|Year : 2013 | Volume
| Issue : 35 | Page : 271-282
Qualitative and quantitative analysis of nine major compounds in the Bozhougyiqi-Tang using a high-performance liquid chromatography coupled with a diode array detector and electrospray ionization mass spectrometer
Jin Bae Weon1, Jin Yeul Ma2, Hye Jin Yang1, Bohyoung Lee1, Bo-Ra Yun1, Choong Je Ma3
1 Department of Medical Biomaterials Engineering, College of Biomedical Science, Kangwon National University, Chuncheon 200-701, Korea
2 TKM Converging Research Division, Korea Institute of Oriental Medicine, 483 Exporo, Yuseong-gu, Daejeon 305-811, Korea
3 Department of Medical Biomaterials Engineering, College of Biomedical Science; Research Institute of Biotechnology, Kangwon National University, Chuncheon 200-701, Korea
|Date of Submission||29-Jun-2012|
|Date of Acceptance||28-Jul-2013|
|Date of Web Publication||11-Jun-2013|
Choong Je Ma
Department of Biomaterials Engineering, Division of Bioscience and Biotechnology, Kangwon National University, Hyoja-2 Dong, Chuncheon 200-701
Source of Support: Supported by a grant [K11050] from the Korea Institute
of Oriental Medicine., Conflict of Interest: None
| Abstract|| |
Background: Bozhougyiqi-Tang (BZYQT) is of traditional herbal medicine used for enhancement of digestive capacity. Objective: An accurate and reliable simultaneous determination using a HPLC-DAD and ESI-MS was developed and validated for the qualitative and quantitative analysis of 9 major compounds, ferulic acid (1), naringin (2), hesperidin (3), decursinol (4), glycyrrhizin (5), saikosaponin A (6), 6-gingerol (7), ginsenoside Rg3 (8), decursin (9), in traditional herbal medicine 'Bozhougyiqi-Tang.' Materials and Methods: The chromatographic separation of 9 compounds was performed on a SHISEIDO C 18 column (250 mm × 4.6 mm i.d., S-5 μm) using gradient elution with 0.1% trifluoroacetic acid and acetonitrile at a flow rate of 1.0 ml/min. The 9 compounds were identified based on peak retention time and UV spectrum and MS data of these compounds. Results: This developed method showed good linearity (R 2 > 0.999). The LOD and LOQ of the major compounds were less than 0.09 and 0.28 μg/ml, respectively. The intra - day and inter - day RSD values were within 2.06% and 1.64%, respectively. The mean recoveries were from 92.10% to 108.56% with less than 1.88%. The results indicated that established method had good precision and accuracy. Conclusion: The new method was successfully applied to the simultaneous analysis of 9 compounds in Bozhougyiqi-Tangs samples.
Keywords: Bozhougyiqi-Tang, simultaneous determination, high-performance liquid chromatography-diode array detector, liquid chromatography-mass spectrometry, major compounds
|How to cite this article:|
Weon JB, Ma JY, Yang HJ, Lee B, Yun BR, Ma CJ. Qualitative and quantitative analysis of nine major compounds in the Bozhougyiqi-Tang using a high-performance liquid chromatography coupled with a diode array detector and electrospray ionization mass spectrometer. Phcog Mag 2013;9:271-82
|How to cite this URL:|
Weon JB, Ma JY, Yang HJ, Lee B, Yun BR, Ma CJ. Qualitative and quantitative analysis of nine major compounds in the Bozhougyiqi-Tang using a high-performance liquid chromatography coupled with a diode array detector and electrospray ionization mass spectrometer. Phcog Mag [serial online] 2013 [cited 2022 Dec 1];9:271-82. Available from: http://www.phcog.com/text.asp?2013/9/35/271/113291
| Introduction|| |
Bozhougyiqi-Tang (BZYQT) is one of traditional herbal medicines consisting of Panax ginseng, Astragalus membranaceus, Angelica gigas, Bupleurum falcatum, Citrus unshiu, Glycyrrhiza uralensis, Atractylodes japonica, Zingiber officinale, Zizyphus jujube, Cimicifuga heracleifolia. This herbal medicine has been widely used for enhancement of digestive capacity and clinical treatment of pulmonary tuberculosis. Recently, BZYQT has been reported various activities, including anti-allergy, anti-inflammatory, anti-cancer, skin protection and so on. , Traditional herbal medicines have a long history and have been widely used in many countries. Research about the multiple therapeutic effects of the traditional herbal medicines has been increasing. The therapeutic effects of herbal medicines are attributed to various compounds in it. ,, However, because of change in content of many compounds in constituent herbs by many factors, such as cultural environment, processing and storage, it is difficult to evaluate quality and therapeutic efficacy of traditional herbal medicines. Thus, stable and reliable analytical method for simultaneous determination of various compounds will be helpful for evaluation of the quality of the traditional herbal medicine.
Generally, high-performance liquid chromatography (HPLC), one of the chromatography and relative techniques, is used to analyze the traditional herbal medicine. HPLC is able to separate complex compounds mixture.  HPLC is quantitative analyzed and qualitative determined by combined various equipment such as diode array detector (DAD), evaporative light scattering detection (ELSD), and MS spectrum. 
In this study, rapid, sensitive, and reliable simultaneous determination for the qualitative and quantitative analysis of 9 major compounds (ferulic acid in Cimicifuga heracleifolia, naringin and hesperidin in Citrus unshiu, decursinol and decursin in Angelica gigas, glycyrrhizin in Glycyrrhiza uralensis, saikosaponin A in Bupleurum falcatum, 6-gingerol in Zingiber officinale, ginsenoside Rg3 in Panax ginseng) of each herb in BZYQT was developed by HPLC coupled with DAD and ESI-MS [Figure 1]. In this technology, the major compounds were quantitatively determined by HPLC-DAD, and LC-MS is used to direct identification of major compounds. This established method was applied to BZYQT sample for quantitative analysis of their 9 compounds. In addition, BZYQT sample were fermented to obtain the improved bioactivity by Lactobacillus strains, and to determine the quantitative change of contents of 9 compounds in fermented BZYQT samples, this HPLC method was applied.
| Experimental|| |
Reference compounds, decursin, saikosaponin A, hesperidin, naringin, glycyrrhizin, and 6-gingerol were obtained from the Korea Food and Drug Administration. Ferulic acid was purchased from Sigma (USA). Ginsenoside Rg3 was purchased from Chromadex (USA), and decursinol was purchased from Elcomscience (Korea). The purity of each compound was higher than 98% by HPLC. HPLC-grade waster, acetonitrile, and methanol were purchased from J.T. Baker (USA). Analytical grade trifluoroacetic acid used buffer solution was purchased from DAE JUNG (Korea). BZYQT preparation (B-0) and fermented BZYQT samples (B-1~7) were supplied by the Korea Institute of Oriental Medicine.
Standard solutions and sample preparation
Standard stock solutions of accurately weighed 9 reference compounds were prepared at concentration of 250.00 μg/ml for ferulic acid (1), 143.75 μg/ml for naringin (2), 175.00 μg/ml for hesperidin (3), 210.00 μg/ml for decursinol (4), 400.00 μg/ml for glycyrrhizin (5), 330.00 μg/ml for saikosaponin A (6), 265.00 μg/ml for 6-gingerol (7), 250.00 μg/ml for ginsenoside Rg3 (8), 280.00 μg/ml for decursin (9) in methanol. Standard solutions of 9 reference compounds for HPLC-MS analysis were prepared in methanol at concentration of 10 μg/ml. These standard solutions were stored at 4°C until use. The standard working solution mixtures were obtained by diluting each stock solution to appropriate concentration.
The powder of BZYQT preparation (20.5 mg) was weighed precisely and dissolved in 10 ml of 60% methanol. Obtained fermented BZYQT preparations were prepared by same method. These sample solutions were filtered through a 0.45 μm membrane filter prior to HPLC analysis.
HPLC - DAD conditions
The HPLC analysis was performed on a Dionex Ultimate 3000 HPLC system (Dionex, Germany) consisting of a pump (LPG 3X00), an auto sampler (ACC-3000), a column oven (TCC-3000SD), and diode array UV/VIS detector (DAD-3000(RS)). Dionex ChromelonTM Chromatography Data System was used for instrument control and the chromatographic data acquisition. For chromatographic separation, SHISHEDO C 18 column (250 × 4.6 mm i.d., 5 μm) was used. The column temperature was set at 35°C. A linear gradient system consisted of mobile phase A (0.1% (v/v) trifluoroacetic acid) and mobile phase B (acetonitrile). The gradient program was conducted as follows: 15% (B) in 0-5 min, 15-35% (B) in 5-20 min, 35-50% (B) in 20-25 min, 50% (B) in 25-40 min, and 50-70% (B) in 40-50 min. The flow rate of mobile phase was 1.0 ml/min, and sample volume injected was 20 μL. The DAD detector recorded UV spectra in the range from 190 to 400 nm for peak characterization.
LC-MS analysis was performed by using the TSQ Quantum Ultra Triple Stage Quadrupole Mass Spectrometer (Thermo) equipped with electrospray ionization (ESI) ion source in positive ion mode. The chromatographic separation was achieved on an Atlantis dC18 column (150 × 2.0 mm i.d., 3 μm) at a column temperature of at 25°C with the same elution program of HPLC-DAD analysis. The injection volume was 20 μl. The flow rate was 200 μl/min. The MS operating condition were optimized as follows: The ion spray voltage set at 4,000 V, the vaporizer temperature at 100°C, capillary temperature at 320°C, sheath gas pressure at 50 psi, aux gas pressure at 30 psi. Full scan mass spectra were recorded from m/z 100 to 1000 in positive ion modes.
| Results and Discussion|| |
Optimization of the chromatographic conditions
Four different columns, Dionex C 18 column (150 mm × 4.6 mm i.d., 5 μm), LUNA C 18 column (250 × 4.6 mm i.d., 5 μm,), SHISHEDO C 18 column (250 × 4.6 mm i.d., 5 μm), and XTerra TM RP18 (250 mm × 4.6 mm i.d., 5 μm) were compared to select the best column. The SHISHEDO C 18 column (250 × 4.6 mm i.d., 5 μm) exhibited good separation. The different mobile phases, such as water - acetonitrile and water - methanol were tested with various gradient programs to obtain the optical separation condition. Water-acetonitrile had better solution and peak shape than water-methanol. Low concentration of trifluoroacetic acid was added to improve the peak shape and reduce the peak tailing.  According to the higher UV absorption based on literature of 9 major compounds, we set a UV wavelength on HPLC analysis to obtain more detectable peaks. UV wavelengths of each compound were selected such as 205 nm for naringin (2), hesperidin (3), decursinol (4), saikosaponin A (6), 6-gingerol (7), ginsenoside Rg3 (8), and decursin (9), 250 nm for glycyrrhizin (5) and 330 nm for ferulic acid (1). The typical chromatograms for the standard mixtures separated using the optimized analysis condition are shown in [Figure 2]a and obtained a good separation. [Figure 2]b showed BZYQT sample HPLC chromatogram with UV spectrum of each standard peak.
|Figure 2: HPLC-DAD chromatograms of 9 standard compounds in BZYQT preparation and UV spectrum. (a) Mixed standards and (b) BZYQT preparation sample at 205, 250 and 330 nm. (1) ferulic acid, (2) naringin, (3) hesperidin, (4) decursinol, (5) glycyrrhizin, (6) saikosaponin A, (7) 6-gingerol, (8) ginsenoside Rg3, and (9) decursin|
Click here to view
Identification of standard compounds
Nine reference substances and BZYQT sample were qualitative analysis by using the LC-ESI-MS. The TIC chromatograms of 9 standards and BZYQT sample were shown in [Figure 3]. In MS spectra, the obtained molecular mass of the compounds applied for the structural identification of compounds. In positive ESI mode, the 9 compounds exhibited quasi-molecular ion [M+H] + . The protonated molecular ions [M+H] + at m/z 195.13, 581.13, 611.14, 247.14, 823.30, 781.37, 277.22, and 329.17 for ferulic acid (1), naringin (2), hesperidin (3), decursinol (4), glycyrrhizin (5), saikosaponin A (6), 6-gingerol (7) and decursin (8), respectively, were observed, and ginsenoside Rg3 showed characteristic ion [M+H-2glu] + at m/z 459.46 [Table 1]. And these results were shown in [Figure 4].
|Table 1: MS data for identification of the 9 components by LC-MS method |
Click here to view
|Figure 3: Total ion chromatogram of (I) mixed 9 standards and (II) BZYQT preparation sample. Representative SIM and PDA chromatograms of 9 standards in BZYQT preparation; (II)-a ferulic acid, (II)-b naringin, (II)-c hesperidin, (II)-d decursinol, (II)-e glycyrrhizin, (II)-f saikosaponin A, (II)-g 6-gingerol, (II)-h ginsenoside Rg3, and (II)-i decursin|
Click here to view
|Figure 4: The MS spectra of 9 components in BZYQT preparation. For peak identification: (a) ferulic acid, (b) naringin, (c) hesperidin, (d) decursinol, (e) glycyrrhizin, (f) saikosaponin A, (g) 6-gingerol, (h) ginsenoside Rg3, and (i) decursin|
Click here to view
| Method Validation|| |
Calibration curves, limits of detection, and quantification
Calibration curves were constructed by plotting the peak area against the corresponding 6 concentrations from the following concentration ranges; 0.347 - 27.778 μg/ml for ferulic acid (1), 0.200 - 15.972 μg/ml for naringin (2), 0.243 - 19.444 μg/ml for hesperidin (3), 0.292 - 23.333 μg/ml for decursinol (4), 0.556 - 44.444 μg/ml for glycyrrhizin (5), 0.458 - 36.667 μg/ml for saikosaponin A (6), 0.368 - 29.444 μg/ml for 6-gingerol (7), 0.347 - 27.777 μg/ml for ginsenoside Rg3 (8), and 0.389 - 31.111 μg/ml for decursin (9).  Correlation coefficient values indicated good linearity (R 2 > 0.999). The limits of detection (LOD) and limits of quantification (LOQ) were determined at the signal-to-noise ratio equal to 3 and 10, respectively.  The range of LOD and LOQ for all compounds was from 0.0074 to 0.0931 ng/ml and 0.0224 to 0.2821 ng/ml, respectively. The detailed contents were exhibited in [Table 2].
|Table 2: Linear regression data, limit of detection (LOD), and limit of quantification (LOQ) of 9 compounds |
Click here to view
Precision and accuracy
The precision test of developed method was conducted by the intra-day and inter-day variation on 3 concentrations of 9 compounds.  Intra-day variation was determined by analyzing 5 replicates within 1 day, and inter-day variation was assayed in 5 replicates for 3 sequential days (1, 3, 5 days). Variations were expressed by relative standard deviation (RSD). The intra-day precision for 9 compounds ranged from 0.19% to 1.91%, and the inter-day precision ranged from 0.05% to 1.64% [Table 3]. The recovery test was measured to evaluate the accuracy by the method of standard addition.  Three different concentrations of 9 standard solutions were added to BZYQT sample and analyzed in triplicate. The accuracy varied between 92.57 and 108.56% with a maximum RSD of 1.98% [Table 4]. These results indicated that the established HPLC method was very sensitive, reliable, and accurate for simultaneous determination of the 9 compounds in BZYQT sample.
Analysis of samples
The developed method was subsequently applied to quantitative and qualitative analysis of 9 compounds, ferulic acid (1), naringin (2), hesperidin (3), decursinol (4), glycyrrhizin (5), saikosaponin A (6), 6-gingerol (7), ginsenoside Rg3 (8), and decursin (9) in obtained BZYQT samples (B-0) and 7 fermented BZYQT samples (B-1~7) from Korea Institute of Oriental Medicine. The sample chromatogram was shown [Figure 2]B, and the quantitative analytical results are summarized in [Table 5]. The contents of 9 compounds varied in different BZYQT samples (B-0~7). Glycyrrhizin (5) was abundant in the B-3, B-4, and B-5. Hesperidin (3) was not detected in the B-2, B-3, B-4, and B-5. Decursin (9) was not detected in the B-2. Decursinol was existed in B-0 and B-1. Ginsenoside Rg3 (8) was existed only in B-0. Ferulic acid (1), naringin (2), saikosaponin A (6), and 6-gingerol (7) were not detected in all samples (B-0~7). "Not detected" was defined as amount of each compound was lower than LOD. The results suggested that the differences of the content between BZYQT sample (B-0) and fermented BZYQT samples (B-1~7) were observed by bio-conversion such as fermentation of BZYQT preparation. In addition, absence of ferulic acid (1), naringin (2), saikosaponin A (6), and 6-gingerol (7) may attribute to the various factors, including preparation process, condition of medicinal herbs that compose BZYQT.
|Table 5: Contents (µ g/mg) of the 9 compounds in the 8 BZYQT samples (B-0~7) |
Click here to view
| Conclusion|| |
In this study, we established a reliable, accurate, and sensitive analysis method using HPLC-DAD/LC-MS for simultaneous determination of BZYQT sample. Nine compounds, ferulic acid (1), naringin (2), hesperidin (3), decursinol (4), glycyrrhizin (5), saikosaponin A (6), 6-gingerol (7), ginsenoside Rg3 (8), and decursin (9) in BZYQT successfully identified by UV wavelength and MS data. The method showed good linearity, precision, and accuracy by validation test. The developed method used for quantitative analysis of BZYQT sample and fermented BZYQT samples and proved that the differences in the content for compounds among different samples were obvious. Previous determination of glycyrrhizic acid in BZYQT by HPLC-DAD was reported.  But, our analysis method analyzed more various compounds than previous method and used LC-MS detector. This study could provide effect quality control method of BZYQT and information on influence of the content differences of compounds on therapeutic effect of BZYQT sample. Further research on analysis of more novel compounds is required.
| Acknowledgement|| |
This research was supported by a grant [K11050] from the Korea Institute of Oriental Medicine.
| References|| |
|1.||Kang DS, Kam CW, Park DI. The experimental study on the anti-allergic effect of Boiungikgi-tang. Korean J Life Sci 2003;13:73-82. |
|2.||Seo M, Lee K, Park J, Hong S. The Current Trend of Research about Bojungikki-tang. Korean J Orient Med 2010;16:83-90. |
|3.||Firenzuoli F, Gori L. Herbal Medicine Today: Clinical and Research Issues. Evid Based Complement Alternat Med 2007;4:37-40. |
|4.||Lu AP, Jia HW, Xiao C, Lu Q-P. Theory of traditional Chinese medicine and therapeutic method of diseases. World J Gastroenterol 2004;10:1854-6. |
|5.||Jiang WY. Therapeutic wisdom in traditional Chinese medicine: A perspective from modern science. Trends Pharmacol Sci 2005;26:558-63. |
|6.||Steinmann D, Ganzera M. Recent advances on HPLC/MS in medicinal plant analysis. J Pharm Biomed Anal 2010;55:744-57. |
|7.||Drasar R, Moravcova J. Recent advances in analysis of Chinese medical plants and traditional medicines. J Chromatogr B 2004;812:3-21. |
|8.||Heyrman AN, Henry RA. Importance of controlling mobile phase pH in reversed phase HPLC. Keystone Tech Bull 1999;99:1-7. |
|9.||Jiang Z, Yu QH, Cheng Y, Guo XJ. Simultaneous quantification of 8 major constituents in Herba Siegesbeckiae by liquid chromatography coupled with electrospray ionization time-of-flight tandem mass spectrometry. J Pharm Biomed Anal 2011;55:452-7. |
|10.||Peng JB, Jia HM, Liu YT, Zhang HW, Dong S, Zou ZM. Qualitative and quantitative characterization of chemical constituents in Xin-Ke-Shu preparations by liquid chromatography coupled with a LTQ Orbitrap mass spectrometer. J Pharm Biomed Anal 2011;55:984-95. |
|11.||Liu N, Yu NJ, Li J, Yang Y, Guo JF, Zhao YM. Simultaneous qualitative analysis of coumarins in the rhizome of Angelica polymorpha Maxim. by LC-DAD-ESI-MS. Chromatographia 2011;73:1121-9. |
|12.||Kim MK, Yang DH, Jung M, Jung EH, Eom HY, Suh JH, et al. Simultaneous determination of chromones and coumarins in Radix Saposhnikoviae by high performance liquid chromatography with diode array and tandem mass detectors. J Chromatogr A 2011;1218:6319-30. |
|13.||Baek JH, Kim SM, Ahn JW, Cho CH, Oh MH, Cho JH, et al. Validation and Determination of Glycyrrhizic Acid as a Marker Substance in Bu-Zhong-Yi-Qi-Tang by HPLC/DAD. Yakhak Hoeji 2008;52:7-11. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]