating QC samples for three consecutive 15%. days. -ree diﬀerent concentrations (low, medium, and high) of QC samples were prepared, and each concentration 2.7. Pharmacokinetic Study. Six healthy beagles (gender: was prepared for six portions (n � 6). Quantitative de- half male, half female; body weight: 7–9 kg; age: 2-3 years termination of each concentration was performed daily to old) were provided by the Laboratory Animal Center of calculate interday precision, and then the intraday accuracy Henan University of Science and Technology (Luoyang, and standard curve were calculated for three consecutive China) and were authorized by the Animal Ethics days. Precision and accuracy were expressed by relative Committee of Henan University of Science and Tech- standard deviation (RSD, %) and relative error (RE, %), nology and were cared in accordance with the National respectively. RSD � standard deviation/mean × 100%, Institutes of Health Guide for the Care and Use of Lab- RE � (average concentration − theoretical average concen- oratory Animals. All experimental animals were kept tration)/theoretical average concentration × 100%. -e RSD separately in the same conditions (room temperature, values were required to be less than 15%, and the RE values ° ° 15 C∼28 C; humidity, 35%∼6/0%; light time, 12 h) and fed were within ±15%. twice daily without limiting the amount of water. One day -e recovery of this experiment was calculated by before the experiment, the animals were fasted for 12 h but comparing the peak areas obtained in QC samples using were free to drink water. After giving CHSGS (1 g/kg), ethyl acetate extraction method; the peak areas of the ﬁve 2 mL of blood was collected from the foreleg vein at 0.17, analytes were obtained using the ethyl acetate extraction 0.33, 0.5, 0.75, 1, 1.5, 2, 3, 4, and 6 h and was took into method with those obtained from the equal amounts of heparinized polyethylene tubes. All blood samples were compounds spiked into the postextraction supernatant at placed in a centrifuge and centrifuged at 10,000 ×g for three QC concentration levels (n � 6). -e above method was 10 min to obtained 200 μL plasma, which was immediately also used for the recovery of IS (100 ng/mL). -e matrix frozen at −20 C. Using the established method to detect eﬀects (ME) were calculated by A/B, where A was the peak the concentration of ﬁve components in plasma, the data area of a blank sample spiked with ﬁve analytes after ex- of drug concentration of ﬁve analytes were dealt with DAS traction and B was the peak area of a standard solution 2.0 and then expressed as the mean± standard deviation containing equal amounts of the ﬁve analytes. -e ME of IS (mean± SD). (100 ng/mL) were treated in the same way. In order to test the stability of plasma samples, three 3. Results and Discussion diﬀerent concentrations of QC (n � 6) samples were pre- pared for measurement in 4 diﬀerent environments (room 3.1. Method Development and Optimization. -e IS method ° ° temperature for 4 h, 4 C for 12 h, −20 C for 4 weeks, and is a common method in biological sample analysis. -e ideal ° ° −20 C∼25 C for three freeze-thaw cycles). Short-term sta- IS should have the same physical and chemical properties as bility was calculated by keeping the QC samples at room the analyte. When IS was selected in this experiment, a series temperature for 4 h and 4 C for 12 h. Long-term stability was of IS, mainly including baicalein, geniposide, paeonol, and Journal of Analytical Methods in Chemistry 5 naringenin, were analyzed and compared. It was found that -e precision and accuracy results of each analyte are when naringenin was used as IS, the reproducibility was shown in Table 5. -e intraday precision (RSD, %) values of good and naringenin did not interfere with the endogenous the ﬁve analytes were all below 8.80%, while the intraday substances. accuracy (RE, %) values were within ±6.67%. Interday Considering the large number of components in the precision (RSD, %) and accuracy (RE, %) values were less plasma, and each component is diﬀerent, we used gradient than 4.11% and within ±5.11%. Both the RSD and RE values elution for separation. When selecting the mobile phase, of the intraday and interday met the requirements we tested methanol-water and acetonitrile-water, re- (RSD< 15% and RE within ±15%), indicating that the spectively. -e results showed that when the mobile phase method was accurate and reliable. was acetonitrile-water, the resolution between the analytes Table 6 shows that the recoveries ranged from 85.42% to was higher and the peak shape was better. Additionally, we 90.85% at three concentrations (RSD from 2.97% to 8.93%, also tried to add formic acid and acetic acid to the mobile n � 6), and the recovery of IS (100 ng/mL) was 88.96% phase to adjust the PH or polarity. -e results showed that (RSD � 4.77%, n � 6), which indicated that the method was the addition of formic acid could improve the peak shape reproducible. -e ME of the ﬁve analytes were all between and resolution. Meanwhile, when the formic acid con- 94.52% and 103.91% (RSD from 3.24% to 7.89%, n � 6), and centration was 0.1%, the results were best. Finally, ace- the ME of IS was 97.19% (RSD � 3.24%, n � 6). -e results tonitrile −0.1% formic acid-water was selected as the showed that the ME of plasma did not aﬀect the de- mobile phase. -e use of gradient elution procedure ef- termination of analytes in this method. fectively improved the analysis sensitivity and accuracy -e stability results of this method showed that, at three and signiﬁcantly reduced analysis time (the total mea- concentration levels (n � 6), the RE of all analytes ranged surement time only needs 4 min). from −4.71% to 2.97% (RSD≤ 7.24%) for 4 h at room Common plasma treatment methods include direct temperature, −7.29% to 5.23% (RSD≤ 6.07%) for 24 h at 4 C, dilution method, protein precipitation method, ultraﬁl- −5.30% to 1.94% (RSD≤ 7.75%) for three freeze-thaw cycles, tration method, liquid-liquid extraction method, and and −7.00% to 3.64% (RSD≤ 7.38%), which indicated that solid-liquid extraction method . When treating there was no signiﬁcant degradation under the experimental plasma, we compared the use of ethyl acetate extraction, conditions. -erefore, this method established in the ex- methanol precipitation, and acetonitrile precipitation to periment was stable. process each analyte under medium concentrations, re- spectively. -e results, which were shown in supple- 3.3. Pharmacokinetics of Five Analytes. -e methodological mentary material, showed that when the ethyl acetate extraction method was used, the sample recovery was data meet the requirements of FDA, indicating that our method can be used to study the pharmacokinetics of the higher and the reproducibility was better. Although the methanol precipitation method and acetonitrile pre- ﬁve analytes. After giving a single dose of CHSGS (1 g/ kg), the mean plasma drug concentration-time curves of cipitation method were simple in operation and short in processing time, they had low recovery rates and poor ﬁve analytes in plasma are shown in Figure 3. Additionally, reproducibility. After the samples were treated by the Table 7 shows the main pharmacokinetic parameters of ethyl acetate extraction method, the LLOQ could be as low the ﬁve analytes, mainly including t , C , T , AUC , 1/2 max max 0−t as 0.5 ng/mL for naringin and hesperidin and 1 ng/mL for AUC , MRT , and MRT . 0−∞ (0−t) (0−∞) neohesperidin, paeoniﬂorin, and liquiritin, respectively. After giving a single dose of CHSGS (1 g/kg), the T max -erefore, the method has high sensitivity under the values of liquiritin were less than 3 h, and the t values were 1/2 less than 0.55 h, which indicated that liquiritin was absorbed experimental conditions. slowly in the blood in beagles but eliminated quickly. Due to the double peak of liquiritin between 2 h and 3 h, the T max 3.2. Method Validation. Figure 2 shows the results of values of liquiritin were signiﬁcantly larger than the T max speciﬁcity. -e chromatographic peaks of ﬁve analytes and values of other analytes. Without double peak, liquiritin IS in plasma were well separated, indicating that the en- might be absorbed faster in beagles. By analyzing the C , max dogenous substances in the beagle plasma did not aﬀect the AUC , and AUC values, the concentrations of the (0 − t) (0 −∞) determination of ﬁve analytes and IS. Meanwhile, the re- ﬁve analytes in plasma were low (all values were less than tention time of each analyte and IS was 1.24 min for nar- 297.87± 93.01 ng/mL), which might indicate that the ingin, 1.26 min for paeoniﬂorin, 1.26 min for liquiritin, pharmacological eﬀect of CHSGS was derived from the 1.28 min for hesperidin, 1.84 min for neohesperidin, and superimposed eﬀect after long-term administration. In this 2.73 min for naringenin (IS), respectively. experiment, since six beagles were given a single dose of -e typical regression equations, correlation coeﬃcient, CHSGS, the C values of the analytes were not very high. max and LLOQ of each analyte are shown in Table 5. X represents After a long-term administration, the steady-state plasma the plasma concentration and Y represents the ratio of the concentration of the analytes may be higher. -e pharma- peak area to the peak area of IS. -e results showed that the cokinetic parameters of hesperidin and neohesperidin were standard calibration curves of the ﬁve analytes had a satis- extremely close, mainly because hesperidin and neo- factory linear relationship and all R values were higher than hesperidin are a pair of structural isomers, and their met- 0.9923. abolic characteristics should also be similar. 6 Journal of Analytical Methods in Chemistry 1.28 1.43 90 150 190 1.14 150 60 100 0.49 3.85 3.93 0.59 2.03 2.32 50 1.54 3.31 1.59 3.00 3.16 3.55 0.85 2.28 3.17 15 4.00 2.86 1.80 2.72 0.74 3.79 0 0 0 1.0 2.0 3.0 4.0 1.0 2.0 3.0 4.0 1.0 2.0 3.0 4.0 Time (min) Time (min) Time (min) a-Naringin a-Paeoniflorin a-Liquiritin 250 50 240 48 230 46 220 44 210 42 200 40 190 38 180 36 170 34 160 32 0.65 150 30 140 28 130 26 120 24 110 22 1.53 2.86 0.24 1.33 90 18 1.20 80 16 3.81 3.22 60 12 50 10 2.47 2.67 1.05 40 0.50 8 30 6 20 4 10 2 0 0 1.0 2.0 3.0 4.0 1.0 2.0 3.0 4.0 Time (min) Time (min) a-Hesperidin a-Neohesperidin (a) Figure 2: Continued. Journal of Analytical Methods in Chemistry 7 1.26 1.26 1.24 1050 6670 550 3500 100 50 0 0 1.0 2.0 3.0 4.0 1.0 2.0 3.0 4.0 1.0 2.0 3.0 4.0 Time (min) Time (min) Time (min) b-Naringin b-Paeoniflorin b-Liquiritin 1.84 2.73 1.28 568 5200 3800 5000 500 4600 1400 200 150 1400 1.17 0 0 0 1.0 2.0 3.0 4.0 1.0 2.0 3.0 4.0 1.0 2.0 3.0 4.0 Time (min) Time (min) Time (min) b-Hesperidin b-Neohesperidin b-Naringenin (IS) 1.25 1.25 1.25 3600 1300 1400 500 100 200 0 0 1.0 2.0 3.0 4.0 1.0 2.0 3.0 4.0 1.0 2.0 3.0 4.0 Time (min) Time (min) Time (min) c-Naringin c-Paeoniflorin c-Liquiritin (b) Figure 2: Continued. 8 Journal of Analytical Methods in Chemistry 1.84 1.30 3200 700 1600 350 0 0 1.0 2.0 3.0 4.0 1.0 2.0 3.0 4.0 Time (min) Time (min) c-Hesperidin c-Neohesperidin (c) Figure 2: Representative MRM chromatograms: (a) blank plasma; (b) blank plasma spiked with ﬁve analytes and IS; (c) real plasma samples after giving a single dose of CHSGS. Table 5: -e typical regression equations, correlation coeﬃcient, and LLOQ in beagle plasma determined by HPLC-MS/MS. Analyte Linear range (ng/ml) Regression equation R LLOQ (ng/mL) −3 −3 Neohesperidin 0.5–100 y � 1.79 ×10 x + 4.76 ×10 0.9993 0.5 −2 −2 Hesperidin 0.5–100 y � 1.40 ×10 x + 2.32 ×10 0.9953 0.5 −3 −3 Naringin 1–200 y � 5.09 ×10 x + 8.59 ×10 0.9980 1 −3 −2 Paeoniﬂorin 1–200 y � 7.54 ×10 x + 2.85 ×10 0.9937 1 −2 −2 Liquiritin 1–200 y � 1.57 ×10 x + 4.73 ×10 0.9923 1 Table 6: -e precision, accuracy, recovery, and matrix eﬀects of ﬁve analytes in beagle plasma. Intraday Interday Added Recovery Analytes ME (%) Detected Detected (ng/mL) (%) RSD (%) RE (%) RSD (%) RE (%) (ng/mL) (ng/mL) 1 0.99± 0.05 5.32 −1.50 0.98± 0.02 2.35 −1.56 89.97± 8.02 99.90± 3.62 Neohesperidin 25 25.28± 1.52 6.02 1.13 25.40± 0.85 3.35 1.58 85.42± 3.74 102.79± 5.37 75 74.92± 3.29 4.39 −0.11 75.47± 2.47 3.27 0.63 87.21± 5.95 100.66± 7.42 1 0.97± 0.03 3.45 −3.00 0.99± 0.04 3.90 −1.28 89.09± 7.62 98.27± 4.14 Hesperidin 25 24.58± 1.37 5.59 −1.67 24.89± 0.35 1.42 −0.43 90.70± 6.24 99.41± 7.38 75 76.28± 3.40 4.45 1.70 74.50± 2.64 3.55 −0.67 89.42± 4.52 100.89± 5.56 2.5 2.47± 0.13 5.29 −1.13 2.49± 0.04 1.55 −0.51 88.57± 5.90 101.86± 7.47 Naringin 50 50.67± 3.85 7.61 1.33 51.15± 2.10 4.11 2.29 86.69± 6.47 103.53± 5.87 150 150.65± 8.51 5.65 0.44 149.39± 2.27 1.52 −0.41 90.85± 5.51 103.45± 3.98 2.5 2.39± 0.17 6.98 −4.53 2.37± 0.06 2.68 −5.11 89.37± 4.99 103.91± 6.70 Paeoniﬂorin 50 48.27± 3.38 7.01 −3.46 48.44± 0.93 1.92 −3.12 87.18± 7.05 94.52± 5.97 150 147.59± 9.13 6.18 −1.61 151.61± 2.90 1.91 1.08 89.27± 5.25 100.68± 4.65 2.5 2.33± 0.18 7.51 −6.67 2.49± 0.04 1.66 −0.29 90.12± 6.41 102.03± 8.05 Liquiritin 50 49.29± 4.34 8.80 −1.41 51.16± 1.63 3.19 −1.60 89.82± 6.38 100.62± 7.64 150 147.60± 5.78 3.91 −1.60 153.97± 6.00 3.90 2.64 87.41± 5.81 99.29± 6.46 Journal of Analytical Methods in Chemistry 9 40 50 0 0 0 2468 0 2468 Time (h) Time (h) Neohesperidin Hesperidin (a) (b) 80 150 0 0 0 2468 0 2468 Time (h) Time (h) Naringin Paeoniflorin (c) (d) 0 2468 Time (h) Liquiritin (e) Figure 3: Mean plasma concentration: time curves of ﬁve analytes after a single dose of CHSGS (n � 6). –1 –1 Concentration (ng·mL ) Concentration (ng·mL ) –1 Concentration (ng·mL ) –1 –1 Concentration (ng·mL ) Concentration (ng·mL ) 10 Journal of Analytical Methods in Chemistry Table 7: Main pharmacokinetic parameters of ﬁve analytes after giving a single dose of CHSGS (n � 6). Parameters Neohesperidin Hesperidin Naringin Paeoniﬂorin Liquiritin t (h) 0.69± 0.17 0.71± 0.18 0.97± 0.12 0.75± 0.34 0.55± 0.06 1/2 T (h) 1.58± 0.38 1.75± 0.27 0.92± 0.13 2.67± 0.52 3.00± 0.00 max MRT (h) 1.88± 0.12 1.95± 0.12 1.92± 0.08 2.94± 0.18 2.91± 0.07 (0−t) MRT (h) 1.93± 0.13 2.00± 0.15 2.07± 0.17 2.97± 0.17 2.95± 0.07 (0−∞) C (ng/mL) 31.22± 2.52 36.53± 5.44 67.92± 3.00 111.24± 17.77 45.00± 7.90 max AUC (ng·h/mL) 75.48± 7.00 75.01± 9.91 150.21± 15.18 282.07± 69.49 85.37± 18.81 (0−t) AUC (ng·h/mL) 76.29± 7.27 76.04± 10.35 154.02± 16.40 297.87± 93.01 86.70± 19.04 (0−∞)  N. Yang, X. Jiang, X. Qiu, Z. Hu, L. Wang, and M. Song, 4. Conclusion “Modiﬁed chaihu shugan powder for functional dyspepsia: meta-analysis for randomized controlled trial,” Evidence- In this experiment, we established a HPLC-MS/MS method Based Complementary and Alternative Medicine, vol. 2013, that can simultaneously detect naringin, neohesperidin, Article ID 791724, 10 pages, 2013. hesperidin, paeoniﬂorin, and liquiritin in beagle plasma.  C. R. Yang, “Clinical application of chaihu shugan san in Meanwhile, the method was ﬁrst successfully applied to treating digestive system disease,” Clinical Journal of Chinese a pharmacokinetic study of CHSGS. Our method showed Medicine, vol. 23, pp. 19-20, 2015. a good linearity for all analytes within acceptable speciﬁcity,  R. Q. Tan, Z. Zhang, J. Ju, and J. H. 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Journal of Analytical Methods in Chemistry – Hindawi Publishing Corporation
Published: Aug 19, 2020
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