nps Natural Product Sciences Natural Product Sciences 1226-3907 2288-9027 The Korean Society of Pharmacognosy 10.20307/nps.2017.23.2.84 nps-23-84 Original Article Quantitative Analysis of Eleven Bioactive Constituents of a Traditional Herbal Medicine, Yeonggyechulgam-tang using, Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry SeoChang-Seob ShinHyeun-Kyoo ∗ K-herb Research Center, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon 34054, Korea ∗Author for correspondence Dr. Hyeun-Kyoo Shin, K-herb Research Center, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon 34054, Korea. Tel: +82-42-868-9464; E-mail: hkshin@kiom.re.kr 01 2017 20 01 2017 23 2 84 91 25112016 19012017 23012017 Copyright © 2017 The Korean Society of Pharmacognosy 2017 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract

This study proposes a sensitive and selective liquid chromatography-electrospray ionization tandem mass spectrometry method of efficiently assessing the quality of a traditional herbal medicine called Yeonggyechulgam-tang (YGCGT). The following compounds 1 – 11, namely, liquiritin apioside (1), liquiritin (2), liquiritigene (3), coumarin (4), cinnamic acid (5), cinnamaldehyde (6), glycyrrhizin (7), atractylenolide III (8), atractylenolide II (9), atractylenolide I (10), and pachymic acid (11) were separated on a UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) at a column temperature of 45oC eluted with a gradient condition of 0.1% (v/v) formic acid in distilled water and acetonitrile. The correlation coefficient of the calibration curve of the eleven constituents was ≥ 0.9936. The limits of detection and quantification of the compounds 1 – 11 were 0.06 – 4.73 ng/mL and 0.17–14.20 ng/ mL, respectively. Using this analytical method, the compound 11 in lyophilized YGCGT decoction extract was not detected, while the compounds 1 – 10 were detected 0.13–166.43 mg/g.

 Quantitative analysis Yeonggyechulgam-tang LC-MS/MS References Zhang J. Qian C. C. Hao W. S.; Shang Han Lun; People's Medical Publishing House: Beijing, 2005;p.4142 Heo, J.; Dongeuibogam; Namsandang: Seoul,2004;p128 Xi F. Sang F. Zhou C. Ling Y. Neural Regener. Res. 2012 7 28672873 Yuanyuan W. Minghua J. Lina Z. Suhua L. Jiayu Z. Yongzhi S. Chunyu C. Jian Q. J. Tradit. Chin. Med. 2015 35 218221 Fu S. Zhang J. Gao X. Xia Y. Ferrelli R. Fauci A. Guerra R. Hu L. Heart Asia 2010 2 2427 Huang J. Wang L. Shi H. Hou X. J. Tradit. Chin. Med. 2013 33 343348 Kim T. H. Yang K. S. Park S. A. Kor. J. Pharmacogn. 1999 30 1217 Park, S. A; Kim T. H. Yang K. S. Kor. J. Pharmacogn. 2000 31 364372 Kim S. H.; The guideline on the visual and organoleptic examination of herbal medicine II; National Institute of Food and Drug Safety Evaluation: Seoul, 2007;p19 Moon C. J.; The guideline on the visual and organoleptic examination of herbal medicine I; Ministry of Food and Drug Safety: Seoul, 2006;p17, 52, 54. Li L. Liang S. Du, F; Li C. J. Am. Soc. Mass Spectrom. 2007 18 778782 Tan G. Zhu Z. Jing J. Lv L. Lou Z. Zhang G. Chai Y. Biomed. Chromagogr. 2011 25 913924 Concannon S. Ramachandran V. N. Smyth W. F. Rapid Commun. Mass Spectrom. 2000 14 11571166 Wu L. F. Wang K. F. Mao X. Liang W. Y. Chen W. J. Li S. Qi Q. Cui Y. P. Zhang L. Z. Molecules 2016 21, 227,doi: 10.3390/molecules21020227. Gardana C. Scaglianti M. Pietta P. Simonetti P. J. Pharm. Biomed. Anal. 2007 45 390399 Flamini R. Vedova, A. D,; Cancian D. Panighel A. De Rosso M. J. Mass Spectrom. 2007 42 641646 Chen L. Qi J. Chang Y. Zhu D. Yu B. J. Pharm. Biomed. Anal. 2009 50 127137 Figures and Tables

Chemical structures of the compounds 1 – 11 in Yeonggyechulgam-tang.

Mass spectra of the precursor ion (Q1, A) and product ion (Q3, B) for LC-MS/MS MRM mode of the compounds 1 – 11. Liquiritin apioside (1), liquiritin (2), liquiritigene (3), coumarin (4), cinnamic acid (5), cinnamaldehyde (6), glycyrrhizin (7), atractylenolide III (8), atractylenolide II (9), atractylenolide I (10), and pachymic acid (11).

Total ion chromatograms of the reference standard (A) and lyophilized Yeonggyechulgam-tang extract (B) by LC-MS/MS MRM mode. Liquiritin apioside (1), liquiritin (2), liquiritigene (3), coumarin (4), cinnamic acid (5), cinnamaldehyde (6), glycyrrhizin (7), atractylenolide III (8), atractylenolide II (9), atractylenolide I (10), and pachymic acid (11, not detected).

Composition of Yeonggyechulgam-tang

Herbal medicine Scientific name Origin Amount (g)
  Poria Sclerotium Poria cocos Wolf Pyeongchang, Korea 7.500
  Cinnamomi Ramulus Cinnamomum cassia Presl Vietnam 5.625
  Atractylodis Rhizoma Alba Atractylodes macrocephala Koidzumi China 5.625
  Glycyrrhizae Radix et Rhizoma Glycyrrhiza uralensis Fischer China 3.750
Total amount     22.500

Linearities, regression equation, correlation coefficients, LOD, and LOQ for the compounds 1 – 11

Analyte Linear range (ng/mL) Regression equationa Correlation coefficient LODb (ng/mL) LOQc (ng/mL)
1 10–500 y = 8.21x – 27.85 0.9997 0.55 1.65
2 10–500 y = 9.01x – 19.10 1.0000 1.14 3.41
3 10–500 y = 29.58x – 29.21 1.0000 0.11 0.34
4 10–500 y = 38.59x + 51.41 0.9999 0.91 2.74
5 10–500 y = 21.35x – 67.24 0.9999 0.87 2.60
6 10–500 y = 1.15x – 18.20 0.9936 4.73 14.20
7 10–500 y = 3.08x – 60.81 0.9961 1.42 4.27
8 10–500 y = 52.12x – 97.00 1.0000 0.15 0.44
9 10–500 y = 56.54x + 33.78 1.0000 0.06 0.17
10 10–500 y = 53.42x + 63.53 1.0000 0.12 0.37
11 10–500 y = 1.46x – 10.67 1.0000 1.05 3.16

y: peak area of compounds; x: concentration (ng/mL) of compounds.

LOD = 3 × signal-to-noise ratio.

LOQ = 10 × signal-to-noise ratio.

Mass detection condition of the compounds 1 – 11

Analyte Molecular weight (Da) Ionization mode Retention time (min) Precursor ion (m/z) Product ion (m/z) Cone voltage (V) Collision energy (eV)
1 550.5 [M−H]− 1.53 549.3 255.0 45 30
2 418.4 [M−H]− 1.65 417.4 255.2 30 15
3 256.3 [M+H]+ 2.99 257.2 137.0 35 25
4 146.1 [M+H]+ 3.06 147.1 91.0 30 20
5 148.1 [M+H]+ 3.74 148.9 130.9 20 20
6 132.1 [M+H]+ 4.42 133.1 115.0 25 15
7 822.9 [M−H]− 5.25 821.9 351.2 45 40
8 248.3 [M+H]+ 6.73 249.3 231.2 25 10
9 248.3 [M+H]+ 8.26 233.2 187.1 35 15
10 230.3 [M+H]+ 9.34 231.2 185.1 35 20
11 528.8 [M−H]− 11.97 527.6 465.4 45 35

Amounts of the compounds 1 – 11 in lyophilized Yeonggyechulgam-tang extract (n = 3)

Compound Amount (mg/g) Source
Mean SD RSD (%)
1 81.58 1.28 1.56 G. uralensis
2 79.88 0.31 0.39 G. uralensis
3 8.96 0.27 3.02 G. uralensis
4 54.52 1.45 2.65 C. cassia
5 10.18 0.25 2.50 C. cassia
6 121.09 1.11 0.92 C. cassia
7 166.43 11.17 6.71 G. uralensis
8 2.47 0.04 1.58 A. macrocephala
9 0.88 0.03 2.94 A. macrocephala
10 0.13 0.01 4.44 A. macrocephala
11 N.D.a – – P. cocos

N.D. means not detected.