Phytochemical Analysis

Journal of Chromatography A, 1216 (2009) 2045–2062

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Journal of Chromatography A

journa l homepage: www.e lsev ier .com/ locate /chroma

Review

Phytochemical analysis of traditional Chinese medicine using liquidchromatography coupled with mass spectrometry

Min Yanga, Jianghao Sunb, Zhiqiang Lua, Guangtong Chena, Shuhong Guana,Xuan Liua, Baohong Jianga, Min Yeb, De-An Guoa,b,∗

a Shanghai Research Center for TCM Modernization, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, Chinab The State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China

a r t i c l e i n f o

Article history:Available online 3 September 2008

Keywords:Phytochemical analysisTraditional Chinese medicineHPLC–MSTandem mass spectrometryQuality control

a b s t r a c t

Traditional Chinese medicine (TCM) is commonly considered to operate due to the synergistic effects ofall the major and minor components in the medicines. Hence sensitive and comprehensive analyticaltechniques are needed to acquire a better understanding of the pharmacological basis of the herb and toenhance the product quality control. The present review mainly focuses on the phytochemical analysisof TCMs using high-performance liquid chromatography coupled with mass spectrometry (HPLC–MS).Atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) are the two commonlyused ion sources. Triple quadrupole, ion trap (IT), Fourier transform ion cyclotron resonance (FTICR) andtime-of-flight (TOF) mass spectrometers are used as on-line analyzer. The relationship between struc-tural features and fragmentation patterns should be investigated as thoroughly as possible and hence beapplied in the on-line analysis to deduce the structures of detected peaks. Characteristic fragmentationbehaviors of the reference standards, as well as information regarding polarity obtained from retentiontime data, on-line UV spectra, data from the literature and bio-sources of the compounds allowed the iden-tification of the phytochemical constituents in the crude extracts. Although a mass spectrometer is nota universal detector, high-performance liquid chromatography coupled with multistage mass spectrom-etry (HPLC–MSn) technique was still proved to be a rapid and sensitive method to analyze the majority
of the many constituents in herbal medicines, particularly for the detection of those present in minor ortrace amounts. The methods established using HPLC–MS techniques facilitate the convenient and rapidquality control of traditional medicines and their pharmaceutical preparations. However, the quantitative
C

0d

analysis is not the topic of this review.© 2008 Elsevier B.V. All rights reserved.

ontents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20462. Identification of known and unknown constituents in the extracts of traditional Chinese medicines and their derived products . . . . . . . . . . . . . 2047

2.1. Phenolic compounds (including flavonoids) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20472.1.1. Danshen: root of Salvia miltiorrhiza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20472.1.2. Dengzhanxixin: whole plant of Erigeron breviscapus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20472.1.3. Rhubarb: root and rhizome of Rheum species. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20482.1.4. Lianqiao: fruits of Forsythia suspense . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20482.1.5. Huangqin: roots of Scutellaria baicalensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2048
2.1.6. Maidong: tuber of Ophiopogon japonicus . . . . . . . . . . . . . . . . . .2.1.7. Ginger: rhizome of Zingiber officinale . . . . . . . . . . . . . . . . . . . . .2.1.8. Propolis (Fengjiao) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.1.9. Kushen: roots of Sophora flavescens . . . . . . . . . . . . . . . . . . . . . . .
∗ Corresponding author at: Shanghai Research Center for TCM Modernization, Shangha199, Zhangjiang, Shanghai 201203, China. Tel.: +86 21 50271516; fax: +86 21 50272789.

E-mail address: [email protected] (D.-A. Guo).

021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2008.08.097

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2048. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2049. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2049. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2050

i Institute of Materia Medica, Chinese Academy of Sciences, Guo Shoujing Road

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2046 M. Yang et al. / J. Chromatogr. A 1216 (2009) 2045–2062

2.1.10. Dandelion (Pugongying): whole plant of Taraxacum officinale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20502.1.11. Jiangxiang: heartwood of Dalbergia odorifera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20502.1.12. Artichoke: Cynara scolymus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20502.1.13. Honghuayanhuangqi: roots of Hedysarum multijugum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20502.1.14. Tusizi: seeds of Cuscuta chinensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20502.1.15. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2051

2.2. Saponins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20512.2.1. Ginseng: root and rhizome of Panax spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20512.2.2. Sanqi: root and rhizome of Panax notoginseng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20512.2.3. Ciwujia: roots of Acanthopanax senticosus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20522.2.4. Huashanfan: roots and leaves of Symplocos chinensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20522.2.5. Huangqi: roots of Astragalus spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20522.2.6. Cynanchum: Radix Cynanchi atrati (Baiwei) and Cynanchum chekiangense (Manjiancao) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20522.2.7. Chuanshanlong: rhizomes of Dioscorea nipponica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2052

2.3. Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20522.3.1. Aconite: Aconitum sinomontanum, Aconitum kusnezoffii, Aconitum carmichaeli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20532.3.2. Liangmianzhen: roots of Zanthoxylum nitidium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20532.3.3. Baibu: root tuber of Stemona tuberosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20532.3.4. Lilu: root and rhizome of Veratrum nigrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20532.3.5. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2054

2.4. Monoterpene glycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20542.4.1. Shaoyao: dried roots of Paeonia lactiflora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20542.4.2. Zhizi: fruits of Gardenia jasminoides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20542.4.3. Jishiteng: whole plant of Paederia scandens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20542.4.4. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2054

2.5. Diterpenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20542.5.1. Danshen: roots of S. miltiorrhiza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2054

2.6. Triterpenoids (aglycone) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20542.6.1. Lingzhi: fruit body of Ganoderma lucidum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2055

2.7. Steroids (aglycone) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20552.7.1. Chansu: skin secretions of giant toads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2056

2.8. Others and miscellaneous natural products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20562.8.1. Mudanpi (Cortex Moutan): root bark of Paeonia suffruticosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20562.8.2. Jinguolan: roots of Tinospora sagittata and Tinospora capillipes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20562.8.3. Shengma: rhizome and root of Cimicifuga species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20582.8.4. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2059

2.9. Complex traditional Chinese medicine prescription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20602.9.1. Shuanghuanglian oral liquid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20602.9.2. Shuangdan granule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20602.9.3. Qingkailing injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20602.9.4. Compound Danshen Dripping Pill . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2060

3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2061Acknowledgement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2061

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

Traditional Chinese medicine (TCM) played a significant rolen the health of the Chinese people for thousands of years. Overhe past decades, TCMs have always been the most importantesources for screening lead compounds. Fast analysis of natu-al products in bioactive crude extracts attracted the attention ofost investigators. Natural products gained prominence through

ntibiotics earlier in the last century; today they have been devel-ped for a variety of medicinal uses such as immunosuppressivegents, hypocholesterolemic agents, enzyme inhibitors, antimi-rane agents, herbicides, antiparasitic agents and ruminant growthromoters, and bioinsecticides [1]. Natural products are also theost important resources of anticancer agents, where over 60% of

he approved and pre-new drug application candidates are eitheratural products or synthetic molecules based upon the natural
roduct molecular skeletons.
Herbal medicines and their derived products are widely useds therapeutic products in many countries. Their worldwide useas increased in the last decade [2]. Most herbal medicines andheir derivative products were often prepared from the crude plant

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xtracts, which comprise a complex mixture of different phyto-hemical constituents (plant secondary metabolites). There maye hundreds of active components in these herbs. The chemicaleatures of these constituents differ considerably among the dif-erent species. Even the same herbal extracts may vary dependingpon the harvest season, plant origin, drying process and other fac-ors. Therefore, the quality control of the herbal medicines and theirerived products is difficult. Recently, the chromatographic finger-rinting of the components, especially by high-performance liquidhromatography–diode array detection (HPLC–DAD), is a power-ul and widely used technique to analyze plant extracts becausehis technique could systematically profile the composition of sam-les and it focuses on the identification and consistency assessmentf the components [3]. However, a valuable and convincing chro-atographic fingerprint should have most of its peaks assigned,

specially those corresponding to the active constituents and toxic
ngredients. Unfortunately, such routine techniques as HPLC–DADould only provide very limited structural information like UV spec-rum; standard compounds, which are commercially unavailable in
ost cases, are usually necessary for the characterization of indi-idual constituents. As a result, the isolation and purification from

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rude plant extracts of adequate amounts (at least 5–10 mg) of pureompounds (>90% purity) for nuclear magnetic resonance (NMR)dentification was needed before they could serve as referenceompounds. The whole process is tedious, laborious and expen-ive. Moreover, some constituents are only present in raw plantaterials in very low amount or are quite unstable under normal

onditions, and their enrichment and purification are laborious.The inherent variety of natural product extracts has shown

ignificant challenges for separation and detection techniques tonable rapid characterization of the biologically active componentn the mixture. Except for paper chromatography and thin-layerhromatography, gas chromatography (GC) is a powerful separationechnique that has been utilized since the 1960s for the analysis ofolatile natural products or derivatives. However, the role that hasecently been played by HPLC for the work of this nature has beennvaluable, considering that approximately 80% of all known natu-al compounds are nonvolatile or thermally unstable and thereforencompatible with GC. When coupled with a variety of detections,PLC began to serve as a powerful tool for the rapid characterizationf natural product extracts. In addition, some developing chromato-raphic techniques, such as capillary electrophoresis (CE), highpeed countercurrent chromatography (HSCCC), gradually foundheir application in the separation of phytochemical constituentsf herbal medicines.

Mass spectrometry (MS) is the most selective technique for theapid qualitative determination of known compounds as well ashe identification of unknown compounds from the extracts of nat-ral products. The analysis of natural products has been especiallyffective following the successful interface with a mass spectrome-er detection system in the chromatographic method. Atmosphericressure ionization (API), which consists of atmospheric pressurehemical ionization (APCI) and electrospray ionization (ESI), is theost successful interface used in HPLC–MS configuration. There-

ore, HPLC–MS combines the efficient separation capabilities ofPLC and the great power in structural characterization of MS, androvides a new powerful approach to identify the constituents inlant extracts rapidly and accurately. Otherwise, diode array detec-ion techniques could also be used in combination with HPLC–MS.AD and MS can provide on-line UV and MS information at theame time for each individual peak in a chromatogram. One canharacterize some of the peaks directly on-line by comparison withtandard compounds or with literature data. In the 1990s, somenvestigators made some preliminary attempts on the comprehen-ive analysis of phytochemical constituents in botanical extracts. Heelivered a review in 2000 [4]. The present review focuses on thepplication of HPLC–MS in the analysis of Chinese herbal medicinesfter 1999.

The mentioned compounds’ numbers are only used in each indi-idual sub-section. Only qualitative analysis is discussed in thiseview.

. Identification of known and unknown constituents inhe extracts of traditional Chinese medicines and theirerived products

The soft ionization methods [ESI, APCI, thermospray (TSP), etc.]o not typically produce many fragments. The collision-inducedissociation (CID) or collision activated dissociation (CAD) meth-ds can overcome this disadvantage. Quadrupole, ion trap (IT),ourier transform ion cyclotron resonance (FTICR), time-of-flight
TOF) mass spectrometer are the frequently used detectors for thehytochemical analysis of herbal medicines. The multistage masspectra (MSn, n ≥ 2) provided by IT-MS could confirm the relation-hip between precursor and daughter ions. This property is veryelpful for the structural determination of unknown compounds.
(dsip

1216 (2009) 2045–2062 2047

TICR and TOF could give high-resolution mass spectra of all theons and allow the definition of the ions’ elemental composition.ere, a number of examples were used to describe how HPLC–MSould be effectively applied to perform component identificationn complex mixtures.

.1. Phenolic compounds (including flavonoids)

Phenolic compounds are the major bioactive constituents ofhe herbs. Nearly half of the literatures reported on the on-linenalysis of phytochemical constituents of the bioactive pheno-ic compounds. The following sections are some of the exampletudies. Flavonoids appeared with very high frequency in thoseiteratures.

.1.1. Danshen: root of Salvia miltiorrhizaThe dried root of Salvia miltiorrhiza Bunge (Chinese name ‘Dan-

hen’) is one of the most well-known traditional Chinese medicines.t is widely used to treat coronary heart diseases, cerebrovasculariseases, bone loss, hepatitis, hepatocirrhosis and chronic renal fail-re, dysmenorrheal and neurasthenic insomnia. Phenolic acids arehe water soluble active constituents of Danshen. Therefore, it hasncited a great deal of research on the analysis of the phenolic acidsn this herb [5–7]. Electrospray ionization in negative ion mode wasdopted in all the reported studies.

Zeng et al. have studied phenolic acids in S. miltiorrhiza byPLC/ESI-MS/MS [7]. They have applied triple quadrupole mass

pectrometer as the detector. It was found that caffeic acid andts monomeric analogues containing a carboxyl group readily loseO2, while dimers, trimers and tetramers of caffeic acid eliminateduccessively danshensu or caffeic acid or their esters. Twenty-eighthenolic compounds in the extract of S. miltiorrhiza were charac-erized and eight of them were positively identified by comparingith the reference standards.

The phenolic compounds in S. miltiorrhiza were investigated byiu et al. using IT-MS [6], which has been considered more suit-ble for qualitative analysis than quadrupole mass spectrometer.he major fragmentations were very similar to those reported byeng et al. [7]. However, the genetic relationships between the pre-ursor and daughter ions are more affirmative. Based on the MSragmentation rules, the extract of Danshen was analyzed. In total,2 phenolic acids were identified or tentatively identified in onePLC–MSn run, and 16 of them were identified for the first time. Inddition, some isomers and close analogues could be distinguishedrom each other by comparing their MS/MS and MSn spectra.

In addition to IT, TOF was also employed for the study of phe-olic compounds in S. miltiorrhiza by Zhu et al. [5]. The accurateolecular weights of the constituents should be determined byPLC/ESI-TOF-MS and most of the compounds in Danshen coulde identified by TOF-MS from the formula database. HPLC–DAD,PLC/ESI-TOF-MS and HPLC/ESI-MSn provided complementary

nformation for the identification of the constituents in Danshen.sing the established methods, 22 phenolic compounds and 18 tan-

hinones were simultaneously characterized in 30 min based onheir negative and positive ion mass spectrometry. The accurate

olecular weights obtained by on-line TOF mass spectra are a greatelp for the determination of molecular formulas.

.1.2. Dengzhanxixin: whole plant of Erigeron breviscapusThe whole plant of Erigeron breviscapus (Vant.) Hand.-Mazz.

Compositae), known as Dengzhanxixin, is an important herbalrug in China for the treatment of cardiovascular and cerebral ves-el diseases. The main active components identified from this herbncluded flavonoids, coumarins, lignins, hydroxycinnamic acids,yromeconic acids and erigesides.

2 ogr. A 1216 (2009) 2045–2062

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Table 1Comparison of phenolic compounds in rhubarbs from Sect. Palmata and Sect. Rheum

Compounds Sect. Palmata Sect. Rheum

Sennosides + −Free anthraquinones + +Anthraquinone glycosides ++ +Stilbenes ± ++Glucose galloyl esters ++ +N

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Qu et al. identified seven O-glycosides and their aglyconesscutellarin, apigenin 7-O-glucuronide, quercetin-3-O-glucuronidend their aglycones and baicalin) in the extract of E. breviscapus8]. The sample was analyzed by a HPLC/ESI-MS/MS method on ariple-quadrupole mass spectrometer.

More comprehensive analysis was performed by Zhang etl. [9]. A total of 53 compounds, including caffeoylquinic acidsCQAs), CQA glucosides, malonyl-CQAs, acetyl-CQAs, caffeoyl-2, 7-nhydro-3-deoxy-2-octulopyranosonic acids (CDOAs), caffeoyl-2,-anhydro-2-octulo-pyranosonic acids (COAs), flavones, flavonolsnd flavonones, were identified or tentatively characterized basedn their UV and mass spectra. For HPLC/DAD/ESI-MSn analyses, ainnigan LCQ Deca XPplus IT mass spectrometer was used in neg-tive ion mode. The authors assigned most of the peaks in thehromatogram and the results may be useful for the quality controlf E. breviscapus and its related preparations.

.1.3. Rhubarb: root and rhizome of Rheum speciesRhubarb is one of the most well-known herbal medicines for the

reatment of constipation, inflammation, and cancer. As describedn the Chinese Pharmacopoeia, rhubarb consists of the roots andhizomes of Rheum officinale Baill., Rheum palmatum L., and Rheumanguticum Maxim. ex Balf., all of which belong to Sect. Palmata. Itas been found that this medicine comprised complex constituentsnd the major bioactive components were phenolic compoundsuch as anthraquinone derivatives, phenylbutanone glucopyrano-ides, stilbenes, tannins, naphthalene derivatives. Among theseompounds, sennosides (bianthrones) and anthraquinone glyco-ides are considered to be the main purgative components, whileree anthraquinones possess anti-inflammatory effects. In addition,lucose gallates, naphthalenes and catechins, which were isolatedrom rhubarbs, exhibited potent antioxidant and anticancer activi-ies.

In the extract of R. tanguticum, 41 different constituentsncluding 16 anthraquinone derivatives, 7 phenylbutanone glu-opyranosides, 4 stilbenes and 14 tannins were unambiguouslydentified or tentatively characterized based on their retentionimes, UV spectra and mass spectra in comparison with the datarom the reference standards [10]. For HPLC/DAD/ESI-MS analysis,

Q-TOF mass spectrometer was connected to HPLC instrumentia an ESI interface. In addition to the tandem mass spectra,igh-resolution mass spectra could be provided by the mass spec-rometry. Nine compounds were unambiguously identified byomparing with the pure standards.

Using the HPLC/DAD/ESI-IT-MSn method, a total of 107 com-ounds including 20 anthraquinones, 28 sennosides, 34 stilbenes,9 glucose gallates, 3 naphthalenes, and 3 catechins were identifiedrom the six Rheum species, three official (R. officinale, R. palmatum,nd R. tanguticum) and three unofficial (R. franzenbachii Munt., R.otaoense C.Y. Cheng et C.T. Kao, and R. emodi Wall) by Ye et al. [11].y comparing the chemical profiles of the Rheum species, it was

ound that their phenolic patterns showed significant difference.. officinale contained very different phenolic compounds from thether two official species. Sennoside A, which has been considereds the major purgative component of rhubarb, was only detectedn R. officinale, while its close isomers were observed in R. palma-um and R. tanguticum. In addition, the predominant anthraquinonelycosides in R. officinale were found to be rhein 8-O-glucosidend emodin 1-O-glucoside, whereas those in R. palmatum and R.anguticum were rhein 1-O-glucoside and emodin 8-O-glucoside.
tilbenes, which are the major constituents of unofficial rhubarbs,ere also different among the investigated species (Table 1). Due
o the significant differences in chemical components of Rheumpecies, the authors suggested that different Rheum species be usedeparately in clinical practice.

2

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aphthalenes + +

rom Ref. [11] with permission. Note: ++, major components; +, present; ±, presentn minute amounts; −, absent.

.1.4. Lianqiao: fruits of Forsythia suspenseThe fruit of Forsythia suspense (Thunb.) Vahl (Oleaceae), named

ianqiao in Chinese, is a well-known traditional Chinese medicine,hich has been widely used as an antipyretic, detoxicant and anti-

nflammatory agent for the treatment of various infectious diseases.t has also been shown that the herb is able to suppress vom-ting, resist hepatic injury, inhibit elastase activity, and exhibitiuretic, analgesic, antioxidant, antiendotoxin and antiviral effects.he Chinese Pharmacopoeia listed more than 40 Chinese medic-nal preparations containing Lianqiao, such as Shuanghuanglianral Solution, Yinqiao Jiedu Tablet and Qinlian Tablet. The pheno-

ic compounds, including phenylethanoid glycosides, lignans andavonols, are responsible for the major biological activities of thiserbal medicine.

Fragmentation behaviors of seventeen phenolic compoundsncluding phenylethanoid glycosides, lignans and flavonols werenvestigated by tandem mass spectrometry (MSn, IT). ESI masspectra in both negative and positive modes were examinedn the study. The obtained rules of the fragmentations werepplied to characterize the bioactive compounds in the fruits of. suspense by HPLC/DAD/ESI-MS analysis [12]. A total of 51 com-ounds were identified or tentatively characterized, including 24henylethanoid glycosides, 21 lignans and 6 flavonols. Amonghem, 17 compounds are new and most of them are reported from. suspense for the first time.

.1.5. Huangqin: roots of Scutellaria baicalensisHuangqin, the roots of Scutellaria baicalensis Georgi (Labiatae), is

n important traditional Chinese medicine used for the treatmentf hepatitis, tumors, diarrhea, and inflammatory diseases. It acts askey ingredient in a number of formulas such as Shuanghuanglianral liquid, Compound Huangqin granule, and Yinhuang Tablet,mployed for detoxication and relief of fever. Flavonoids are theajor active components and more than 60 flavonoids have been

eported from Huangqin. These compounds are obviously criticalor the quality control of Huangqin.

A HPLC/DAD/ESI-MSn method was developed by Han et al. [13]o analyze flavonoids in the roots of S. baicalensis. A total of 26ompounds were identified or tentatively characterized, including

C-glycosides, 12 O-glycosides and 9 free aglycones. Two C-lycosides, apigenin-6-C-glucosyl-8-C-arabinoside and chrysin-6,-di-C-glucoside, together with some O-glycosides, were reportedrom S. baicalensis for the first time. Furthermore, the comparativenvestigation of Huangqin collected from different regions was per-ormed by the established method. Only three standard compoundsere studied in this work. Therefore, deduction of the fragmenta-

ion rules was not possible.

.1.6. Maidong: tuber of Ophiopogon japonicusOphiopogon japonicus (Thunb.) Ker-Gawler, a traditional Chi-

ese medicine (Maidong) frequently used as a tonic drug in thelinic, is abundant in homoisoflavonoids, which are a type of spe-

M. Yang et al. / J. Chromatogr. A 1216 (2009) 2045–2062 2049

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ig. 1. Proposed MS fragmentation pathway for the [M−H]− ions of compounds 3ermission.

ial flavonoids with their B- and C-rings connected by an additionalH2 group. These compounds are rare in plants and few reportsould be found on their HPLC–MS analysis. The ESI-MSn monitoredn negative ion mode was used to study the fragmentation patternsf homoisoflavonoids [14]. The [M−H]− ions of homoisoflavonoidsith a saturated C2–3 bond underwent C3–9 bond cleavage to lose

he B-ring, which was followed by the loss of a molecule of CO. TheM−H]− ions of homoisoflavonoids with a C2–3 double bond usuallyliminated a CO molecule first, and then underwent the cleavage of3–9 or C9–1′ bonds. For homoisoflavonoids with a C-6 formyl group,owever, the neutral loss of CO was the first fragmentation step; theresence of a methoxyl group at C-8 could lead to the cleavage of-ring. No retro Diels-Alder (RDA) fragmentation characteristic forormal flavonoids was observed (Fig. 1).

The obtained fragmentation rules were implemented for thenalysis of homoisoflavonoids in the CHCl3–MeOH extract of O.aponicus [14]. A total of 18 homoisoflavonoids, including sevenew minor constituents, were identified or tentatively charac-erized based on the UV spectra and tandem mass spectra ofhe HPLC peaks. The paper reported the fragmentation rules ofomoisoflavonoids and the results were helpful to distinguishomoisoflavonoids and normal flavonoids.

.1.7. Ginger: rhizome of Zingiber officinaleGinger, the rhizome of Zingiber officinale Rosc., Zingiberaceae,

as long served culinary and medicinal uses. Gingerol-relatedompounds and diarylheptanoids are the two major groups of com-ounds and have been reported to be the bioactive components ofhis plant. Jiang et al. developed HPLC/ESI-MS/MS methods in bothositive and negative ion modes using an IT mass spectrometer

2

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mylisoophiopogonanone A) and 4 (methylophiopogonone A). From Ref. [14] with

oupled to HPLC in the continuing work to identify gingerol-relatedompounds [15] and diarylheptanoids [16] in Zingiberaceae plants,espectively.

Gingerol-related compounds comprise distinct groups (homol-gous series), which are differentiated by the length of theirnbranched alkyl chains, and have recently gained attention in

variety of biological activity studies. Using the establishedPLC/ESI(±)-MS/MS method, a total of 31 gingerol-related com-ounds, including three new compounds, were identified from theethanolic crude extract of fresh ginger rhizomes [15].Diarylheptanoids have been found to possess antioxidant,

ntihepatotoxic, anti-inflammatory, antiproliferative, antiemetic,hemopreventive, and antitumor activities, which lead to anncreasing interest in the recent years. In all, the diarylheptanoidsomprise five distinct groups (homologous series), which are dif-erentiated by structural differences on the heptane skeletons,hereas homologs within each group differed by substitution pat-

erns on the aromatic rings. Characteristic fragmentation behaviorn (+)- and (−)ESI-MS/MS analyses for each group of homologs, as

ell as information regarding polarity obtained from retention timeata, allowed the identification of 26 diarylheptanoids in the crudeethanolic extract from fresh ginger rhizomes [16]. Fifteen of themere reported for the first time and 18 of them were acylated, whichas found to be different from the diarylheptanoids of turmeric,

nother member of the Zingiberaceae.

.1.8. Propolis (Fengjiao)Propolis, also called “bee glue,” is a resinous substance that bees

se to construct and maintain their hives and has exhibited a varietyf interesting antimicrobial and antitumor properties in laboratory

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ests. Propolis is a traditional remedy in folk medicine and usuallyontains a variety of different chemical compounds. Phenolic acidsnd flavonoids are the two classes of principal constituents. Theropolis samples from different geographical areas were investi-ated by Gardana et al. [17]. HPLC/DAD-MS/MS in the negative ionode provides an effective fingerprinting method for the screening

f different propolis samples. By comparing the chromatographicnd UV behavior with that of standards, most of the compoundsould be identified. Moreover, tandem mass spectrometry providedy a triple quadrupole mass spectrometer with collision-inducedissociation (CID) allows structural identification, especially whentandard compounds are not available. A total of 60 compoundsere characterized in different samples. The results showed thatropolis from Argentina and Europe (Italy) was comparable, andiffered markedly from most Brazilian propolis.

.1.9. Kushen: roots of Sophora flavescensKushen is the Chinese name for the dried roots of Sophora

avescens Ait. (Leguminosae). It is a well-known traditional Chi-ese medicine widely spread in China, Japan and Korea. Itas been used as an antipyretic, diuretic, antihelmintic andtomachic for the treatment of diarrhea, gastrointestinal hem-rrhage and eczema. The principal phytochemical constituentsere quinolizidine alkaloids and prenylated flavonoids. Zhang

t al. [18] investigated the flavonoid compounds and developedHPLC/DAD/ESI-MS/MS method using an IT mass spectrom-

ter in positive ion mode. A total of 24 flavonoids weredentified. Fourteen of them were unambiguously identified byomparing experimental data for retention time (tR), UV andS spectra with those of the authentic compounds, which

re 3′,7-dihydroxy-4′-methoxy-isoflavone, trifolirhizin, kurarinol,ormononetin, 7,4′-dihydroxy-5-methoxy-8-(�, �-dimethylallyl)-avanone, maackiain, isoxanthohumol, kuraridine, kuraridinol,ophoraflavanone G, xanthohumol, isokurarinone, kurarinone andushenol D, and additional 10 compounds were tentativelydentified as kushenol O, trifolirhizin-6′′-malonate, sophoraisofla-anone A, norkurarinol/kosamol Q, kushenol I/N, kushenol C,′-methoxykurarinone, kosamol R, kushecarpin A and kushenol,espectively. The study provided a feasible approach for the rapidharacterization of flavonoids in the roots of S. flavescens.

.1.10. Dandelion (Pugongying): whole plant of Taraxacumfficinale

Dandelion (Taraxacum officinale WEB. ex. WIGG., Cichoriaceae),common plant in the northern hemisphere, has long been

sed for its choleretic, diuretic, antirheumatic, anti-inflammatory,axative, and appetite-stimulating properties for treating livernd gallbladder disorders, digestive complaints, and arthritic andheumatic diseases. In the present literature [19], a HPLC/DAD/ESI-Sn method was used for the characterization of phenolic acids

nd flavonoids in the extracts from dandelion (T. officinale) root anderb juice. An IT instrument was used as the analyzer. A total of 26ompounds, including five mono- and dicaffeoylquinic acids, fiveartaric acid derivatives, eight flavone and eight flavonol glycosidesere characterized based on their UV spectra and their fragmen-

ation patterns in collision-induced dissociation experiments. Theredominant compound in this herb was found to be chicoric aciddicaffeoyltartaric acid).

.1.11. Jiangxiang: heartwood of Dalbergia odorifera
Jiangxiang, the heartwood of Dalbergia odorifera T. Chen. (Legu-
inosae), is a traditional Chinese medicine, which has been used toreat blood disorders, ischemia, swelling, necrosis, and rheumaticain. It is a main ingredient in a number of formulae such as Guan-in-Er-Hao decoction, Xiangdan injection, and Xinning tablet. As

npt[

1216 (2009) 2045–2062

widely used medicinal herb to treat cardiovascular diseases, D.dorifera is known to be rich in flavonoids, which were reportedo have anti-inflammatory, anticoagulant, antitumor, vasodilative,ntihyperlipidic and antioxidant activities. A method combiningPLC with ESI-MS/MS in negative ion mode was developed for theualitative characterization of flavonoids in D. odorifera [20]. Byomparing their retention times, UV and MS spectra with thosef authentic compounds, twenty-three flavonoids including sixsoflavones, six neoflavones, four isoflavanones, three flavanones,wo chalcones, one isoflavanonol and one pterocarpan were unam-iguously identified. The study provided a simple, reliable andensitive method for phytochemical screening of flavonoids in D.dorifera. But this study did not take full advantage of the large num-ers of reference compounds (23 compounds) and the MSn (‘n’ cane greater than 2) spectra of IT mass spectrometer. Only the peaks ofuthentic compounds were assigned and no other unknown struc-ures were deduced. Hence, more research is necessary.

.1.12. Artichoke: Cynara scolymusThe leaves of Artichoke (Cynara scolymus L.) are higher in

edicinal value than flowers, with antihepatotoxic, choleretic,iuretic, hypocholesterolemic and antilipidemic properties thatre attributed to the phenolic components. A fast and efficientethod HPLC/ESI-MS in tandem mode with negative ion detectionas developed and validated for the qualitative analysis of arti-

hoke waste [21]. Forty-five phenolic compounds were identifiedn the basis of their mass spectra in full scan mode, mass spec-ra in different MS/MS modes, and retention times compared withhose of available reference standards. The major compounds wereound to be both caffeoylquinic and dicaffeoylquinic acids, luteolinlucuronide, luteolin galactoside, quercetin, and some quercetinlycosides.

.1.13. Honghuayanhuangqi: roots of Hedysarum multijugumHedysarum multijugum Maxim. is a medicinal plant of the family

eguminosae, which has been used in folk herbal medicine in Chinand was recorded in many Chinese herbal books for the treatmentf palpitation and chronic nephritis. Phytochemical studies showedhat the plant mainly consisted of pterocarpenes, coumestans,enzofurans and isoflavones. However, more phenolic compoundsere/could not be isolated and identified from the herb. Therefore,

he fragmentation behavior of four types of the phenolic com-ounds was studied using ESI-MSn (n = 2–5) in negative ion modey Yang et al. [22]. The obtained fragmentation rules were applied inhe identification of constituents in methanolic extract of H. mul-ijugum by HPLC/DAD/ESI-MSn method. A total of 29 compoundsere characterized and nine of them were reported for the first

ime. In this study, it was found that the number of methoxy groupsnfluenced the competitive loss of CO (28 Da) and CO2 (44 Da),

hich would be very useful for the identification of flavonoids andhould be confirmed by studying more reference standards.

.1.14. Tusizi: seeds of Cuscuta chinensisTusizi, prepared from the seeds of Cuscuta chinensis Lam. (dod-

er, Convolvulaceae family), is used as a tonic in Chinese medicinend has been shown to be able to improve sexual function, regulatehe body’s endocrine and immune system, and to prevent senes-ence. The chemical constituents mainly comprise flavonols likeuercetin, kaempferol and their glycosides. These compounds maye responsible for the biological activities of this drug.

However, seeds of C. australis R.Br. are also offered under theame of this medicine in the herbal market. In order to make a com-arison of their chemical constituents, the phenolic compounds ofhese two Cuscuta species were analyzed by HPLC/DAD/ESI-MSn

23]. A total of 50 compounds were observed in the methanol


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xtract, including 23 flavonoids, 20 lignans and 7 quinic acid deriva-ives. The phenolic patterns of these two Cuscuta species were foundo be very different (Fig. 2). Kaempferol and astragalin were theredominant constituents of C. australis, while hyperoside was theajor compound in C. chinensis. The large differences observed

etween the phenolic constituents of C. chinensis and C. australistrongly encouraged further comparison of the bioactivities ofhese two species.

.1.15. OthersCitrus species (Zhiqiao and Zhishi in TCM) [24–26], Dendrobium

pecies (Shihu in TCM) [27–29], Hypericum species (Guanyelian-iao in TCM) [30–33], Rhodiola rosea L. [34], Peucedanum palustre L.,ngelica archangelica (L.) var. archangelica [35] and Pueraria lobatahwi (Gegen) [36] were all proved to contain phenolic compoundsy HPLC–MS.

.2. Saponins

Saponins are glycosides of triterpenes and steroids, and com-only occur in higher plants. Saponins exhibit a variety of

iological activities and are widely used in foods, medicines andosmetics. The sugars found in saponins are hexoses (glucose, galac-ose), 6-deoxyhexoses (rhamnose), pentoses (arabinose, xylose),ronic acids (glucuronic acid) or amino sugars (glucosamine). Sug-rs may be linked to the sapogenin at one or multiple glycosylationites. Saponins response only at a short wavelength (200–210 nm)sing an UV detector, therefore, the MS spectrometer is preferablend reliable to be a detector in the chromatographic analysis.

.2.1. Ginseng: root and rhizome of Panax spp.When saponins from the herbal medicines were discussed,

e have to bring to mind the ginsenosides firstly. Ginseng rootas been used traditionally in Chinese medicine for over 2000ears and is now one of the most commonly found Chinese herbssed worldwide. Pharmacological studies indicated that ginsengnd its constituents have multifold bioactivities including anti-itogenic effect, improving impaired memory and inhibition of

umor cell growth. The pharmacological properties of Ginseng
re generally attributed to its triterpene glycosides, called gin-enosides. Ginsenosides are mainly dammarane triterpenes with20S)-protopanaxadiol and (20S)-protopanaxatriol aglycone moi-ties. The only oleanolic acid-type saponin identified in the rootsf P. ginseng C. A. Mey. is ginsenoside Ro.
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scuta australis (a) and Cuscuta chinensis (b). From Ref. [23] with permission.

Fuzzati et al. [37] developed a HPLC/ESI-MS method in negativeon mode for the systematic analysis of ginsenosides in P. ginsengoots. Ion trap analyzer was used to produce full mass and MS/MSpectral for each peak detected from P. ginseng. A total of 25 gin-enosides were separated and characterized.

Malonic esters of ginsenosides have been isolated from P. gin-eng and these may be among the major ginsenosides present ininseng root. Malonyl-ginsenosides are unstable and are readilye-malonylated upon heating. The acidic malonyl-ginsenosides areore difficult to analyze directly by HPLC than their neutral coun-

erparts. Consequently, a single HPLC–MS analysis method, coupledith automatic MS/MS scanning and post-acquisition neutral lossata analysis, was established for profiling of the malonylated andcetylated ginsenosides in ginseng extract [38]. Comparative inves-igation showed that the profiles of malonyl-ginsenosides appearo be different in P. quinquefolius L. (American), P. ginseng (Asian)nd P. notoginseng (Burk.) F. H. Chen (Sanchi ginseng). The resultsould provide useful additional information for the authenticationf these different ginseng species. The relative content of malony-ated ginsenosides is reduced in the red form of Asian ginsengompared with the white form and there is a concomitant increasen the levels of the corresponding acetylated ginsenosides.

Leung et al. [39] tried to differentiate three ginseng species usingHPLC/APCI-MS/MS method in negative ion mode. Notoginseno-

ide R1 is observed in both P. notoginseng and Chinese ginseng, whileseudoginsenoside F11 is found exclusively in the American species.he results permit the definitive identification of the species.

.2.2. Sanqi: root and rhizome of Panax notoginsengPanax notoginseng (Burk.) F. H. Chen, named Sanqi in Chinese, is a

ighly valued and important Chinese medicinal herb, belonging tohe same genus as Chinese and Korean ginseng (Panax ginseng) andmerican ginseng (Panax quinquefolium) and the saponin profilesf them are very similar. However, the function of Sanqi is differentrom that of Ginseng in tradition Chinese medicine. Quality controlf this herb is hence paramount. A HPLC/ESI-MS method was devel-ped for the analyses and identification of saponins in plant extractrom Sanqi [40]. An IT mass spectrometer with an electrospray ionource was used for detection of the ions. A CID experiment wasarried out to produce fragment ions. Eight saponins in the crude
xtract of P. notoginseng have been identified.
Furthermore, the raw form of Sanqi is traditionally used in Chi-ese medicine for its haemostatic and cardiovascular properties;he steamed form is used as a tonic to ‘nourish’ blood and toncrease production of various blood cells in anemic conditions.

2 ogr. A

DcCrdcrwptilstc

2

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2

autwHacorbThca

2

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ld

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awuari

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ue to their contrasting pharmacological actions and clinical indi-ations, the authentic identification of the forms is very important.han et al. [41] presented an ultra-performance liquid chromatog-aphy (UPLC)/TOF-MS method for direct detection of down-streamerivatives of metabolites, arising from the herbal formulation pro-ess. The UPLC, which can provide perfect chromatogram with highesolution and high retention time reproducibility in 11 min, alongith the accurate mass measurement by TOF is significantly com-lementary for the tentative identification of the biomarkers. Usinghe marker ion selection criteria of ‘present in steamed and absentn raw samples (vice versa)’ and ‘higher abundance in steamed andower in raw samples (vice versa)’, 74 and 146 marker ions wereubsequently obtained for the raw and steamed samples, respec-ively. The result confirmed that the steaming process led to thehanges in the levels and occurrence of ginsenosides.

.2.3. Ciwujia: roots of Acanthopanax senticosusAcanthopanax senticosus Harms (Ciwujia in Chinese) is a typi-

al Chinese herb, its root is an important Chinese folk medicine forhe treatment of ischemic heart diseases, hypertension, theumaticnd tumor, etc. The principal bioactive constituents of Ciwujia areaponins. Guo et al. investigated the saponins in crude extractrom leaves of A. senticosus by electrospray ionization multistageandem mass spectrometry (IT) and high-resolution mass spec-rometry (TOF) [42]. Eighteen compounds were identified from theaponin mixtures. The authors only used ESI-MSn in combinationith structural correlations existing in the plant to characterize the

tructures of underivatized saponins from medicinal herbs. If HPLCechnique was introduced, the established method would be better.

.2.4. Huashanfan: roots and leaves of Symplocos chinensisSymplocos chinensis (Lour.) Druce (Huashanfan in Chinese) is

toxic herb distributed in southern China and has been widelysed as a folk medicine to treat several diseases. Some newriterpenoid saponins isolated and characterized from this plantere found to have significant cytotoxic activities. Therefore, aPLC–MSn method was developed to rapidly identify and char-cterize triterpenoid saponins in this herb [43]. A total of 14onstituents in the crude extract were structurally characterizedn the basis of their retention time and tandem mass spectromet-ic analysis. There are no less than five pairs of isomers detectedy HPLC–MSn study and three pairs of those were differentiated.he authors indicated that the established analytical method stillas some limitations in differentiating compounds with geometri-ally isomeric substituting acyl groups and in locating them on theglycones.

.2.5. Huangqi: roots of Astragalus spp.Radix Astragali, called Huangqi in Chinese, is the dried roots

f Astragalus mongholicus Bge. or A. membranceus (Fisch.) Bge. Its traditionally used as an antiperspirant, a diuretic or a tonic.he Polysaccharides, triterpene saponins (astragalosides and soyas-ponins), flavonoids and �-aminobutyric acid were reported to behe major active components. The saponins isolated from Astragaluspp. could protect the liver of mice from chemical injury induced byarbon tetrachloride, d-galactosamine and acetaminophen. Astra-alosides usually have a 9, 19-cyclolanostane cycloastragenol ashe aglycone except for astragaloside VIII, which possesses anleanene-type soyasapogenol B as the aglycone. Xu et al. inves-igated the analysis of saponins in the roots of Astragalus spp. and
eveloped a HPLC/APCI-MS for on-line characterization [44]. Tripleuadrupole mass spectrometer was used in negative ion mode.welve astragalosides in the extract of Radix Astragali obtainedrom Shandong in China were separated and identified. Obviously,
ore effort should be made to differentiate the isomers. The estab-

i(LWm

1216 (2009) 2045–2062

ished method could also be used to distinguish Astragalus spp. fromifferent habitat regions.

.2.6. Cynanchum: Radix Cynanchi atrati (Baiwei) andynanchum chekiangense (Manjiancao)

Radix Cynanchi atrati, called Baiwei in Chinese, is traditionallysed as an antifebrile and diuretic in Chinese orthodox medicine.ynanchum versicolor Bge. and C. atratum Bge. are listed as sourcesf Baiwei in Chinese Pharmacopoeia (Committee for the Phar-acopoeia of China, 2005). C21 steroids and their saponins are

eported to be the main components in C. versicolor (Manshengbai-ei) and considered to be the bioactive components with cytotoxic,

ntimicrobial, herbicidal and anti-inflammatory activities. The C21teroidal saponins were studied in positive and negative ESI-MSn.he obtained fragmentation rules were applied in the HPLC/ESI-Sn approach for the identification of the saponins in 90% ethanolic

xtract from the root and rhizome of C. versicolor by Zheng et al.45]. Nine C21 steroidal saponins and an isomer of atratoglaucosidewere detected and elucidated simultaneously. It was obvious that

he principal peaks in the chromatogram were mostly character-zed.

Cynanchum chekiangense M. Cheng ex Tsiang et PT Li. (Asclepi-daceae) belongs to the same genus as Baiwei and is distributedildely in southern China. The roots of the plant have beensed for the treatment of rheumatoid arthritis and rheumaticches in Chinese folk medicine. The CHCl3 soluble extract of theoots, comprising C21 steroidal glycosides, was reported to possessmmunosuppressive.

Thus, it is necessary to identify their chemical structures to theurther investigation of pharmaceutical activity and quality controlf the medicine. HPLC/ESI-MS/MS was used by Tai et al. for on-lineharacterization of the C21 steroidal glycosides with immunologicalctivities in roots of C. chekiangense [46]. In the MS/MS spectra,ragmentations of the [M+Na]+ were recorded to provide structuralnformation about the glycosyl and aglycone moieties.

Four known steroidal glycosides cynascyroside C, chekiangen-osides A and B, glaucoside H, and four new steroidal glycosideshekiangensosides C, D, E and chekiangensoside A isomer weredentified based on mass spectral data. The results were confirmedy off-line FTICR-MS/MS and NMR spectral data.

.2.7. Chuanshanlong: rhizomes of Dioscorea nipponicaThe dried rhizome of Dioscorea nipponica Makino, called Chuan-

hanlong in Chinese, is the raw material of Di-ao-xin-xue-kang,hich has been used to cure coronary heart disease for more than

0 years in China. Steroidal saponins are the principal bioactiveomponents. A HPLC/ESI-MSn method was developed for the anal-ses and characterization of steroidal saponins in plant extractrom the rhizomes of D. nipponica [47]. Twelve saponins wereetected and three of them were characterized as pseudoproto-ioscin, methyl protodioscin and dioscin. The authors suggestedhat further investigation should be conducted to characterize theeaks mostly.

.3. Alkaloids

Alkaloids are usually described as the cyclic compounds thatontain negative valent nitrogen atoms and they exist in organ-sm. Over 10,000 alkaloids were isolated from nature so far andistributed widely in the plants. Some important plant medicines

ncluding opium, Mahuang (Herba Ephedrae), cinchona, MaqianziStrychnos nux-vomica L.), Hanfangji (Stephania tetrandra S. Moore),angdang (Hyoscyamus niger L.), Yanhusuo (Corydalis yanhusuo. T. Wang), Kushen (Sophora flavescens Ait.), Yangjinhua (Daturaetel L.), Qiushuixian (Colchicum autumnale L.), Changchunhua

ogr. A

(H

2A

tcsAutaiastm

oaRawlXmk[

rccadaadat

bdfsasd

tAWgptbf

2

Lduat

eCbar

[abaodhwcacacoc

2

nttsh

cautaaocfiodaHsoS

2

bdCTeaiUtsimultaneously [56]. A total of 21 steroidal alkaloids (5 protoverine-


Catharanthus roseus (L.) G. Don), Sanjianshan (Cephalotaxus fortuneook.f.), Fuzi (Aconitum carmichaeli Debx) are rich in alkaloids.

.3.1. Aconite: Aconitum sinomontanum, Aconitum kusnezoffii,conitum carmichaeli

The plants of Aconitum genus are widely distributed acrosshe Northern Asia and Northern America and have been found toomprise abundant natural alkaloids. The roots of Aconitum, theo-called Fuzi (Radix Aconiti laterals praeparata.), Chuanwu (Radixconiti) and Caowu (Radix Aconiti kusnezoffii) have been widelysed for analgesic, cardiotonic and antirheumatism treatment inraditional Chinese medicine. Aconitum alkaloids have analgesic,nti-inflammatory and cardiotonic activities and can be dividednto four groups according to the structure of their skeleton: i.e.,conitines, lycoctonines, atisines and veatchines. The rapid andensitive analysis of alkaloids in Aconitum plants will be of advan-age to the pharmaceutical studies and quality control of the herbal

edicines and their preparations.The group of Shuying Liu has performed a series of research

n the analysis of alkaloids in Aconitum species [48–51]. Aconitinend its analogues are the main active components of A. kusnezoffiieichb. Wang et al. found that the ESI-MSn spectra could providesimple method for the direct analysis of alkaloid mixtures, byhich about 70 alkaloids (aconitines, lipo-aconitines and triester

ipo-aconitines) were detected in the flowers of A. kusnezoffii [48].u et al. performed the analysis of alkaloinds in the roots of A. sino-antanurn Nakai using the ESI-MSn technique. Structures of six

nown norditerpenoid alkaloids were simultaneously determined49].

The herbal medicines are always used as decoctions, whicheadily lead to the change of the constituents compared with therude drugs. Wang et al. [50] checked the diversified aconitines-ontaining samples, including crude aconite, decoction of crudeconite, residues from decoction of crude aconite, prepared aconite,ecoction of prepared aconite, decoction of prepared aconite withdded palmitic acid, and decoction of a mixture of mesaconitinend hypaconitine standards with liquorice root, and found thatiester-diterpenoid aconitines (DDA) can be converted into lipo-lkaloids other than monoester-diterpenoid aconitines (MDA) inhe process of decocting aconite.

Furthermore, the stability of DDA in different solutions and pHuffers was studied by HPLC/ESI-MS [51]. In different solvents, theecomposition pathways of DDA are quite different and their dif-erence in stabilities depends on the substituents at the N atom andubstituents at C-3. The decomposition pathways of DDA in buffersre related to the substituent on the C-3 position. The decompo-ition pathway of aconitine is similar to that of mesaconitine, butifferent from that of hypaconitine.

Wu et al. introduced matrix-assisted laser desorption ioniza-ion (MALDI)-TOF-MS technique for direct analysis of alkaloids in. carmichaeli Debx. (Fuzi in Chinese) along with C. yanhusuo W.T.ang (Yanhusuo in Chinese) and Coptis chinensis Franch. (Huan-

lian in Chinese) [52]. MALDI-TOF-MS proved valuable for thereliminary study of plant component profiles. The rapid collec-ion of information from the direct analysis on plant tissues coulde valuable for supporting the discovery of new compounds andor the quality control of medicinal herbs.

.3.2. Liangmianzhen: roots of Zanthoxylum nitidiumThe root of Zanthoxylum nitidum (Roxb.) DC., locally called

iangmianzhen, is one of the traditional Chinese medicines widelyistributed throughout the southeastern part of China. It has beensed for removing rheumatoid arthralgia, resolutiving turgescencend controlling pain, and so on. Pharmacological studies indicatedhat Z. nitidum has antitumor, antibacterial and relieving pain prop-

taHfa

1216 (2009) 2045–2062 2053

rties. The extract of Z. nitidium has been added to toothpaste inhina due to its strong antibacterial activity. Alkaloids, includingenzo[c]phenanthridine alkaloids, protopine alkaloids, aporphinelkaloids and quinoline alkaloids, are considered to be mainlyesponsible for the activity.

An HPLC/ESI-MS/MS method was developed by Liang et al.53] and used to study the separation and detection of ninelkaloids in Z. nitidium, but no structural characterization haseen discussed. Cai et al. [54] studied the fragmentation behaviornd the corresponding fragmentation decomposition mechanismsf six benzo[c]phenanthridine alkaloids, dihydrochelerythrin,ihydronitidine, 8-acetonyldihydrochelerythrine, 8-acetonyldi-ydronitidine, nitidine and 1,3-bis(8-dihydronitidinyl)-acetoneere studied in detail by positive ion ESI-MSn. Furthermore, the

rude alkaloid extract from the roots of Z. nitidium was rapidlynalyzed by HPLC–MSn, and ten constituents were identified byomparing the retention times and ESI-MSn spectra with theuthentic standards. The authors suggested that not only theharacteristic fragments but also the characteristic abundancesf the fragment ions can be used for the detailed structuralharacterization.

.3.3. Baibu: root tuber of Stemona tuberosaThe root tubers of Stemona tuberosa Lour., named Baibu in Chi-

ese, belonging to family Stemonaceae, are widespread throughouthe tropical Asia and have long been used in Chinese and Japaneseraditional medicine for the treatment of respiratory diseasesuch as bronchitis, pertussis and tuberculosis, and against entericelminthes and ectoparasites in humans and cattle.

Members of the Stemonaceae family contain interesting poly-yclic alkaloids with an unusual molecular architecture bearingpyrrolo or a pyrido[1,2-a]azepine nucleus. These alkaloids are

nique to this family and are called Stemona alkaloids. Some ofhese alkaloids have been found to have a variety of biologicalctivities. The alkaloids profiles of the herb were found to be vari-ble, which demonstrated that a careful chemical authenticationf S. tuberosa is needed to assure the consistency, safety and effi-acy in pharmaceutical applications. An HPLC–MS chromatogramngerprint was developed for selective and reliable authenticationf this species [55]. Six different samples of S. tuberosa and threeifferent species of Stemona (S. tuberose, S. sessilifolia (Miq.) Miq.nd S. japonica (Bl.) Miq.) were investigated comparatively. ThePLC–MS fingerprints of these alkaloids, though variable among

amples, were proved to be useful for the authenticity and qualityf this species and help to differentiate it from S. japonica and. sessilifolia.

.3.4. Lilu: root and rhizome of Veratrum nigrumLilu, the roots and rhizomes of several Veratrum species, has

een used to treat aphasia arising from apoplexy, wind-typeysentery, jaundice, scabies and chronic malaria for centuries inhina. Nevertheless, Veratrum nigrum L. is a very poisonous plant.he steroidal alkaloids isolated from this plant were reported toxert teratogenic effects in several laboratory animals. Therefore,rapid and reliable method for characterization of the alkaloids

n this plant is necessary to ensure the safety of the medicine.sing the established HPLC/ESI-MSn method, different cevanine-

ype alkaloids in herbal extract were detected and identified

ype alkaloids, 14 germinetype alkaloids and 2 zygadenin-typelkaloids) were selectively identified from 27 detected peaks.owever, the authors suggested that further studies should be per-

ormed to obtain more detailed structural information on steroidallkaloids.

2 ogr. A

2

fteamM(

2

cdTpia

2

doisraa

ystaatfm

2

imbgsa

euch

2

bodPpaHti

2

dLpco[

2

agts

mnuStgofammt[

2

omaiegm[motTS

tc

2

tLaa(acid and ganoderic acids) were found from TCMs. HPLC–MS anal-


.3.5. OthersCui et al. studied the phenanthroindolizidine alkaloids in San-

endan (Tylophora atrofolliculata Metc.) with HPLC/ESI(±)-MSn

echnique [57]. Nine compounds were characterized in the crudextract. In a reported study, the variation in the profile of imidazolelkaloids in different seasons and in different parts of the Pilocarpusicrophyllus Stapf plant during the summer was analyzed by ESI(+)-S [58]. MSn (n = 2–3, IT) and high-resolution mass spectrometry

TOF) were used for the structural determination.

.4. Monoterpene glycosides

Monoterpenoids are constructed from two units of isoprene,ontaining ten carbon atoms. Iridoids are acetal metabolites of iri-oidial. The structure of iridoids contains the units of cyclopentane.he chemical feature of iridoids is somewhat like that of monoter-enes. To our knowledge, the most famous monoterpene glycoside

s paeoniflorin. However, most of the iridoids were found to exists glycosides in the plants.

.4.1. Shaoyao: dried roots of Paeonia lactifloraThe root of Paeoniae lactiflora Pall. is a common Chinese herb

rug that is widely used in both Japan and China for the treatmentf a variety of diseases, including cleansing heat, cooling blood,nvigorating blood circulation, alleviating pain, regulating men-truation, treating liver disease and cancer. The major constituentseported from P. lactiflora Pall. were monoterpene glycosides suchs paeoniflorin, albiflorin, oxypaeoniflorin, benzoylpaeoniflorinnd galloylpaeoniflorin.

Dong et al. reported a HPLC/ESI(−)-MS/MS method for the anal-sis of constituents in P. lactiflora Pall [59]. IT and FTICR masspectrometer were used in the study. Five compounds were charac-erized in the crude extract. The application of ESI-FTICR-MS/MS ishighlight in this paper. But it is a pity that this technique was notpplied in the on-line study and only five compounds were charac-erized. More available data from authentic standards are neededor the characterization of more unknown constituents of the herbal

edicine.

.4.2. Zhizi: fruits of Gardenia jasminoidesThe fruit of Gardenia jasminoides Ellis (named Zhizi in Chinese)

s a widely used traditional Chinese medicine for treatment ofany diseases, such as hepatitis, inner fever, hypertension, and dia-

etes. Zhizi contains a large amount of iridoid glycosides such aseniposide, gardenoside, genipin gentibioside, gardoside, shanzhi-ide, geniposidic acid, scandoside methyl ester and methyl deacetylsperulosidate.

Ren et al. developed a HPLC–MSn method to analyze the crudextract of G. jasminoides fruit [60]. ESI(−)-IT mass spectrometer wassed to provide full scan and MSn spectra. Five constituents wereharacterized. Obviously, further studies were needed to compre-ensively profile the iridoid glycosides of Zhizi.

.4.3. Jishiteng: whole plant of Paederia scandensThe whole plant of Paederia scandens (Lour) Merrill has long

een used as a Chinese traditional medicine for the treatmentf toothache, chest pain, piles, and inflammation of the spleen,iuretic, emetic, rheumatic arthritis and curing bacillary dysentery.. scandens produces sulfur-containing iridoid glucosides, in which
aederoside exhibited an inhibitory effect on Epstein-Barr virusctivation. Zhou et al. studied the iridoid glucosides of Jishiteng byPLC/ESI(+)-MSn technique [61]. IT-MS and Q-TOF-MS were used in
he on-line analysis. Five sulfur-containing iridoid glucosides weredentified in the crude extract.

yCia[

1216 (2009) 2045–2062

.4.4. OthersAn HPLC/ESI-MS analysis was performed to determine the iri-

oid composition of Lamium album L., Lamium amplexicaule L.,amium garganicum L., Lamium maculatum L., and Lamium pur-ureum L. by Alipieva et al. The study showed that the HPLC/ESI-MSould be used as a quick method for species authenticationr to assess the chemodiversity and phylogeny of the genus62].

.5. Diterpenoids

The most famous diterpenoid in the last century is the taxols an antitumor medicine found from a plant. Ye and Guo investi-ated the fragmentation behaviors of taxoids by ESI- and APCI-MSn

echniques [63]. The results could facilitate the rapid screening andtructural characterization of taxoids in plant extract by HPLC–MS.

Tanshinones and ginkgolides as well as bilobalide are the activeembers in diterpenoids, too. Ginkgo biloba Linne (Yinxing in Chi-

ese) is one of the oldest living tree species. In China, medicinalses of Ginkgo were first described in Ben Cao Gang Mu by Lihizhen in 1596. The fruits (Baiguo in Chinese) were used for thereatment of cough, asthma, enuresis, alcohol misuse and pyo-enic infection of skin. Recently, Ginkgo leaf extracts have becomene of the top selling phytopharmaceuticals in the US and Europeor mental alertness, enhanced vitality level, circulatory healthnd blood vessel health. The terpene trilactones are one of theajor active components of Ginkgo leaf extract. Some analyticalethods using HPLC–MS techniques were developed for the quan-

itative determination of major active components in G. biloba64–66].

.5.1. Danshen: roots of S. miltiorrhizaTanshinones, possessing the skeleton of abietane, is one class

f the active components present in the roots of Danshen (S.iltiorrhiza). Tanshinones are reported to have pharmacological

ctions of anti-inflammation, dilating coronary artery and increas-ng coronary flow, antioxidation, cytotoxic activity and modulatingffect on mutagenic activity. We reported the HPLC–MSn investi-ation for separation and characterization of tanshinones in theethanol–chloroform (7:3) extract from the root of Danshen (Fig. 3)

67]. Ion trap spectrometer was used as the mass analyzer. Theajor fragmentation behaviors of the compounds were the losses

f H2O, CO, propylene and CH3. A total of 27 tanshinones were iden-ified or tentatively characterized, including five new constituents.anshinones consist of both ortho- and para-quinone structures.tudies on para-quinone compounds are lacking in this paper.

Zhu et al. developed a simple method to separate and charac-erize tanshinones and phenolic compounds simultaneously in onehromatogram [5] (see Section 2.1.1).

.6. Triterpenoids (aglycone)

Triterpenoids are derived from squalene in biogenesis and con-ain 30 carbon atoms. Many plant families such as Araliaceae,eguminosae, Campanulaceae and Scrpophulariaceae containedbundant triterpenoids. In addition, some animals and fungi arelso the sources of triterpenoids. A number of active triterpenoidse.g. meliacane, quassinane, bruceantin, oleanolic acid, glycyrrhizic

sis of toosendanin in Melia toosendan Sieb. et Zucc. (Chuanlian inhinese) [68], azadirachtin and related triterpenoids in Azadirachta

ndica A. Juss (Yinglian, in Chinese) [69], oleanolic acid and ursoliccid in Anoectochilus roxburghii (wall.) Lindl (Jinxianlan in Chinese)70] was reported recently.


F -sheni

2

cCthoacadIliaa

wecbewGOiv

Fc

ig. 3. HPLC/DAD/ESI-MSn analysis of the chloroform–methanol (3:7) extract of Danon current profile of Dan-shen. From Ref. [67] with permission.

.6.1. Lingzhi: fruit body of Ganoderma lucidumTriterpenic acids were found to be the most important active

omponents in Ganoderma lucidum (Leyss. ex Fr.) Karst. (Lingzhi inhinese), which has long been used as a folk remedy for the promo-ion of health and longevity in China and other oriental countries. Itas prominent immunomodulatory and antitumor activities. Somef the triterpenoids isolated from Lingzhi showed antiandrogenic,ntihepatitis B, antioxidant, antitumor, anticomplement, antimi-robial, anti-HIV-1, selectively inhibit eukaryotic DNA polymerase,nd angiotensin converting enzyme-inhibitory activities. Thus, weeveloped a valuable and convincing method using HPLC/ESI(−)-

TMSn technique for the characterization of triterpenoids of G.ucidum for quality control of the herb (Fig. 4) [71]. Interestingly,t was found that some characteristic signals were not always thebundant peaks in MSn spectra (Fig. 5). When the method waspplied, a total of 32 triterpenoids, including six new compounds,

2

ih

ig. 4. HPLC/DAD/ESI-MSn analysis of the CHCl3 extract of Ganoderma lucidum. (a) HPLCurrent (TIC) profile. From Ref. [71] with permission.

crude drug. (a) HPLC-UV chromatogram monitored at 270 nm and (b) ESI-MS total

ere identified or tentatively characterized from the chloroformxtract of G. lucidum (Fig. 6). Most of the reference standards areompounds of type A (Fig. 6). Hence, more type A constituents coulde characterized. Fortunately, the main components in chloroformxtract of the studied G. lucidum are the triterpenic acids of type A,hich make most of the chromatographic peaks assigned. But someanoderma species mainly contain other types of triterpenic acids.bviously, further research should be carried out for the compar-

son of species differences and sufficient reference standards areery important in such an investigation.

.7. Steroids (aglycone)

Steroid is one of the familiar components occurring naturallyn plants and animals, possessing a nuclear of cyclopentanoper-ydrophenanthrene. Sterols, bile acid, steroid hormones, cardiac

-UV chromatogram monitored at 252 nm. (b) HPLC-negative ion ESI-MS total ion


F indicaA at m

gs

2

coXhtwcahGiomuts

2

Fgcmwcf

2

Paflb

poolwptpl1iiidPosos

ig. 5. ESI-MS spectra and major fragmentation pathways for ganoderic acids, theM1 (a) and ganoderic acid J (b); MS/MS spectrum at m/z 497 (c) and MS3 spectrum

lycosides, toad poison, steroidal saponins are some importantteroids.

.7.1. Chansu: skin secretions of giant toadsChanSu, prepared from the skin secretions of giant toads, also

alled toad venom or toad poison, is one of the major componentsf many well-known patent TCM drugs like Liu-Shen-Wan and She-iang-Bao-Xin-Wan, which is frequently used in clinics to treateart failure, sores, pains, and various cancers. The major effec-ive constituents of ChanSu are bufadienolides, a type of steroidsith a characteristic a-pyrone ring at C-17, and show significant

ardiotonic, blood-pressure-stimulating, anesthetic, and antitumorctivities. In the past several years, we have performed a compre-ensive investigation of this important Chinese medicine. Ye anduo achieved the rapid and accurate identification of the steroids

n Chansu [72], which is of great significance in the quality controlf this natural drug and its formulations. HPLC/DAD/APCI(+)-MSethod was developed for the analysis. IT mass spectrometer was

sed to provide full scan and multistage mass spectra (MSn). Aotal of 35 bufadienolides were identified, including four new con-tituents.

.8. Others and miscellaneous natural products

Many other types of natural products exist in herbal medicines.urthermore, herbal medicines usually do not contain only one sin-le type of compounds. Simultaneous analysis of miscellaneous
onstituents is very important in the investigation of traditionaledicines. This can also be preformed by HPLC–MS technique, inhich ionization of different types of compounds under the same
ondition was the difficulty. Some cases on the characterization ofarraginous compounds are shown below.

2c

G

ted peaks are characteristic ions: MS3 spectra for ion at m/z 451 of ganoderic acid/z 453 (d) of ganoderic acid B. From Ref. [71] with permission.

.8.1. Mudanpi (Cortex Moutan): root bark of Paeonia suffruticosaCortex Moutan, called Mudanpi in Chinese, is the root bark of

aeonia suffruticosa (Paeoniaceae) and known traditionally as annalgesic, a sedative, an anti-inflammatory agent, and remedy foremale disorders in China. Monoterpenes, acetophenones and gal-oyl glucoses are found to be the main constituents and thought toe responsible for the biological activities of this crude drug.

Recently, the root cortices of P. delavayi Franch. and P. decom-osita Hand.-Mazz. are found to be used in the southwest provincesf China as Cortex Moutan. In order to evaluate these three speciesf Paeonia, a HPLC/DAD/ESI(−)-MS method was established to ana-

yze the multiple constituents in the herbal plant [73]. Q-TOF-MSas applied for the detection. Full scan MS and MS/MS spectra wererovided in the negative ion mode to investigate the fragmenta-ion behavior of the compounds. From the root cortices of the threelants, a total of 50 compounds, including 17 monoterpenes, 14 gal-

oyl glucoses, 10 acetophenones, 5 phenolic acids, 3 flavonoids andtriterpene, were characterized based on their retention behav-

or, UV spectra and MS fragmentation patterns. Great differencesn the chemical constituents among the three species were foundn the comparative analysis. Paeonol was found to be the pre-ominant constituent of P. suffruticosa and P. decomposita, while. delavayi contained albiflorin and more galloyl glucoses than thether two species. The authors suggested that further compari-on of the bioactivities of these three species should be carriedut considering the significant differences in their chemical con-tituents.

.8.2. Jinguolan: roots of Tinospora sagittata and Tinosporaapillipes

Jinguolan, prepared from the roots of Tinospora sagittata (Oliv.)agnep and T. capillipes Gagnep. (Menispermaceae family), is


ed fro

cttgahTe

be

Fig. 6. Chemical structures of the triterpenoids identifi

ommonly used in traditional Chinese medicine (TCM) for thereatment of sore throat, laryngitis, gastralgia and diarrhea. Phy-ochemical studies revealed that they comprised three major
roups of compounds including protoberberine and aporphinelkaloids, clerodane-type diterpenes and botanic steroids, whichave been reported as bioactive components from this herb.he ethanolic extract of Jinguolan was investigated using mod-rn pharmacological methods and found to possess broader
bacmT

m Ganoderma lucidum. From Ref. [71] with permission.

ioactivities, such as antibiosis, anti-inflammatory and antiviralffects.

Zhang et al. studied the constituents in the extract of Jinguolan
y HPLC/DAD/ESI(±)-MSn [74]. A total of 24 compounds were char-cterized. The obtained results suggested the necessity for furtheromparison of the bioactivities of the two species due to the dra-atic difference in the contents of the compounds in the roots of
. sagittata and T. capillipes.


F n oraE

2

(dTCap

H

ticCp

TC

P

2

22222

F

ig. 7. HPLC/DAD/ESI-MSn analysis of the methanolic extract of Shuang-Huang-LiaSI-MS total ion current (TIC). From Ref. [81] with permission.

.8.3. Shengma: rhizome and root of Cimicifuga speciesThe dried rhizomes and roots of Cimicifuga racemosa L. Nutt

Ranunculaceae) (syn. Actaea racemosa L.) are widely used as herbalietary supplement for the alleviation of menopausal symptoms.he Asian Cimicifuga species, including C. dahurica (Turcz.) Maxim.,
. foetida L., and C. heracleifolia Kom., namely Shengma in Chinese,re used for their antipyretic, analgesic and anti-inflammatoryroperties.
He et al. reported a fingerprinting approach based onPLC–DAD/MS/evaporative light scattering detection (ELSD) for

as3ms

able 2haracterization of compounds in Shuang-Huang-Lian oral liquid by HPLC/DAD/ESI-MS

eak no. Retention time (min) [M−H]−

1 2.94 401a

2 5.46 4613 5.95 3534 8.97 3535 9.83 3536 15.37 6397 18.03 7558 18.41 6539 18.68 623

10 19.16 62311 22.37 62112 22.93 51513 23.30 51914 25.32 51515 25.73 579a

16 26.69 51517 29.18 44518 32.86 593a

19 36.71 4590 39.22 459

21 39.81 1457a

2 40.79 1295a

3 41.90 1133a

4 44.73 9115 45.40 10736 45.64 911

27 47.97 283

rom Ref. [81] with permission. Glc: glucose; ara: arabinose; rha: rhamnose; glu acid: glua [M+CH3COO]− ion.

l liquid. (A) HPLC-UV chromatogram monitored at 280 nm. (B) HPLC-negative ion

he identification of a total of 10 Cimicifuga species [75]. Thesenclude three North American species, C. racemosa, C. ameri-ana, C. rubifolia, and seven Asian species, C. acerina, C. biternat,. dahurica, C. heracleifolia, C. japonica, C. foetida, and C. sim-lex. The chemotaxonomic difference of the HPLC fingerprints
llows identification of all 10 Cimicifuga species. The triterpeneaponins, cimigenol-3-O-arabinoside, cimifugin, and cimifugin--O-glucoside, were considered to be suitable species-specificarkers for the distinction of C. racemosa from the other Cimicifuga
pecies.

m/z Identification

Caffeic acid glucosideForsythoside E3-Caffeoylquinic acidChlorogenetic acid4-Caffeoylquinic acidSuspensasideForsythoside BSuspensaside methyl etherActeosideForsythoside ASuspensaside A3,4-Dicaffeoylquinic acidPinoresinol-4′-O-�-d-glucopyranoside3,5-Dicaffeoylquinic acidEpipinoresinol-4′′-O-�-d-glucopyranoside4,5-Dicaffeoylquinic acidBaicalin3′ ,4′ ,5′-Trimethoxyl-4′′-hydroxyllignan-O-glucosideOroxylin A-7-O-glu acidWogonosideMacranthoidin BMacranthoidin ADipsacoside BHederagenin-28-O-ara-rha-glc esterMacranthoside BMacranthoside AWogonin

curonic acid.


PAL a

2

QmteHzc

T(CD

TM

C

STC1TPT

F

Fig. 8. Proposed scheme of possible metabolic pathway of

.8.4. OthersHerrero et al. studied the Spirulina platensis using HPLC/ESI(±)-

-TOF-MS/MS technique [76]. Four free fatty acids, fouronogalactosyl monoacylglycerols, three phosphatidyl-glycerols,

wo sulfoquinovosyl diacylglycerols were characterized. Zhaot al. developed a reliable and rapid method based on thePLC/DAD/ESI(±)-TOF-MS technique for isolation and characteri-ation of multiple constituents in the extract from Langdu (Stellerahamaejasme L.) [77]. Twenty-two obvious peaks appeared in the

tndtd

able 3etabolic reactions characterized of the seven tanshinones

ompounds Metabolic reactions

Monohydroxylation Dihydroxylation

odium tanshinone IIA sulfonate + −anshinone IIA + −ryptotanshinone + +5,16-Dihydrotanshinone I + −anshinone IIB + +rzewaquinone A + −anshinone I − −rom Ref. [93] with permission. +, positive; −, negative.

dministered orally in rats. From Ref. [94] with permission.

IC and nine of them were characterized by TOF/MS. DangguiAngelica sinensis Diels) is also a commonly used traditionalhinese medicine. Wang et al. investigated the constituents ofanggui using HPLC/APCI(±)-MS [78]. Fifteen compounds were

entatively identified. Chen et al. studied another famous TCMamed Chuangxiong (Rhizoma Chuanxiong) in Chinese [79]. Two-imension liquid chromatographic separation system was used forhe separation of components of Chuanxiong. The effluents wereetected by both DAD and APCI mass spectrometer. More than 50

Dehydrogenation Oxidation in side chain D-ring cleavage

+ − −+ + −+ − ++ − +− + −− + −− − −

2060 M. Yang et al. / J. Chromatogr. A

FR

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atcbmst

oc

2

Ruof[cflbecrq

2

ptZtwswulcsfc

2

aGCfiHaatias

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Dartbmsww

ig. 9. The proposed metabolic pathway of the total phenolic acids in danshen. Fromef. [95] with permission.

eaks were resolved and 11 of them were preliminarily identified.hou et al. identified two aminoethylphenyl oligoglycosides fromingucao (Schnabelia tetradonta (Sun) C.Y. Wu et C. Chen) usingPLC/ESI(±)-ITMSn [80].

.9. Complex traditional Chinese medicine prescription

In fact, TCMs are famous as complex prescription, which playedn important role in the public health of Chinese people forhousands of years. The Chinese doctors and some investigators
onsidered that the therapeutic effects of complex prescriptionenefited from the synergistic effect of all the componentialedicines. Obviously, it is more difficult to profile the chemical con-
tituents and control the quality of the complex prescription thanhose of single herbals. In the recent years, our group and some

dSeM1

1216 (2009) 2045–2062

ther groups have tried to use HPLC–MS technique to analyze theonstituents of some complex prescriptions [81–88].

.9.1. Shuanghuanglian oral liquidShuang-Huang-Lian oral liquid (SHL) consists of three herbs:

adix Scutellariae, Flos Lonicerae and Fructus Forsythiae. It is oftensed to treat acute upper respiratory tract infection caused by virusr bacteria. In our group, Han et al. developed a HPLC–MS methodor the quality control of Shuang-Huang-Lian oral liquid (Fig. 7)81]. A total of 27 compounds, including seven phenylethanoid gly-osides, three lignans, seven quinic acids, six saponins and fouravonoids, in the extract of Shuang-Huang-Lian oral liquid haveeen identified or tentatively characterized (Table 2). It set a goodxample for the rapid identification of bioactive constituents inomplex herbal preparations and made it possible to fulfill theequirements for a modern drug characterized as safe, effective anduality controllable.

.9.2. Shuangdan granuleShuangdan granule, one of the widely used Chinese complex

rescriptions prepared from Radix Salviae miltiorrhizae and Cor-ex Moutan, was authorized to marketing by SFDA of China (No.10960044) for treating coronary heart disease, myocardial infarc-ion, angina and atherosclerosis. An HPLC/DAD/ESI-MSn methodas developed for the simultaneous analysis of the chemical con-

tituents in Shuangdan granules [82]. Ion trap mass spectrometeras applied and both the positive and negative ion modes weresed in the analysis. A total of 28 compounds, including 12 pheno-

ic acids, 6 diterpenoid quinones, 8 monoterpenoids and two otheromponents, were characterized by their retention behaviors, UVpectra and multistage mass spectra. The results would be help-ul to ensure the safety and efficiency, and to optimize the qualityontrol of the medicine.

.9.3. Qingkailing injectionQingkailing injection is prepared from eight medicinal materi-

ls or their extracts, including Radix Isatidis, Flos Lonicerae, Fructusardenise, Cornu Bubal, Concha Margaritifera, Baicalinum, Acidumholicum and Acidum Hyodesoxy-cholicum. An approach to pro-le the main constituents in Qingkailing injection by combiningPLC/TOF-MS and HPLC–MSn (IT) had been described by Zhang etl. [83]. Both the positive and negative ions were detected in thenalysis and a total of thirty-three compounds were identified. Allhe components identified were surveyed and classified accord-ng to their medicinal material origins. The study is expected to beccepted as an effective and reliable pattern for comprehensive andystematic characterization of the complex TCM systems.

.9.4. Compound Danshen Dripping Pill“Compound Danshen Dripping Pill”, called Fufang-Danshen-

iwan in Chinese, is prepared from S. miltiorrhiza and P. notoginsengnd widely used for the prevention and treatment of coronary arte-iosclerosis, angina pectoris and hyperlipaemia. It has been clarifiedhat salvianolic acids, tanshinones and saponins are the majorioactive components. Zhang et al. developed an HPLC/ESI(−)-MSn

ethod for the identification of the saponins in Compound Dan-hen Dripping Pill [84]. By comparing the multistage mass spectraith those of reference standards and literature data, 19 saponinsere characterized in all.

Although the above four examples were investigated to some
epth, they could not be considered as classical complex formulas.ome classical traditional formulas have also been investigated. Forxample, Zhang et al. [85] studied Si-Wu-Tang using HPLC/DAD/ESI-S, 12 compounds were identified on-line; Lin et al. [86] identified
4 components in Gan-Lu-Yin by HPLC/ESI-MS; Liu et al. [87]

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nalyzed Xue-Fu-Zhu-Yu-Tang using HPLC/DAD/ESI-MS and 22omponents were identified or tentatively characterized. But thereere no MSn studies in these three studies.

. Conclusion

Over the past years, HPLC–MS has been used frequently inhytochemical analysis studies of traditional Chinese medicine.mergence of selected ion monitoring (SIM), selected reac-ion monitoring (SRM) and multiple reaction monitoring (MRM)echniques largely promoted the application of HPLC–MS in quan-itative investigation of TCMs and their in vivo process. But theualitative study of TCMs using HPLC–MS is still not as matures that by GC–MS. Analysis of mass spectra requires superior pro-essional knowledge and skill. The wide application of GC–MS inhe study of essential oil benefited from the commercial databasef abundant EI-MS spectra, which have been accumulated forany years and made it possible to obtain the initial structures

epending on computer search results. But the frequently usedon sources in HPLC–MS are mainly ESI and APCI. The fragmentsroduced by soft ionization sources and CID are much differentrom those by EI. Thus, the widespread uses of HPLC–MS in thenalysis of traditional Chinese medicines mainly rely on the eluci-ation of the fragmentation regular patterns of the constituents inhe herbal medicines using MSn spectra. Furthermore, the devel-pment of HPLC–MS database is a necessity. But this is a long-termroject and is currently in the groping stage since ESI and APCI-MSre easily affected by ionization and LC conditions. A univer-al mass spectrometer for natural products may be the ultimateesirability. But currently, a high-resolution mass spectrometer,hich could provide on-line MSn spectra, will be a great help to

he structural characterization of the constituents in the herbaledicines.Recently, an advanced HPLC technique called ultra-performance

iquid chromatography (UPLC) was developed. UPLC can providehromatograms with high resolution and high retention timeeproducibility, which may improve the separating ability of thePLC–MS technique. UPLC–MS was also applied in a study of TCM

41]. However, the high cost makes it difficult for the universalpplication. Additionally, capillary electrophoresis coupled to masspectrometry was also tried in the investigations of herbs [89,90].

In the phytochemical analysis of traditional Chinese medicinessing HPLC–MS technique, the relationship between structural

eatures and fragmentation patterns should be investigated ashoroughly as possible. Characteristic fragmentation behaviors ofhe reference standards, as well as information regarding polar-ty obtained from retention time data, on-line UV spectra, datarom literatures and biogenetic origin of the compounds allowedhe identification or tentative characterization of the chemical con-tituents in crude extracts. Undoubtedly, if there were no sufficienteference standards, this kind of study would be very difficult toroceed.

The methods established by HPLC–MS facilitated the convenientnd rapid quality control of traditional medicines and their phar-aceutical preparations. Moreover, the HPLC–MS technique could

lso be applied in the in vivo studies of herbal medicines [91–95].ome papers investigated the metabolism of phenolic compoundsnd tanshinones from the roots of S. miltiorrhiza in rats [92–95].he obtained results (Figs. 8 and 9, Table 3) are helpful for profilinghe in vivo process of Danshen.

When performing the literature search, hundreds of papersegarding the analysis of herbal medicines using HPLC–MS coulde traced. Nevertheless, majority of these papers are just tamettempts. More effort should be made for exploring the componentsn the herbal medicines and their preparations using HPLC–MS.

[[[[

1216 (2009) 2045–2062 2061

cknowledgement

This research was supported by the National Supporting Pro-ram for Traditional Chinese Medicine from the ministry ofciences and Technology of China (No. 2006BAI08B03-03).

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