Phytochemical Analysis

Published on July 2016 | Categories: Documents | Downloads: 36 | Comments: 0 | Views: 664
of 18
Download PDF   Embed   Report

Comments

Content

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

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Review

Phytochemical analysis of traditional Chinese medicine using liquid chromatography coupled with mass spectrometry
Min Yang a , Jianghao Sun b , Zhiqiang Lu a , Guangtong Chen a , Shuhong Guan a , Xuan Liu a , Baohong Jiang a , Min Ye b , De-An Guo a,b,∗
a b

Shanghai Research Center for TCM Modernization, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China 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

a b s t r a c t
Traditional Chinese medicine (TCM) is commonly considered to operate due to the synergistic effects of all the major and minor components in the medicines. Hence sensitive and comprehensive analytical techniques are needed to acquire a better understanding of the pharmacological basis of the herb and to enhance the product quality control. The present review mainly focuses on the phytochemical analysis of TCMs using high-performance liquid chromatography coupled with mass spectrometry (HPLC–MS). Atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) are the two commonly used ion sources. Triple quadrupole, ion trap (IT), Fourier transform ion cyclotron resonance (FTICR) and time-of-flight (TOF) mass spectrometers are used as on-line analyzer. The relationship between structural features and fragmentation patterns should be investigated as thoroughly as possible and hence be applied in the on-line analysis to deduce the structures of detected peaks. Characteristic fragmentation behaviors of the reference standards, as well as information regarding polarity obtained from retention time data, on-line UV spectra, data from the literature and bio-sources of the compounds allowed the identification of the phytochemical constituents in the crude extracts. Although a mass spectrometer is not a universal detector, high-performance liquid chromatography coupled with multistage mass spectrometry (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 or trace amounts. The methods established using HPLC–MS techniques facilitate the convenient and rapid quality control of traditional medicines and their pharmaceutical preparations. However, the quantitative analysis is not the topic of this review. © 2008 Elsevier B.V. All rights reserved.

Article history: Available online 3 September 2008 Keywords: Phytochemical analysis Traditional Chinese medicine HPLC–MS Tandem mass spectrometry Quality control

Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification of known and unknown constituents in the extracts of traditional Chinese medicines and their derived products . . . . . . . . . . . . . 2.1. Phenolic compounds (including flavonoids) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Danshen: root of Salvia miltiorrhiza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Dengzhanxixin: whole plant of Erigeron breviscapus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Rhubarb: root and rhizome of Rheum species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.4. Lianqiao: fruits of Forsythia suspense . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.5. Huangqin: roots of Scutellaria baicalensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2046 2047 2047 2047 2047 2048 2048 2048 2048 2049 2049 2050

∗ Corresponding author at: Shanghai Research Center for TCM Modernization, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Guo Shoujing Road 199, Zhangjiang, Shanghai 201203, China. Tel.: +86 21 50271516; fax: +86 21 50272789. E-mail address: [email protected] (D.-A. Guo). 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.08.097

2046

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

3.

2.1.10. Dandelion (Pugongying): whole plant of Taraxacum officinale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.11. Jiangxiang: heartwood of Dalbergia odorifera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.12. Artichoke: Cynara scolymus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.13. Honghuayanhuangqi: roots of Hedysarum multijugum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.14. Tusizi: seeds of Cuscuta chinensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.15. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Saponins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Ginseng: root and rhizome of Panax spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Sanqi: root and rhizome of Panax notoginseng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Ciwujia: roots of Acanthopanax senticosus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.4. Huashanfan: roots and leaves of Symplocos chinensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.5. Huangqi: roots of Astragalus spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.6. Cynanchum: Radix Cynanchi atrati (Baiwei) and Cynanchum chekiangense (Manjiancao) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.7. Chuanshanlong: rhizomes of Dioscorea nipponica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Aconite: Aconitum sinomontanum, Aconitum kusnezoffii, Aconitum carmichaeli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. Liangmianzhen: roots of Zanthoxylum nitidium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3. Baibu: root tuber of Stemona tuberosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.4. Lilu: root and rhizome of Veratrum nigrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.5. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Monoterpene glycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. Shaoyao: dried roots of Paeonia lactiflora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2. Zhizi: fruits of Gardenia jasminoides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.3. Jishiteng: whole plant of Paederia scandens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.4. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Diterpenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.1. Danshen: roots of S. miltiorrhiza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Triterpenoids (aglycone) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.1. Lingzhi: fruit body of Ganoderma lucidum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Steroids (aglycone) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7.1. Chansu: skin secretions of giant toads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. Others and miscellaneous natural products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8.1. Mudanpi (Cortex Moutan): root bark of Paeonia suffruticosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8.2. Jinguolan: roots of Tinospora sagittata and Tinospora capillipes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8.3. Shengma: rhizome and root of Cimicifuga species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8.4. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9. Complex traditional Chinese medicine prescription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9.1. Shuanghuanglian oral liquid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9.2. Shuangdan granule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9.3. Qingkailing injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9.4. Compound Danshen Dripping Pill . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2050 2050 2050 2050 2050 2051 2051 2051 2051 2052 2052 2052 2052 2052 2052 2053 2053 2053 2053 2054 2054 2054 2054 2054 2054 2054 2054 2054 2055 2055 2056 2056 2056 2056 2058 2059 2060 2060 2060 2060 2060 2061 2061 2061

1. Introduction Traditional Chinese medicine (TCM) played a significant role in the health of the Chinese people for thousands of years. Over the past decades, TCMs have always been the most important resources for screening lead compounds. Fast analysis of natural products in bioactive crude extracts attracted the attention of most investigators. Natural products gained prominence through antibiotics earlier in the last century; today they have been developed for a variety of medicinal uses such as immunosuppressive agents, hypocholesterolemic agents, enzyme inhibitors, antimigrane agents, herbicides, antiparasitic agents and ruminant growth promoters, and bioinsecticides [1]. Natural products are also the most important resources of anticancer agents, where over 60% of the approved and pre-new drug application candidates are either natural products or synthetic molecules based upon the natural product molecular skeletons. Herbal medicines and their derived products are widely used as therapeutic products in many countries. Their worldwide use has increased in the last decade [2]. Most herbal medicines and their derivative products were often prepared from the crude plant

extracts, which comprise a complex mixture of different phytochemical constituents (plant secondary metabolites). There may be hundreds of active components in these herbs. The chemical features of these constituents differ considerably among the different species. Even the same herbal extracts may vary depending upon the harvest season, plant origin, drying process and other factors. Therefore, the quality control of the herbal medicines and their derived products is difficult. Recently, the chromatographic fingerprinting of the components, especially by high-performance liquid chromatography–diode array detection (HPLC–DAD), is a powerful and widely used technique to analyze plant extracts because this technique could systematically profile the composition of samples and it focuses on the identification and consistency assessment of the components [3]. However, a valuable and convincing chromatographic fingerprint should have most of its peaks assigned, especially those corresponding to the active constituents and toxic ingredients. Unfortunately, such routine techniques as HPLC–DAD could only provide very limited structural information like UV spectrum; standard compounds, which are commercially unavailable in most cases, are usually necessary for the characterization of individual constituents. As a result, the isolation and purification from

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

2047

crude plant extracts of adequate amounts (at least 5–10 mg) of pure compounds (>90% purity) for nuclear magnetic resonance (NMR) identification was needed before they could serve as reference compounds. The whole process is tedious, laborious and expensive. Moreover, some constituents are only present in raw plant materials in very low amount or are quite unstable under normal conditions, and their enrichment and purification are laborious. The inherent variety of natural product extracts has shown significant challenges for separation and detection techniques to enable rapid characterization of the biologically active component in the mixture. Except for paper chromatography and thin-layer chromatography, gas chromatography (GC) is a powerful separation technique that has been utilized since the 1960s for the analysis of volatile natural products or derivatives. However, the role that has recently been played by HPLC for the work of this nature has been invaluable, considering that approximately 80% of all known natural compounds are nonvolatile or thermally unstable and therefore incompatible with GC. When coupled with a variety of detections, HPLC began to serve as a powerful tool for the rapid characterization of natural product extracts. In addition, some developing chromatographic techniques, such as capillary electrophoresis (CE), high speed countercurrent chromatography (HSCCC), gradually found their application in the separation of phytochemical constituents of herbal medicines. Mass spectrometry (MS) is the most selective technique for the rapid qualitative determination of known compounds as well as the identification of unknown compounds from the extracts of natural products. The analysis of natural products has been especially effective following the successful interface with a mass spectrometer detection system in the chromatographic method. Atmospheric pressure ionization (API), which consists of atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI), is the most successful interface used in HPLC–MS configuration. Therefore, HPLC–MS combines the efficient separation capabilities of HPLC and the great power in structural characterization of MS, and provides a new powerful approach to identify the constituents in plant extracts rapidly and accurately. Otherwise, diode array detection techniques could also be used in combination with HPLC–MS. DAD and MS can provide on-line UV and MS information at the same time for each individual peak in a chromatogram. One can characterize some of the peaks directly on-line by comparison with standard compounds or with literature data. In the 1990s, some investigators made some preliminary attempts on the comprehensive analysis of phytochemical constituents in botanical extracts. He delivered a review in 2000 [4]. The present review focuses on the application of HPLC–MS in the analysis of Chinese herbal medicines after 1999. The mentioned compounds’ numbers are only used in each individual sub-section. Only qualitative analysis is discussed in this review. 2. Identification of known and unknown constituents in the extracts of traditional Chinese medicines and their derived products The soft ionization methods [ESI, APCI, thermospray (TSP), etc.] do not typically produce many fragments. The collision-induced dissociation (CID) or collision activated dissociation (CAD) methods can overcome this disadvantage. Quadrupole, ion trap (IT), Fourier transform ion cyclotron resonance (FTICR), time-of-flight (TOF) mass spectrometer are the frequently used detectors for the phytochemical analysis of herbal medicines. The multistage mass spectra (MSn , n ≥ 2) provided by IT-MS could confirm the relationship between precursor and daughter ions. This property is very helpful for the structural determination of unknown compounds.

FTICR and TOF could give high-resolution mass spectra of all the ions and allow the definition of the ions’ elemental composition. Here, a number of examples were used to describe how HPLC–MS could be effectively applied to perform component identification in complex mixtures. 2.1. Phenolic compounds (including flavonoids) Phenolic compounds are the major bioactive constituents of the herbs. Nearly half of the literatures reported on the on-line analysis of phytochemical constituents of the bioactive phenolic compounds. The following sections are some of the example studies. Flavonoids appeared with very high frequency in those literatures. 2.1.1. Danshen: root of Salvia miltiorrhiza The dried root of Salvia miltiorrhiza Bunge (Chinese name ‘Danshen’) is one of the most well-known traditional Chinese medicines. It is widely used to treat coronary heart diseases, cerebrovascular diseases, bone loss, hepatitis, hepatocirrhosis and chronic renal failure, dysmenorrheal and neurasthenic insomnia. Phenolic acids are the water soluble active constituents of Danshen. Therefore, it has incited a great deal of research on the analysis of the phenolic acids in this herb [5–7]. Electrospray ionization in negative ion mode was adopted in all the reported studies. Zeng et al. have studied phenolic acids in S. miltiorrhiza by HPLC/ESI-MS/MS [7]. They have applied triple quadrupole mass spectrometer as the detector. It was found that caffeic acid and its monomeric analogues containing a carboxyl group readily lose CO2 , while dimers, trimers and tetramers of caffeic acid eliminated successively danshensu or caffeic acid or their esters. Twenty-eight phenolic compounds in the extract of S. miltiorrhiza were characterized and eight of them were positively identified by comparing with the reference standards. The phenolic compounds in S. miltiorrhiza were investigated by Liu et al. using IT-MS [6], which has been considered more suitable for qualitative analysis than quadrupole mass spectrometer. The major fragmentations were very similar to those reported by Zeng et al. [7]. However, the genetic relationships between the precursor and daughter ions are more affirmative. Based on the MS fragmentation rules, the extract of Danshen was analyzed. In total, 42 phenolic acids were identified or tentatively identified in one HPLC–MSn run, and 16 of them were identified for the first time. In addition, some isomers and close analogues could be distinguished from each other by comparing their MS/MS and MSn spectra. In addition to IT, TOF was also employed for the study of phenolic compounds in S. miltiorrhiza by Zhu et al. [5]. The accurate molecular weights of the constituents should be determined by HPLC/ESI-TOF-MS and most of the compounds in Danshen could be identified by TOF-MS from the formula database. HPLC–DAD, HPLC/ESI-TOF-MS and HPLC/ESI-MSn provided complementary information for the identification of the constituents in Danshen. Using the established methods, 22 phenolic compounds and 18 tanshinones were simultaneously characterized in 30 min based on their negative and positive ion mass spectrometry. The accurate molecular weights obtained by on-line TOF mass spectra are a great help for the determination of molecular formulas. 2.1.2. Dengzhanxixin: whole plant of Erigeron breviscapus The whole plant of Erigeron breviscapus (Vant.) Hand.-Mazz. (Compositae), known as Dengzhanxixin, is an important herbal drug in China for the treatment of cardiovascular and cerebral vessel diseases. The main active components identified from this herb included flavonoids, coumarins, lignins, hydroxycinnamic acids, pyromeconic acids and erigesides.

2048

M. Yang et al. / J. Chromatogr. A 1216 (2009) 2045–2062 Table 1 Comparison of phenolic compounds in rhubarbs from Sect. Palmata and Sect. Rheum Compounds Sennosides Free anthraquinones Anthraquinone glycosides Stilbenes Glucose galloyl esters Naphthalenes Sect. Palmata + + ++ ± ++ + Sect. Rheum − + + ++ + +

Qu et al. identified seven O-glycosides and their aglycones (scutellarin, apigenin 7-O-glucuronide, quercetin-3-O-glucuronide and their aglycones and baicalin) in the extract of E. breviscapus [8]. The sample was analyzed by a HPLC/ESI-MS/MS method on a triple-quadrupole mass spectrometer. More comprehensive analysis was performed by Zhang et al. [9]. A total of 53 compounds, including caffeoylquinic acids (CQAs), CQA glucosides, malonyl-CQAs, acetyl-CQAs, caffeoyl-2, 7anhydro-3-deoxy-2-octulopyranosonic acids (CDOAs), caffeoyl-2, 7-anhydro-2-octulo-pyranosonic acids (COAs), flavones, flavonols and flavonones, were identified or tentatively characterized based on their UV and mass spectra. For HPLC/DAD/ESI-MSn analyses, a Finnigan LCQ Deca XPplus IT mass spectrometer was used in negative ion mode. The authors assigned most of the peaks in the chromatogram and the results may be useful for the quality control of E. breviscapus and its related preparations. 2.1.3. Rhubarb: root and rhizome of Rheum species Rhubarb is one of the most well-known herbal medicines for the treatment of constipation, inflammation, and cancer. As described in the Chinese Pharmacopoeia, rhubarb consists of the roots and rhizomes of Rheum officinale Baill., Rheum palmatum L., and Rheum tanguticum Maxim. ex Balf., all of which belong to Sect. Palmata. It has been found that this medicine comprised complex constituents and the major bioactive components were phenolic compounds such as anthraquinone derivatives, phenylbutanone glucopyranosides, stilbenes, tannins, naphthalene derivatives. Among these compounds, sennosides (bianthrones) and anthraquinone glycosides are considered to be the main purgative components, while free anthraquinones possess anti-inflammatory effects. In addition, glucose gallates, naphthalenes and catechins, which were isolated from rhubarbs, exhibited potent antioxidant and anticancer activities. In the extract of R. tanguticum, 41 different constituents including 16 anthraquinone derivatives, 7 phenylbutanone glucopyranosides, 4 stilbenes and 14 tannins were unambiguously identified or tentatively characterized based on their retention times, UV spectra and mass spectra in comparison with the data from the reference standards [10]. For HPLC/DAD/ESI-MS analysis, a Q-TOF mass spectrometer was connected to HPLC instrument via an ESI interface. In addition to the tandem mass spectra, high-resolution mass spectra could be provided by the mass spectrometry. Nine compounds were unambiguously identified by comparing with the pure standards. Using the HPLC/DAD/ESI-IT-MSn method, a total of 107 compounds including 20 anthraquinones, 28 sennosides, 34 stilbenes, 19 glucose gallates, 3 naphthalenes, and 3 catechins were identified from the six Rheum species, three official (R. officinale, R. palmatum, and R. tanguticum) and three unofficial (R. franzenbachii Munt., R. hotaoense C.Y. Cheng et C.T. Kao, and R. emodi Wall) by Ye et al. [11]. By comparing the chemical profiles of the Rheum species, it was found that their phenolic patterns showed significant difference. R. officinale contained very different phenolic compounds from the other two official species. Sennoside A, which has been considered as the major purgative component of rhubarb, was only detected in R. officinale, while its close isomers were observed in R. palmatum and R. tanguticum. In addition, the predominant anthraquinone glycosides in R. officinale were found to be rhein 8-O-glucoside and emodin 1-O-glucoside, whereas those in R. palmatum and R. tanguticum were rhein 1-O-glucoside and emodin 8-O-glucoside. Stilbenes, which are the major constituents of unofficial rhubarbs, were also different among the investigated species (Table 1). Due to the significant differences in chemical components of Rheum species, the authors suggested that different Rheum species be used separately in clinical practice.

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

2.1.4. Lianqiao: fruits of Forsythia suspense The fruit of Forsythia suspense (Thunb.) Vahl (Oleaceae), named Lianqiao in Chinese, is a well-known traditional Chinese medicine, which has been widely used as an antipyretic, detoxicant and antiinflammatory agent for the treatment of various infectious diseases. It has also been shown that the herb is able to suppress vomiting, resist hepatic injury, inhibit elastase activity, and exhibit diuretic, analgesic, antioxidant, antiendotoxin and antiviral effects. The Chinese Pharmacopoeia listed more than 40 Chinese medicinal preparations containing Lianqiao, such as Shuanghuanglian Oral Solution, Yinqiao Jiedu Tablet and Qinlian Tablet. The phenolic compounds, including phenylethanoid glycosides, lignans and flavonols, are responsible for the major biological activities of this herbal medicine. Fragmentation behaviors of seventeen phenolic compounds including phenylethanoid glycosides, lignans and flavonols were investigated by tandem mass spectrometry (MSn , IT). ESI mass spectra in both negative and positive modes were examined in the study. The obtained rules of the fragmentations were applied to characterize the bioactive compounds in the fruits of F. suspense by HPLC/DAD/ESI-MS analysis [12]. A total of 51 compounds were identified or tentatively characterized, including 24 phenylethanoid glycosides, 21 lignans and 6 flavonols. Among them, 17 compounds are new and most of them are reported from F. suspense for the first time. 2.1.5. Huangqin: roots of Scutellaria baicalensis Huangqin, the roots of Scutellaria baicalensis Georgi (Labiatae), is an important traditional Chinese medicine used for the treatment of hepatitis, tumors, diarrhea, and inflammatory diseases. It acts as a key ingredient in a number of formulas such as Shuanghuanglian oral liquid, Compound Huangqin granule, and Yinhuang Tablet, employed for detoxication and relief of fever. Flavonoids are the major active components and more than 60 flavonoids have been reported from Huangqin. These compounds are obviously critical for the quality control of Huangqin. A HPLC/DAD/ESI-MSn method was developed by Han et al. [13] to analyze flavonoids in the roots of S. baicalensis. A total of 26 compounds were identified or tentatively characterized, including 5 C-glycosides, 12 O-glycosides and 9 free aglycones. Two Cglycosides, apigenin-6-C-glucosyl-8-C-arabinoside and chrysin-6, 8-di-C-glucoside, together with some O-glycosides, were reported from S. baicalensis for the first time. Furthermore, the comparative investigation of Huangqin collected from different regions was performed by the established method. Only three standard compounds were studied in this work. Therefore, deduction of the fragmentation rules was not possible. 2.1.6. Maidong: tuber of Ophiopogon japonicus Ophiopogon japonicus (Thunb.) Ker-Gawler, a traditional Chinese medicine (Maidong) frequently used as a tonic drug in the clinic, is abundant in homoisoflavonoids, which are a type of spe-

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

2049

Fig. 1. Proposed MS fragmentation pathway for the [M−H]− ions of compounds 3 (6-formylisoophiopogonanone A) and 4 (methylophiopogonone A). From Ref. [14] with permission.

cial flavonoids with their B- and C-rings connected by an additional CH2 group. These compounds are rare in plants and few reports could be found on their HPLC–MS analysis. The ESI-MSn monitored in negative ion mode was used to study the fragmentation patterns of homoisoflavonoids [14]. The [M−H]− ions of homoisoflavonoids with a saturated C2–3 bond underwent C3–9 bond cleavage to lose the B-ring, which was followed by the loss of a molecule of CO. The [M−H]− ions of homoisoflavonoids with a C2–3 double bond usually eliminated a CO molecule first, and then underwent the cleavage of C3–9 or C9–1 bonds. For homoisoflavonoids with a C-6 formyl group, however, the neutral loss of CO was the first fragmentation step; the presence of a methoxyl group at C-8 could lead to the cleavage of C-ring. No retro Diels-Alder (RDA) fragmentation characteristic for normal flavonoids was observed (Fig. 1). The obtained fragmentation rules were implemented for the analysis of homoisoflavonoids in the CHCl3 –MeOH extract of O. japonicus [14]. A total of 18 homoisoflavonoids, including seven new minor constituents, were identified or tentatively characterized based on the UV spectra and tandem mass spectra of the HPLC peaks. The paper reported the fragmentation rules of homoisoflavonoids and the results were helpful to distinguish homoisoflavonoids and normal flavonoids. 2.1.7. Ginger: rhizome of Zingiber officinale Ginger, the rhizome of Zingiber officinale Rosc., Zingiberaceae, has long served culinary and medicinal uses. Gingerol-related compounds and diarylheptanoids are the two major groups of compounds and have been reported to be the bioactive components of this plant. Jiang et al. developed HPLC/ESI-MS/MS methods in both positive and negative ion modes using an IT mass spectrometer

coupled to HPLC in the continuing work to identify gingerol-related compounds [15] and diarylheptanoids [16] in Zingiberaceae plants, respectively. Gingerol-related compounds comprise distinct groups (homologous series), which are differentiated by the length of their unbranched alkyl chains, and have recently gained attention in a variety of biological activity studies. Using the established HPLC/ESI(±)-MS/MS method, a total of 31 gingerol-related compounds, including three new compounds, were identified from the methanolic crude extract of fresh ginger rhizomes [15]. Diarylheptanoids have been found to possess antioxidant, antihepatotoxic, anti-inflammatory, antiproliferative, antiemetic, chemopreventive, and antitumor activities, which lead to an increasing interest in the recent years. In all, the diarylheptanoids comprise five distinct groups (homologous series), which are differentiated by structural differences on the heptane skeletons, whereas homologs within each group differed by substitution patterns on the aromatic rings. Characteristic fragmentation behavior in (+)- and (−)ESI-MS/MS analyses for each group of homologs, as well as information regarding polarity obtained from retention time data, allowed the identification of 26 diarylheptanoids in the crude methanolic extract from fresh ginger rhizomes [16]. Fifteen of them were reported for the first time and 18 of them were acylated, which was found to be different from the diarylheptanoids of turmeric, another member of the Zingiberaceae. 2.1.8. Propolis (Fengjiao) Propolis, also called “bee glue,” is a resinous substance that bees use to construct and maintain their hives and has exhibited a variety of interesting antimicrobial and antitumor properties in laboratory

2050

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

tests. Propolis is a traditional remedy in folk medicine and usually contains a variety of different chemical compounds. Phenolic acids and flavonoids are the two classes of principal constituents. The propolis samples from different geographical areas were investigated by Gardana et al. [17]. HPLC/DAD-MS/MS in the negative ion mode provides an effective fingerprinting method for the screening of different propolis samples. By comparing the chromatographic and UV behavior with that of standards, most of the compounds could be identified. Moreover, tandem mass spectrometry provided by a triple quadrupole mass spectrometer with collision-induced dissociation (CID) allows structural identification, especially when standard compounds are not available. A total of 60 compounds were characterized in different samples. The results showed that propolis from Argentina and Europe (Italy) was comparable, and differed markedly from most Brazilian propolis. 2.1.9. Kushen: roots of Sophora flavescens Kushen is the Chinese name for the dried roots of Sophora flavescens Ait. (Leguminosae). It is a well-known traditional Chinese medicine widely spread in China, Japan and Korea. It has been used as an antipyretic, diuretic, antihelmintic and atomachic for the treatment of diarrhea, gastrointestinal hemorrhage and eczema. The principal phytochemical constituents were quinolizidine alkaloids and prenylated flavonoids. Zhang et al. [18] investigated the flavonoid compounds and developed a HPLC/DAD/ESI-MS/MS method using an IT mass spectrometer in positive ion mode. A total of 24 flavonoids were identified. Fourteen of them were unambiguously identified by comparing experimental data for retention time (tR ), UV and MS spectra with those of the authentic compounds, which are 3 ,7-dihydroxy-4 -methoxy-isoflavone, trifolirhizin, kurarinol, formononetin, 7,4 -dihydroxy-5-methoxy-8-( , -dimethylallyl)flavanone, maackiain, isoxanthohumol, kuraridine, kuraridinol, sophoraflavanone G, xanthohumol, isokurarinone, kurarinone and kushenol D, and additional 10 compounds were tentatively identified as kushenol O, trifolirhizin-6 -malonate, sophoraisoflavanone A, norkurarinol/kosamol Q, kushenol I/N, kushenol C, 2 -methoxykurarinone, kosamol R, kushecarpin A and kushenol, respectively. The study provided a feasible approach for the rapid characterization of flavonoids in the roots of S. flavescens. 2.1.10. Dandelion (Pugongying): whole plant of Taraxacum officinale Dandelion (Taraxacum officinale WEB. ex. WIGG., Cichoriaceae), a common plant in the northern hemisphere, has long been used for its choleretic, diuretic, antirheumatic, anti-inflammatory, laxative, and appetite-stimulating properties for treating liver and gallbladder disorders, digestive complaints, and arthritic and rheumatic diseases. In the present literature [19], a HPLC/DAD/ESIMSn method was used for the characterization of phenolic acids and flavonoids in the extracts from dandelion (T. officinale) root and herb juice. An IT instrument was used as the analyzer. A total of 26 compounds, including five mono- and dicaffeoylquinic acids, five tartaric acid derivatives, eight flavone and eight flavonol glycosides were characterized based on their UV spectra and their fragmentation patterns in collision-induced dissociation experiments. The predominant compound in this herb was found to be chicoric acid (dicaffeoyltartaric acid). 2.1.11. Jiangxiang: heartwood of Dalbergia odorifera Jiangxiang, the heartwood of Dalbergia odorifera T. Chen. (Leguminosae), is a traditional Chinese medicine, which has been used to treat blood disorders, ischemia, swelling, necrosis, and rheumatic pain. It is a main ingredient in a number of formulae such as GuanXin-Er-Hao decoction, Xiangdan injection, and Xinning tablet. As

a widely used medicinal herb to treat cardiovascular diseases, D. odorifera is known to be rich in flavonoids, which were reported to have anti-inflammatory, anticoagulant, antitumor, vasodilative, antihyperlipidic and antioxidant activities. A method combining HPLC with ESI-MS/MS in negative ion mode was developed for the qualitative characterization of flavonoids in D. odorifera [20]. By comparing their retention times, UV and MS spectra with those of authentic compounds, twenty-three flavonoids including six isoflavones, six neoflavones, four isoflavanones, three flavanones, two chalcones, one isoflavanonol and one pterocarpan were unambiguously identified. The study provided a simple, reliable and sensitive method for phytochemical screening of flavonoids in D. odorifera. But this study did not take full advantage of the large numbers of reference compounds (23 compounds) and the MSn (‘n’ can be greater than 2) spectra of IT mass spectrometer. Only the peaks of authentic compounds were assigned and no other unknown structures were deduced. Hence, more research is necessary. 2.1.12. Artichoke: Cynara scolymus The leaves of Artichoke (Cynara scolymus L.) are higher in medicinal value than flowers, with antihepatotoxic, choleretic, diuretic, hypocholesterolemic and antilipidemic properties that are attributed to the phenolic components. A fast and efficient method HPLC/ESI-MS in tandem mode with negative ion detection was developed and validated for the qualitative analysis of artichoke waste [21]. Forty-five phenolic compounds were identified on the basis of their mass spectra in full scan mode, mass spectra in different MS/MS modes, and retention times compared with those of available reference standards. The major compounds were found to be both caffeoylquinic and dicaffeoylquinic acids, luteolin glucuronide, luteolin galactoside, quercetin, and some quercetin glycosides. 2.1.13. Honghuayanhuangqi: roots of Hedysarum multijugum Hedysarum multijugum Maxim. is a medicinal plant of the family Leguminosae, which has been used in folk herbal medicine in China and was recorded in many Chinese herbal books for the treatment of palpitation and chronic nephritis. Phytochemical studies showed that the plant mainly consisted of pterocarpenes, coumestans, benzofurans and isoflavones. However, more phenolic compounds were/could not be isolated and identified from the herb. Therefore, the fragmentation behavior of four types of the phenolic compounds was studied using ESI-MSn (n = 2–5) in negative ion mode by Yang et al. [22]. The obtained fragmentation rules were applied in the identification of constituents in methanolic extract of H. multijugum by HPLC/DAD/ESI-MSn method. A total of 29 compounds were characterized and nine of them were reported for the first time. In this study, it was found that the number of methoxy groups influenced the competitive loss of CO (28 Da) and CO2 (44 Da), which would be very useful for the identification of flavonoids and should be confirmed by studying more reference standards. 2.1.14. Tusizi: seeds of Cuscuta chinensis Tusizi, prepared from the seeds of Cuscuta chinensis Lam. (dodder, Convolvulaceae family), is used as a tonic in Chinese medicine and has been shown to be able to improve sexual function, regulate the body’s endocrine and immune system, and to prevent senescence. The chemical constituents mainly comprise flavonols like quercetin, kaempferol and their glycosides. These compounds may be responsible for the biological activities of this drug. However, seeds of C. australis R.Br. are also offered under the name of this medicine in the herbal market. In order to make a comparison of their chemical constituents, the phenolic compounds of these two Cuscuta species were analyzed by HPLC/DAD/ESI-MSn [23]. A total of 50 compounds were observed in the methanol

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

2051

Fig. 2. HPLC-UV chromatograms (360 nm) of the methanolic extract of seeds of Cuscuta australis (a) and Cuscuta chinensis (b). From Ref. [23] with permission.

extract, including 23 flavonoids, 20 lignans and 7 quinic acid derivatives. The phenolic patterns of these two Cuscuta species were found to be very different (Fig. 2). Kaempferol and astragalin were the predominant constituents of C. australis, while hyperoside was the major compound in C. chinensis. The large differences observed between the phenolic constituents of C. chinensis and C. australis strongly encouraged further comparison of the bioactivities of these two species. 2.1.15. Others Citrus species (Zhiqiao and Zhishi in TCM) [24–26], Dendrobium species (Shihu in TCM) [27–29], Hypericum species (Guanyelianqiao in TCM) [30–33], Rhodiola rosea L. [34], Peucedanum palustre L., Angelica archangelica (L.) var. archangelica [35] and Pueraria lobata Ohwi (Gegen) [36] were all proved to contain phenolic compounds by HPLC–MS. 2.2. Saponins Saponins are glycosides of triterpenes and steroids, and commonly occur in higher plants. Saponins exhibit a variety of biological activities and are widely used in foods, medicines and cosmetics. The sugars found in saponins are hexoses (glucose, galactose), 6-deoxyhexoses (rhamnose), pentoses (arabinose, xylose), uronic acids (glucuronic acid) or amino sugars (glucosamine). Sugars may be linked to the sapogenin at one or multiple glycosylation sites. Saponins response only at a short wavelength (200–210 nm) using an UV detector, therefore, the MS spectrometer is preferable and reliable to be a detector in the chromatographic analysis. 2.2.1. Ginseng: root and rhizome of Panax spp. When saponins from the herbal medicines were discussed, we have to bring to mind the ginsenosides firstly. Ginseng root has been used traditionally in Chinese medicine for over 2000 years and is now one of the most commonly found Chinese herbs used worldwide. Pharmacological studies indicated that ginseng and its constituents have multifold bioactivities including antimitogenic effect, improving impaired memory and inhibition of tumor cell growth. The pharmacological properties of Ginseng are generally attributed to its triterpene glycosides, called ginsenosides. Ginsenosides are mainly dammarane triterpenes with (20S)-protopanaxadiol and (20S)-protopanaxatriol aglycone moieties. The only oleanolic acid-type saponin identified in the roots of P. ginseng C. A. Mey. is ginsenoside Ro.

Fuzzati et al. [37] developed a HPLC/ESI-MS method in negative ion mode for the systematic analysis of ginsenosides in P. ginseng roots. Ion trap analyzer was used to produce full mass and MS/MS spectral for each peak detected from P. ginseng. A total of 25 ginsenosides were separated and characterized. Malonic esters of ginsenosides have been isolated from P. ginseng and these may be among the major ginsenosides present in ginseng root. Malonyl-ginsenosides are unstable and are readily de-malonylated upon heating. The acidic malonyl-ginsenosides are more difficult to analyze directly by HPLC than their neutral counterparts. Consequently, a single HPLC–MS analysis method, coupled with automatic MS/MS scanning and post-acquisition neutral loss data analysis, was established for profiling of the malonylated and acetylated ginsenosides in ginseng extract [38]. Comparative investigation showed that the profiles of malonyl-ginsenosides appear to be different in P. quinquefolius L. (American), P. ginseng (Asian) and P. notoginseng (Burk.) F. H. Chen (Sanchi ginseng). The results could provide useful additional information for the authentication of these different ginseng species. The relative content of malonylated ginsenosides is reduced in the red form of Asian ginseng compared with the white form and there is a concomitant increase in the levels of the corresponding acetylated ginsenosides. Leung et al. [39] tried to differentiate three ginseng species using a HPLC/APCI-MS/MS method in negative ion mode. Notoginsenoside R1 is observed in both P. notoginseng and Chinese ginseng, while pseudoginsenoside F11 is found exclusively in the American species. The results permit the definitive identification of the species. 2.2.2. Sanqi: root and rhizome of Panax notoginseng Panax notoginseng (Burk.) F. H. Chen, named Sanqi in Chinese, is a highly valued and important Chinese medicinal herb, belonging to the same genus as Chinese and Korean ginseng (Panax ginseng) and American ginseng (Panax quinquefolium) and the saponin profiles of them are very similar. However, the function of Sanqi is different from that of Ginseng in tradition Chinese medicine. Quality control of this herb is hence paramount. A HPLC/ESI-MS method was developed for the analyses and identification of saponins in plant extract from Sanqi [40]. An IT mass spectrometer with an electrospray ion source was used for detection of the ions. A CID experiment was carried out to produce fragment ions. Eight saponins in the crude extract of P. notoginseng have been identified. Furthermore, the raw form of Sanqi is traditionally used in Chinese medicine for its haemostatic and cardiovascular properties; the steamed form is used as a tonic to ‘nourish’ blood and to increase production of various blood cells in anemic conditions.

2052

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

Due to their contrasting pharmacological actions and clinical indications, the authentic identification of the forms is very important. Chan et al. [41] presented an ultra-performance liquid chromatography (UPLC)/TOF-MS method for direct detection of down-stream derivatives of metabolites, arising from the herbal formulation process. The UPLC, which can provide perfect chromatogram with high resolution and high retention time reproducibility in 11 min, along with the accurate mass measurement by TOF is significantly complementary for the tentative identification of the biomarkers. Using the marker ion selection criteria of ‘present in steamed and absent in raw samples (vice versa)’ and ‘higher abundance in steamed and lower in raw samples (vice versa)’, 74 and 146 marker ions were subsequently obtained for the raw and steamed samples, respectively. The result confirmed that the steaming process led to the changes in the levels and occurrence of ginsenosides. 2.2.3. Ciwujia: roots of Acanthopanax senticosus Acanthopanax senticosus Harms (Ciwujia in Chinese) is a typical Chinese herb, its root is an important Chinese folk medicine for the treatment of ischemic heart diseases, hypertension, theumatic and tumor, etc. The principal bioactive constituents of Ciwujia are saponins. Guo et al. investigated the saponins in crude extract from leaves of A. senticosus by electrospray ionization multistage tandem mass spectrometry (IT) and high-resolution mass spectrometry (TOF) [42]. Eighteen compounds were identified from the saponin mixtures. The authors only used ESI-MSn in combination with structural correlations existing in the plant to characterize the structures of underivatized saponins from medicinal herbs. If HPLC technique was introduced, the established method would be better. 2.2.4. Huashanfan: roots and leaves of Symplocos chinensis Symplocos chinensis (Lour.) Druce (Huashanfan in Chinese) is a toxic herb distributed in southern China and has been widely used as a folk medicine to treat several diseases. Some new triterpenoid saponins isolated and characterized from this plant were found to have significant cytotoxic activities. Therefore, a HPLC–MSn method was developed to rapidly identify and characterize triterpenoid saponins in this herb [43]. A total of 14 constituents in the crude extract were structurally characterized on the basis of their retention time and tandem mass spectrometric analysis. There are no less than five pairs of isomers detected by HPLC–MSn study and three pairs of those were differentiated. The authors indicated that the established analytical method still has some limitations in differentiating compounds with geometrically isomeric substituting acyl groups and in locating them on the aglycones. 2.2.5. Huangqi: roots of Astragalus spp. Radix Astragali, called Huangqi in Chinese, is the dried roots of Astragalus mongholicus Bge. or A. membranceus (Fisch.) Bge. It is traditionally used as an antiperspirant, a diuretic or a tonic. The Polysaccharides, triterpene saponins (astragalosides and soyasaponins), flavonoids and -aminobutyric acid were reported to be the major active components. The saponins isolated from Astragalus spp. could protect the liver of mice from chemical injury induced by carbon tetrachloride, d-galactosamine and acetaminophen. Astragalosides usually have a 9, 19-cyclolanostane cycloastragenol as the aglycone except for astragaloside VIII, which possesses an oleanene-type soyasapogenol B as the aglycone. Xu et al. investigated the analysis of saponins in the roots of Astragalus spp. and developed a HPLC/APCI-MS for on-line characterization [44]. Triple quadrupole mass spectrometer was used in negative ion mode. Twelve astragalosides in the extract of Radix Astragali obtained from Shandong in China were separated and identified. Obviously, more effort should be made to differentiate the isomers. The estab-

lished method could also be used to distinguish Astragalus spp. from different habitat regions. 2.2.6. Cynanchum: Radix Cynanchi atrati (Baiwei) and Cynanchum chekiangense (Manjiancao) Radix Cynanchi atrati, called Baiwei in Chinese, is traditionally used as an antifebrile and diuretic in Chinese orthodox medicine. Cynanchum versicolor Bge. and C. atratum Bge. are listed as sources of Baiwei in Chinese Pharmacopoeia (Committee for the Pharmacopoeia of China, 2005). C21 steroids and their saponins are reported to be the main components in C. versicolor (Manshengbaiwei) and considered to be the bioactive components with cytotoxic, antimicrobial, herbicidal and anti-inflammatory activities. The C21 steroidal saponins were studied in positive and negative ESI-MSn . The obtained fragmentation rules were applied in the HPLC/ESIMSn approach for the identification of the saponins in 90% ethanolic extract from the root and rhizome of C. versicolor by Zheng et al. [45]. Nine C21 steroidal saponins and an isomer of atratoglaucoside A were detected and elucidated simultaneously. It was obvious that the principal peaks in the chromatogram were mostly characterized. Cynanchum chekiangense M. Cheng ex Tsiang et PT Li. (Asclepiadaceae) belongs to the same genus as Baiwei and is distributed wildely in southern China. The roots of the plant have been used for the treatment of rheumatoid arthritis and rheumatic aches in Chinese folk medicine. The CHCl3 soluble extract of the roots, comprising C21 steroidal glycosides, was reported to possess immunosuppressive. Thus, it is necessary to identify their chemical structures to the further investigation of pharmaceutical activity and quality control of the medicine. HPLC/ESI-MS/MS was used by Tai et al. for on-line characterization of the C21 steroidal glycosides with immunological activities in roots of C. chekiangense [46]. In the MS/MS spectra, fragmentations of the [M+Na]+ were recorded to provide structural information about the glycosyl and aglycone moieties. Four known steroidal glycosides cynascyroside C, chekiangensosides A and B, glaucoside H, and four new steroidal glycosides chekiangensosides C, D, E and chekiangensoside A isomer were identified based on mass spectral data. The results were confirmed by off-line FTICR-MS/MS and NMR spectral data. 2.2.7. Chuanshanlong: rhizomes of Dioscorea nipponica The dried rhizome of Dioscorea nipponica Makino, called Chuanshanlong in Chinese, is the raw material of Di-ao-xin-xue-kang, which has been used to cure coronary heart disease for more than 10 years in China. Steroidal saponins are the principal bioactive components. A HPLC/ESI-MSn method was developed for the analyses and characterization of steroidal saponins in plant extract from the rhizomes of D. nipponica [47]. Twelve saponins were detected and three of them were characterized as pseudoprotodioscin, methyl protodioscin and dioscin. The authors suggested that further investigation should be conducted to characterize the peaks mostly. 2.3. Alkaloids Alkaloids are usually described as the cyclic compounds that contain negative valent nitrogen atoms and they exist in organism. Over 10,000 alkaloids were isolated from nature so far and distributed widely in the plants. Some important plant medicines including opium, Mahuang (Herba Ephedrae), cinchona, Maqianzi (Strychnos nux-vomica L.), Hanfangji (Stephania tetrandra S. Moore), Langdang (Hyoscyamus niger L.), Yanhusuo (Corydalis yanhusuo W. T. Wang), Kushen (Sophora flavescens Ait.), Yangjinhua (Datura metel L.), Qiushuixian (Colchicum autumnale L.), Changchunhua

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

2053

(Catharanthus roseus (L.) G. Don), Sanjianshan (Cephalotaxus fortune Hook.f.), Fuzi (Aconitum carmichaeli Debx) are rich in alkaloids. 2.3.1. Aconite: Aconitum sinomontanum, Aconitum kusnezoffii, Aconitum carmichaeli The plants of Aconitum genus are widely distributed across the Northern Asia and Northern America and have been found to comprise abundant natural alkaloids. The roots of Aconitum, the so-called Fuzi (Radix Aconiti laterals praeparata.), Chuanwu (Radix Aconiti) and Caowu (Radix Aconiti kusnezoffii) have been widely used for analgesic, cardiotonic and antirheumatism treatment in traditional Chinese medicine. Aconitum alkaloids have analgesic, anti-inflammatory and cardiotonic activities and can be divided into four groups according to the structure of their skeleton: i.e., aconitines, lycoctonines, atisines and veatchines. The rapid and sensitive analysis of alkaloids in Aconitum plants will be of advantage to the pharmaceutical studies and quality control of the herbal medicines and their preparations. The group of Shuying Liu has performed a series of research on the analysis of alkaloids in Aconitum species [48–51]. Aconitine and its analogues are the main active components of A. kusnezoffii Reichb. Wang et al. found that the ESI-MSn spectra could provide a simple method for the direct analysis of alkaloid mixtures, by which about 70 alkaloids (aconitines, lipo-aconitines and triester lipo-aconitines) were detected in the flowers of A. kusnezoffii [48]. Xu et al. performed the analysis of alkaloinds in the roots of A. sinomantanurn Nakai using the ESI-MSn technique. Structures of six known norditerpenoid alkaloids were simultaneously determined [49]. The herbal medicines are always used as decoctions, which readily lead to the change of the constituents compared with the crude drugs. Wang et al. [50] checked the diversified aconitinescontaining samples, including crude aconite, decoction of crude aconite, residues from decoction of crude aconite, prepared aconite, decoction of prepared aconite, decoction of prepared aconite with added palmitic acid, and decoction of a mixture of mesaconitine and hypaconitine standards with liquorice root, and found that diester-diterpenoid aconitines (DDA) can be converted into lipoalkaloids other than monoester-diterpenoid aconitines (MDA) in the process of decocting aconite. Furthermore, the stability of DDA in different solutions and pH buffers was studied by HPLC/ESI-MS [51]. In different solvents, the decomposition pathways of DDA are quite different and their difference in stabilities depends on the substituents at the N atom and substituents at C-3. The decomposition pathways of DDA in buffers are related to the substituent on the C-3 position. The decomposition pathway of aconitine is similar to that of mesaconitine, but different from that of hypaconitine. Wu et al. introduced matrix-assisted laser desorption ionization (MALDI)-TOF-MS technique for direct analysis of alkaloids in A. carmichaeli Debx. (Fuzi in Chinese) along with C. yanhusuo W.T. Wang (Yanhusuo in Chinese) and Coptis chinensis Franch. (Huanglian in Chinese) [52]. MALDI-TOF-MS proved valuable for the preliminary study of plant component profiles. The rapid collection of information from the direct analysis on plant tissues could be valuable for supporting the discovery of new compounds and for the quality control of medicinal herbs. 2.3.2. Liangmianzhen: roots of Zanthoxylum nitidium The root of Zanthoxylum nitidum (Roxb.) DC., locally called Liangmianzhen, is one of the traditional Chinese medicines widely distributed throughout the southeastern part of China. It has been used for removing rheumatoid arthralgia, resolutiving turgescence and controlling pain, and so on. Pharmacological studies indicated that Z. nitidum has antitumor, antibacterial and relieving pain prop-

erties. The extract of Z. nitidium has been added to toothpaste in China due to its strong antibacterial activity. Alkaloids, including benzo[c]phenanthridine alkaloids, protopine alkaloids, aporphine alkaloids and quinoline alkaloids, are considered to be mainly responsible 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 nine alkaloids in Z. nitidium, but no structural characterization has been discussed. Cai et al. [54] studied the fragmentation behavior and the corresponding fragmentation decomposition mechanisms of six benzo[c]phenanthridine alkaloids, dihydrochelerythrin, dihydronitidine, 8-acetonyldihydrochelerythrine, 8-acetonyldihydronitidine, nitidine and 1,3-bis(8-dihydronitidinyl)-acetone were studied in detail by positive ion ESI-MSn . Furthermore, the crude alkaloid extract from the roots of Z. nitidium was rapidly analyzed by HPLC–MSn , and ten constituents were identified by comparing the retention times and ESI-MSn spectra with the authentic standards. The authors suggested that not only the characteristic fragments but also the characteristic abundances of the fragment ions can be used for the detailed structural characterization. 2.3.3. Baibu: root tuber of Stemona tuberosa The root tubers of Stemona tuberosa Lour., named Baibu in Chinese, belonging to family Stemonaceae, are widespread throughout the tropical Asia and have long been used in Chinese and Japanese traditional medicine for the treatment of respiratory diseases such as bronchitis, pertussis and tuberculosis, and against enteric helminthes and ectoparasites in humans and cattle. Members of the Stemonaceae family contain interesting polycyclic alkaloids with an unusual molecular architecture bearing a pyrrolo or a pyrido[1,2-a]azepine nucleus. These alkaloids are unique to this family and are called Stemona alkaloids. Some of these alkaloids have been found to have a variety of biological activities. The alkaloids profiles of the herb were found to be variable, which demonstrated that a careful chemical authentication of S. tuberosa is needed to assure the consistency, safety and efficacy in pharmaceutical applications. An HPLC–MS chromatogram fingerprint was developed for selective and reliable authentication of this species [55]. Six different samples of S. tuberosa and three different species of Stemona (S. tuberose, S. sessilifolia (Miq.) Miq. and S. japonica (Bl.) Miq.) were investigated comparatively. The HPLC–MS fingerprints of these alkaloids, though variable among samples, were proved to be useful for the authenticity and quality of this species and help to differentiate it from S. japonica and S. sessilifolia. 2.3.4. Lilu: root and rhizome of Veratrum nigrum Lilu, the roots and rhizomes of several Veratrum species, has been used to treat aphasia arising from apoplexy, wind-type dysentery, jaundice, scabies and chronic malaria for centuries in China. Nevertheless, Veratrum nigrum L. is a very poisonous plant. The steroidal alkaloids isolated from this plant were reported to exert teratogenic effects in several laboratory animals. Therefore, a rapid and reliable method for characterization of the alkaloids in this plant is necessary to ensure the safety of the medicine. Using the established HPLC/ESI-MSn method, different cevaninetype alkaloids in herbal extract were detected and identified simultaneously [56]. A total of 21 steroidal alkaloids (5 protoverinetype alkaloids, 14 germinetype alkaloids and 2 zygadenin-type alkaloids) were selectively identified from 27 detected peaks. However, the authors suggested that further studies should be performed to obtain more detailed structural information on steroidal alkaloids.

2054

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

2.3.5. Others Cui et al. studied the phenanthroindolizidine alkaloids in Sanfendan (Tylophora atrofolliculata Metc.) with HPLC/ESI(±)-MSn technique [57]. Nine compounds were characterized in the crude extract. In a reported study, the variation in the profile of imidazole alkaloids in different seasons and in different parts of the Pilocarpus microphyllus Stapf plant during the summer was analyzed by ESI(+)MS [58]. MSn (n = 2–3, IT) and high-resolution mass spectrometry (TOF) were used for the structural determination. 2.4. Monoterpene glycosides Monoterpenoids are constructed from two units of isoprene, containing ten carbon atoms. Iridoids are acetal metabolites of iridoidial. The structure of iridoids contains the units of cyclopentane. The chemical feature of iridoids is somewhat like that of monoterpenes. To our knowledge, the most famous monoterpene glycoside is paeoniflorin. However, most of the iridoids were found to exist as glycosides in the plants. 2.4.1. Shaoyao: dried roots of Paeonia lactiflora The root of Paeoniae lactiflora Pall. is a common Chinese herb drug that is widely used in both Japan and China for the treatment of a variety of diseases, including cleansing heat, cooling blood, invigorating blood circulation, alleviating pain, regulating menstruation, treating liver disease and cancer. The major constituents reported from P. lactiflora Pall. were monoterpene glycosides such as paeoniflorin, albiflorin, oxypaeoniflorin, benzoylpaeoniflorin and galloylpaeoniflorin. Dong et al. reported a HPLC/ESI(−)-MS/MS method for the analysis of constituents in P. lactiflora Pall [59]. IT and FTICR mass spectrometer were used in the study. Five compounds were characterized in the crude extract. The application of ESI-FTICR-MS/MS is a highlight in this paper. But it is a pity that this technique was not applied in the on-line study and only five compounds were characterized. More available data from authentic standards are needed for the characterization of more unknown constituents of the herbal medicine. 2.4.2. Zhizi: fruits of Gardenia jasminoides The fruit of Gardenia jasminoides Ellis (named Zhizi in Chinese) is a widely used traditional Chinese medicine for treatment of many diseases, such as hepatitis, inner fever, hypertension, and diabetes. Zhizi contains a large amount of iridoid glycosides such as geniposide, gardenoside, genipin gentibioside, gardoside, shanzhiside, geniposidic acid, scandoside methyl ester and methyl deacetyl asperulosidate. Ren et al. developed a HPLC–MSn method to analyze the crude extract of G. jasminoides fruit [60]. ESI(−)-IT mass spectrometer was used to provide full scan and MSn spectra. Five constituents were characterized. Obviously, further studies were needed to comprehensively profile the iridoid glycosides of Zhizi. 2.4.3. Jishiteng: whole plant of Paederia scandens The whole plant of Paederia scandens (Lour) Merrill has long been used as a Chinese traditional medicine for the treatment of toothache, chest pain, piles, and inflammation of the spleen, diuretic, emetic, rheumatic arthritis and curing bacillary dysentery. P. scandens produces sulfur-containing iridoid glucosides, in which paederoside exhibited an inhibitory effect on Epstein-Barr virus activation. Zhou et al. studied the iridoid glucosides of Jishiteng by HPLC/ESI(+)-MSn technique [61]. IT-MS and Q-TOF-MS were used in the on-line analysis. Five sulfur-containing iridoid glucosides were identified in the crude extract.

2.4.4. Others An HPLC/ESI-MS analysis was performed to determine the iridoid composition of Lamium album L., Lamium amplexicaule L., Lamium garganicum L., Lamium maculatum L., and Lamium purpureum L. by Alipieva et al. The study showed that the HPLC/ESI-MS could be used as a quick method for species authentication or to assess the chemodiversity and phylogeny of the genus [62]. 2.5. Diterpenoids The most famous diterpenoid in the last century is the taxol as an antitumor medicine found from a plant. Ye and Guo investigated the fragmentation behaviors of taxoids by ESI- and APCI-MSn techniques [63]. The results could facilitate the rapid screening and structural characterization of taxoids in plant extract by HPLC–MS. Tanshinones and ginkgolides as well as bilobalide are the active members in diterpenoids, too. Ginkgo biloba Linne (Yinxing in Chinese) is one of the oldest living tree species. In China, medicinal uses of Ginkgo were first described in Ben Cao Gang Mu by Li Shizhen in 1596. The fruits (Baiguo in Chinese) were used for the treatment of cough, asthma, enuresis, alcohol misuse and pyogenic infection of skin. Recently, Ginkgo leaf extracts have become one of the top selling phytopharmaceuticals in the US and Europe for mental alertness, enhanced vitality level, circulatory health and blood vessel health. The terpene trilactones are one of the major active components of Ginkgo leaf extract. Some analytical methods using HPLC–MS techniques were developed for the quantitative determination of major active components in G. biloba [64–66]. 2.5.1. Danshen: roots of S. miltiorrhiza Tanshinones, possessing the skeleton of abietane, is one class of the active components present in the roots of Danshen (S. miltiorrhiza). Tanshinones are reported to have pharmacological actions of anti-inflammation, dilating coronary artery and increasing coronary flow, antioxidation, cytotoxic activity and modulating effect on mutagenic activity. We reported the HPLC–MSn investigation for separation and characterization of tanshinones in the methanol–chloroform (7:3) extract from the root of Danshen (Fig. 3) [67]. Ion trap spectrometer was used as the mass analyzer. The major fragmentation behaviors of the compounds were the losses of H2 O, CO, propylene and CH3 . A total of 27 tanshinones were identified or tentatively characterized, including five new constituents. Tanshinones consist of both ortho- and para-quinone structures. Studies on para-quinone compounds are lacking in this paper. Zhu et al. developed a simple method to separate and characterize tanshinones and phenolic compounds simultaneously in one chromatogram [5] (see Section 2.1.1). 2.6. Triterpenoids (aglycone) Triterpenoids are derived from squalene in biogenesis and contain 30 carbon atoms. Many plant families such as Araliaceae, Leguminosae, Campanulaceae and Scrpophulariaceae contained abundant triterpenoids. In addition, some animals and fungi are also the sources of triterpenoids. A number of active triterpenoids (e.g. meliacane, quassinane, bruceantin, oleanolic acid, glycyrrhizic acid and ganoderic acids) were found from TCMs. HPLC–MS analysis of toosendanin in Melia toosendan Sieb. et Zucc. (Chuanlian in Chinese) [68], azadirachtin and related triterpenoids in Azadirachta indica A. Juss (Yinglian, in Chinese) [69], oleanolic acid and ursolic acid in Anoectochilus roxburghii (wall.) Lindl (Jinxianlan in Chinese) [70] was reported recently.

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

2055

Fig. 3. HPLC/DAD/ESI-MSn analysis of the chloroform–methanol (3:7) extract of Dan-shen crude drug. (a) HPLC-UV chromatogram monitored at 270 nm and (b) ESI-MS total ion current profile of Dan-shen. From Ref. [67] with permission.

2.6.1. Lingzhi: fruit body of Ganoderma lucidum Triterpenic acids were found to be the most important active components in Ganoderma lucidum (Leyss. ex Fr.) Karst. (Lingzhi in Chinese), which has long been used as a folk remedy for the promotion of health and longevity in China and other oriental countries. It has prominent immunomodulatory and antitumor activities. Some of the triterpenoids isolated from Lingzhi showed antiandrogenic, antihepatitis B, antioxidant, antitumor, anticomplement, antimicrobial, anti-HIV-1, selectively inhibit eukaryotic DNA polymerase, and angiotensin converting enzyme-inhibitory activities. Thus, we developed a valuable and convincing method using HPLC/ESI(−)ITMSn technique for the characterization of triterpenoids of G. lucidum for quality control of the herb (Fig. 4) [71]. Interestingly, it was found that some characteristic signals were not always the abundant peaks in MSn spectra (Fig. 5). When the method was applied, a total of 32 triterpenoids, including six new compounds,

were identified or tentatively characterized from the chloroform extract of G. lucidum (Fig. 6). Most of the reference standards are compounds of type A (Fig. 6). Hence, more type A constituents could be characterized. Fortunately, the main components in chloroform extract of the studied G. lucidum are the triterpenic acids of type A, which make most of the chromatographic peaks assigned. But some Ganoderma species mainly contain other types of triterpenic acids. Obviously, further research should be carried out for the comparison of species differences and sufficient reference standards are very important in such an investigation. 2.7. Steroids (aglycone) Steroid is one of the familiar components occurring naturally in plants and animals, possessing a nuclear of cyclopentanoperhydrophenanthrene. Sterols, bile acid, steroid hormones, cardiac

Fig. 4. HPLC/DAD/ESI-MSn analysis of the CHCl3 extract of Ganoderma lucidum. (a) HPLC-UV chromatogram monitored at 252 nm. (b) HPLC-negative ion ESI-MS total ion current (TIC) profile. From Ref. [71] with permission.

2056

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

Fig. 5. ESI-MS spectra and major fragmentation pathways for ganoderic acids, the indicated peaks are characteristic ions: MS3 spectra for ion at m/z 451 of ganoderic acid AM1 (a) and ganoderic acid J (b); MS/MS spectrum at m/z 497 (c) and MS3 spectrum at m/z 453 (d) of ganoderic acid B. From Ref. [71] with permission.

glycosides, toad poison, steroidal saponins are some important steroids. 2.7.1. Chansu: skin secretions of giant toads ChanSu, prepared from the skin secretions of giant toads, also called toad venom or toad poison, is one of the major components of many well-known patent TCM drugs like Liu-Shen-Wan and SheXiang-Bao-Xin-Wan, which is frequently used in clinics to treat heart failure, sores, pains, and various cancers. The major effective constituents of ChanSu are bufadienolides, a type of steroids with a characteristic a-pyrone ring at C-17, and show significant cardiotonic, blood-pressure-stimulating, anesthetic, and antitumor activities. In the past several years, we have performed a comprehensive investigation of this important Chinese medicine. Ye and Guo achieved the rapid and accurate identification of the steroids in Chansu [72], which is of great significance in the quality control of this natural drug and its formulations. HPLC/DAD/APCI(+)-MS method was developed for the analysis. IT mass spectrometer was used to provide full scan and multistage mass spectra (MSn ). A total of 35 bufadienolides were identified, including four new constituents. 2.8. Others and miscellaneous natural products Many other types of natural products exist in herbal medicines. Furthermore, herbal medicines usually do not contain only one single type of compounds. Simultaneous analysis of miscellaneous constituents is very important in the investigation of traditional medicines. This can also be preformed by HPLC–MS technique, in which ionization of different types of compounds under the same condition was the difficulty. Some cases on the characterization of farraginous compounds are shown below.

2.8.1. Mudanpi (Cortex Moutan): root bark of Paeonia suffruticosa Cortex Moutan, called Mudanpi in Chinese, is the root bark of Paeonia suffruticosa (Paeoniaceae) and known traditionally as an analgesic, a sedative, an anti-inflammatory agent, and remedy for female disorders in China. Monoterpenes, acetophenones and galloyl glucoses are found to be the main constituents and thought to be responsible for the biological activities of this crude drug. Recently, the root cortices of P. delavayi Franch. and P. decomposita Hand.-Mazz. are found to be used in the southwest provinces of China as Cortex Moutan. In order to evaluate these three species of Paeonia, a HPLC/DAD/ESI(−)-MS method was established to analyze the multiple constituents in the herbal plant [73]. Q-TOF-MS was applied for the detection. Full scan MS and MS/MS spectra were provided in the negative ion mode to investigate the fragmentation behavior of the compounds. From the root cortices of the three plants, a total of 50 compounds, including 17 monoterpenes, 14 galloyl glucoses, 10 acetophenones, 5 phenolic acids, 3 flavonoids and 1 triterpene, were characterized based on their retention behavior, UV spectra and MS fragmentation patterns. Great differences in the chemical constituents among the three species were found in the comparative analysis. Paeonol was found to be the predominant constituent of P. suffruticosa and P. decomposita, while P. delavayi contained albiflorin and more galloyl glucoses than the other two species. The authors suggested that further comparison of the bioactivities of these three species should be carried out considering the significant differences in their chemical constituents. 2.8.2. Jinguolan: roots of Tinospora sagittata and Tinospora capillipes Jinguolan, prepared from the roots of Tinospora sagittata (Oliv.) Gagnep and T. capillipes Gagnep. (Menispermaceae family), is

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

2057

Fig. 6. Chemical structures of the triterpenoids identified from Ganoderma lucidum. From Ref. [71] with permission.

commonly used in traditional Chinese medicine (TCM) for the treatment of sore throat, laryngitis, gastralgia and diarrhea. Phytochemical studies revealed that they comprised three major groups of compounds including protoberberine and aporphine alkaloids, clerodane-type diterpenes and botanic steroids, which have been reported as bioactive components from this herb. The ethanolic extract of Jinguolan was investigated using modern pharmacological methods and found to possess broader

bioactivities, such as antibiosis, anti-inflammatory and antiviral effects. Zhang et al. studied the constituents in the extract of Jinguolan by HPLC/DAD/ESI(±)-MSn [74]. A total of 24 compounds were characterized. The obtained results suggested the necessity for further comparison of the bioactivities of the two species due to the dramatic difference in the contents of the compounds in the roots of T. sagittata and T. capillipes.

2058

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

Fig. 7. HPLC/DAD/ESI-MSn analysis of the methanolic extract of Shuang-Huang-Lian oral liquid. (A) HPLC-UV chromatogram monitored at 280 nm. (B) HPLC-negative ion ESI-MS total ion current (TIC). From Ref. [81] with permission.

2.8.3. Shengma: rhizome and root of Cimicifuga species The dried rhizomes and roots of Cimicifuga racemosa L. Nutt (Ranunculaceae) (syn. Actaea racemosa L.) are widely used as herbal dietary supplement for the alleviation of menopausal symptoms. The Asian Cimicifuga species, including C. dahurica (Turcz.) Maxim., C. foetida L., and C. heracleifolia Kom., namely Shengma in Chinese, are used for their antipyretic, analgesic and anti-inflammatory properties. He et al. reported a fingerprinting approach based on HPLC–DAD/MS/evaporative light scattering detection (ELSD) for
Table 2 Characterization of compounds in Shuang-Huang-Lian oral liquid by HPLC/DAD/ESI-MS Peak no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Retention time (min) 2.94 5.46 5.95 8.97 9.83 15.37 18.03 18.41 18.68 19.16 22.37 22.93 23.30 25.32 25.73 26.69 29.18 32.86 36.71 39.22 39.81 40.79 41.90 44.73 45.40 45.64 47.97

the identification of a total of 10 Cimicifuga species [75]. These include three North American species, C. racemosa, C. americana, C. rubifolia, and seven Asian species, C. acerina, C. biternat, C. dahurica, C. heracleifolia, C. japonica, C. foetida, and C. simplex. The chemotaxonomic difference of the HPLC fingerprints allows identification of all 10 Cimicifuga species. The triterpene saponins, cimigenol-3-O-arabinoside, cimifugin, and cimifugin3-O-glucoside, were considered to be suitable species-specific markers for the distinction of C. racemosa from the other Cimicifuga species.

[M−H]− m/z 401a 461 353 353 353 639 755 653 623 623 621 515 519 515 579a 515 445 593a 459 459 1457a 1295a 1133a 911 1073 911 283

Identification Caffeic acid glucoside Forsythoside E 3-Caffeoylquinic acid Chlorogenetic acid 4-Caffeoylquinic acid Suspensaside Forsythoside B Suspensaside methyl ether Acteoside Forsythoside A Suspensaside A 3,4-Dicaffeoylquinic acid Pinoresinol-4 -O- -d-glucopyranoside 3,5-Dicaffeoylquinic acid Epipinoresinol-4 -O- -d-glucopyranoside 4,5-Dicaffeoylquinic acid Baicalin 3 ,4 ,5 -Trimethoxyl-4 -hydroxyllignan-O-glucoside Oroxylin A-7-O-glu acid Wogonoside Macranthoidin B Macranthoidin A Dipsacoside B Hederagenin-28-O-ara-rha-glc ester Macranthoside B Macranthoside A Wogonin

From Ref. [81] with permission. Glc: glucose; ara: arabinose; rha: rhamnose; glu acid: glucuronic acid. a [M+CH3 COO]− ion.

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

2059

Fig. 8. Proposed scheme of possible metabolic pathway of PAL administered orally in rats. From Ref. [94] with permission.

2.8.4. Others Herrero et al. studied the Spirulina platensis using HPLC/ESI(±)Q-TOF-MS/MS technique [76]. Four free fatty acids, four monogalactosyl monoacylglycerols, three phosphatidyl-glycerols, two sulfoquinovosyl diacylglycerols were characterized. Zhao et al. developed a reliable and rapid method based on the HPLC/DAD/ESI(±)-TOF-MS technique for isolation and characterization of multiple constituents in the extract from Langdu (Stellera chamaejasme L.) [77]. Twenty-two obvious peaks appeared in the
Table 3 Metabolic reactions characterized of the seven tanshinones Compounds Metabolic reactions Monohydroxylation Sodium tanshinone IIA sulfonate Tanshinone IIA Cryptotanshinone 15,16-Dihydrotanshinone I Tanshinone IIB Przewaquinone A Tanshinone I + + + + + + − Dihydroxylation − − + − + − −

TIC and nine of them were characterized by TOF/MS. Danggui (Angelica sinensis Diels) is also a commonly used traditional Chinese medicine. Wang et al. investigated the constituents of Danggui using HPLC/APCI(±)-MS [78]. Fifteen compounds were tentatively identified. Chen et al. studied another famous TCM named Chuangxiong (Rhizoma Chuanxiong) in Chinese [79]. Twodimension liquid chromatographic separation system was used for the separation of components of Chuanxiong. The effluents were detected by both DAD and APCI mass spectrometer. More than 50

Dehydrogenation + + + + − − −

Oxidation in side chain − + − − + + −

D-ring cleavage − − + + − − −

From Ref. [93] with permission. +, positive; −, negative.

2060

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

other groups have tried to use HPLC–MS technique to analyze the constituents of some complex prescriptions [81–88]. 2.9.1. Shuanghuanglian oral liquid Shuang-Huang-Lian oral liquid (SHL) consists of three herbs: Radix Scutellariae, Flos Lonicerae and Fructus Forsythiae. It is often used to treat acute upper respiratory tract infection caused by virus or bacteria. In our group, Han et al. developed a HPLC–MS method for the quality control of Shuang-Huang-Lian oral liquid (Fig. 7) [81]. A total of 27 compounds, including seven phenylethanoid glycosides, three lignans, seven quinic acids, six saponins and four flavonoids, in the extract of Shuang-Huang-Lian oral liquid have been identified or tentatively characterized (Table 2). It set a good example for the rapid identification of bioactive constituents in complex herbal preparations and made it possible to fulfill the requirements for a modern drug characterized as safe, effective and quality controllable. 2.9.2. Shuangdan granule Shuangdan granule, one of the widely used Chinese complex prescriptions prepared from Radix Salviae miltiorrhizae and Cortex Moutan, was authorized to marketing by SFDA of China (No. Z10960044) for treating coronary heart disease, myocardial infarction, angina and atherosclerosis. An HPLC/DAD/ESI-MSn method was developed for the simultaneous analysis of the chemical constituents in Shuangdan granules [82]. Ion trap mass spectrometer was applied and both the positive and negative ion modes were used in the analysis. A total of 28 compounds, including 12 phenolic acids, 6 diterpenoid quinones, 8 monoterpenoids and two other components, were characterized by their retention behaviors, UV spectra and multistage mass spectra. The results would be helpful to ensure the safety and efficiency, and to optimize the quality control of the medicine. 2.9.3. Qingkailing injection Qingkailing injection is prepared from eight medicinal materials or their extracts, including Radix Isatidis, Flos Lonicerae, Fructus Gardenise, Cornu Bubal, Concha Margaritifera, Baicalinum, Acidum Cholicum and Acidum Hyodesoxy-cholicum. An approach to profile the main constituents in Qingkailing injection by combining HPLC/TOF-MS and HPLC–MSn (IT) had been described by Zhang et al. [83]. Both the positive and negative ions were detected in the analysis and a total of thirty-three compounds were identified. All the components identified were surveyed and classified according to their medicinal material origins. The study is expected to be accepted as an effective and reliable pattern for comprehensive and systematic characterization of the complex TCM systems. 2.9.4. Compound Danshen Dripping Pill “Compound Danshen Dripping Pill”, called Fufang-DanshenDiwan in Chinese, is prepared from S. miltiorrhiza and P. notoginseng and widely used for the prevention and treatment of coronary arteriosclerosis, angina pectoris and hyperlipaemia. It has been clarified that salvianolic acids, tanshinones and saponins are the major bioactive components. Zhang et al. developed an HPLC/ESI(−)-MSn method for the identification of the saponins in Compound Danshen Dripping Pill [84]. By comparing the multistage mass spectra with those of reference standards and literature data, 19 saponins were characterized in all. Although the above four examples were investigated to some depth, they could not be considered as classical complex formulas. Some classical traditional formulas have also been investigated. For example, Zhang et al. [85] studied Si-Wu-Tang using HPLC/DAD/ESIMS, 12 compounds were identified on-line; Lin et al. [86] identified 14 components in Gan-Lu-Yin by HPLC/ESI-MS; Liu et al. [87]

Fig. 9. The proposed metabolic pathway of the total phenolic acids in danshen. From Ref. [95] with permission.

peaks were resolved and 11 of them were preliminarily identified. Zhou et al. identified two aminoethylphenyl oligoglycosides from Jingucao (Schnabelia tetradonta (Sun) C.Y. Wu et C. Chen) using HPLC/ESI(±)-ITMSn [80]. 2.9. Complex traditional Chinese medicine prescription In fact, TCMs are famous as complex prescription, which played an important role in the public health of Chinese people for thousands of years. The Chinese doctors and some investigators considered that the therapeutic effects of complex prescription benefited from the synergistic effect of all the componential medicines. Obviously, it is more difficult to profile the chemical constituents and control the quality of the complex prescription than those of single herbals. In the recent years, our group and some

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

2061

analyzed Xue-Fu-Zhu-Yu-Tang using HPLC/DAD/ESI-MS and 22 components were identified or tentatively characterized. But there were no MSn studies in these three studies. 3. Conclusion Over the past years, HPLC–MS has been used frequently in phytochemical analysis studies of traditional Chinese medicine. Emergence of selected ion monitoring (SIM), selected reaction monitoring (SRM) and multiple reaction monitoring (MRM) techniques largely promoted the application of HPLC–MS in quantitative investigation of TCMs and their in vivo process. But the qualitative study of TCMs using HPLC–MS is still not as mature as that by GC–MS. Analysis of mass spectra requires superior professional knowledge and skill. The wide application of GC–MS in the study of essential oil benefited from the commercial database of abundant EI-MS spectra, which have been accumulated for many years and made it possible to obtain the initial structures depending on computer search results. But the frequently used ion sources in HPLC–MS are mainly ESI and APCI. The fragments produced by soft ionization sources and CID are much different from those by EI. Thus, the widespread uses of HPLC–MS in the analysis of traditional Chinese medicines mainly rely on the elucidation of the fragmentation regular patterns of the constituents in the herbal medicines using MSn spectra. Furthermore, the development of HPLC–MS database is a necessity. But this is a long-term project and is currently in the groping stage since ESI and APCI-MS are easily affected by ionization and LC conditions. A universal mass spectrometer for natural products may be the ultimate desirability. But currently, a high-resolution mass spectrometer, which could provide on-line MSn spectra, will be a great help to the structural characterization of the constituents in the herbal medicines. Recently, an advanced HPLC technique called ultra-performance liquid chromatography (UPLC) was developed. UPLC can provide chromatograms with high resolution and high retention time reproducibility, which may improve the separating ability of the HPLC–MS technique. UPLC–MS was also applied in a study of TCM [41]. However, the high cost makes it difficult for the universal application. Additionally, capillary electrophoresis coupled to mass spectrometry was also tried in the investigations of herbs [89,90]. In the phytochemical analysis of traditional Chinese medicines using HPLC–MS technique, the relationship between structural features and fragmentation patterns should be investigated as thoroughly as possible. Characteristic fragmentation behaviors of the reference standards, as well as information regarding polarity obtained from retention time data, on-line UV spectra, data from literatures and biogenetic origin of the compounds allowed the identification or tentative characterization of the chemical constituents in crude extracts. Undoubtedly, if there were no sufficient reference standards, this kind of study would be very difficult to proceed. The methods established by HPLC–MS facilitated the convenient and rapid quality control of traditional medicines and their pharmaceutical preparations. Moreover, the HPLC–MS technique could also be applied in the in vivo studies of herbal medicines [91–95]. Some papers investigated the metabolism of phenolic compounds and tanshinones from the roots of S. miltiorrhiza in rats [92–95]. The obtained results (Figs. 8 and 9, Table 3) are helpful for profiling the in vivo process of Danshen. When performing the literature search, hundreds of papers regarding the analysis of herbal medicines using HPLC–MS could be traced. Nevertheless, majority of these papers are just tame attempts. More effort should be made for exploring the components in the herbal medicines and their preparations using HPLC–MS.

Acknowledgement This research was supported by the National Supporting Program for Traditional Chinese Medicine from the ministry of Sciences and Technology of China (No. 2006BAI08B03-03). References
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] A.L. Demain, Nat. Biotechnol. 16 (1998) 3. B. Stephen, K. Richard, Am. J. Med. 116 (2004) 478. P. Drasar, J. Moravcova, J. Chromatogr. B 812 (2004) 13. X.G. He, J. Chromatogr. A 880 (2000) 203. Z.Y. Zhu, H. Zhang, L. Zhao, X. Dong, X. Li, Y.F. Chai, G.Q. Zhang, Rapid Commun. Mass Spectrom. 21 (2007) 1855. A.H. Liu, H. Guo, M. Ye, Y.H. Lin, J.H. Sun, M. Xu, D.A. Guo, J. Chromatogr. A 1161 (2007) 170. G.F. Zeng, H.B. Xiao, J.X. Liu, X.M. Liang, Rapid Commun. Mass Spectrom. 20 (2006) 499. J. Qu, Y.M. Wang, G.A. Luo, Z.P. Wu, J. Chromatogr. A 928 (2001) 155. Y.F. Zhang, P.Y. Shi, H.B. Qu, Y.Y. Cheng, Rapid Commun. Mass Spectrom. 21 (2007) 2971. W. Jin, Y.F. Wang, R.L. Ge, H.M. Shi, C.Q. Jia, P.F. Tu, Rapid Commun. Mass Spectrom. 21 (2007) 2351. M. Ye, J. Han, H.B. Chen, J.H. Zheng, D.A. Guo, J. Am. Soc. Mass Spectrom. 18 (2007) 82. H. Guo, A.H. Liu, M. Ye, M. Yang, D.A. Guo, Rapid Commun. Mass Spectrom. 21 (2007) 715. J. Han, M. Ye, M. Xu, J.H. Sun, B.R. Wang, D.A. Guo, J. Chromatogr. B 848 (2007) 355. M. Ye, D. Guo, G. Ye, C.G. Huang, J. Am. Soc. Mass Spectrom. 16 (2005) 234. H.L. Jiang, A.M. Sólyom, B.N. Timmermann, D.R. Gang, Rapid Commun. Mass Spectrom. 19 (2005) 2957. H.L. Jiang, B.N. Timmermann, D.R. Gang, Rapid Commun. Mass Spectrom. 21 (2007) 509. C. Gardana, M. Scaglianti, P. Pietta, P. Simonetti, J. Pharm. Biomed. Anal. 45 (2007) 390. L. Zhang, L. Xu, S.S. Xiao, Q.F. Liao, Q. Li, J. Liang, X.H. Chen, K.S. Bi, J. Pharm. Biomed. Anal. 44 (2007) 1019. K. Schütz, D.R. Kammerer, R. Carle, A. Schieber, Rapid Commun. Mass Spectrom. 19 (2005) 179. R.X. Liu, M. Ye, H.Z. Guo, K.S. Bi, D.A. Guo, Rapid Commun. Mass Spectrom. 19 (2005) 1557. F. Sánchez-Rabaneda, O. Jáuregui, R.M. Lamuela-Raventós, J. Bastida, F. Viladomat, C. Codina, J. Chromatogr. A 1008 (2003) 57. M. Yang, W. Wang, J.H. Sun, Y.Y. Zhao, Y. Liu, H. Liang, D.A. Guo, Rapid Commun. Mass Spectrom. 21 (2007) 3833. M. Ye, Y.N. Yan, D.A. Guo, Rapid Commun. Mass Spectrom. 19 (2005) 1469. M.A. Anagnostopoulou, P. Kefalas, E. Kokkalou, A.N. Assimopoulou, V.P. Papageorgiou, Biomed. Chromatogr. 19 (2005) 138. P.Y. Shi, Q. He, Y. Song, H.B. Qu, Y.Y. Cheng, Anal. Chim. Acta 598 (2007) 110. D.Y. Zhou, Q. Xu, X.Y. Xue, F.F. Zhang, X.M. Liang, J. Pharm. Biomed. Anal. 42 (2006) 441. L. Yang, N. Nakamura, M. Hattori, Z.T. Wang, S.W.A. Bligh, L.S. Xu, Rapid Commun. Mass Spectrom. 21 (2007) 1833. G.N. Zhang, F. Zhang, L. Yang, E.Y. Zhu, Z.T. Wang, L.S. Xu, Z.B. Hu, Anal. Chim. Acta 571 (2006) 17. L. Yang, Y. Wang, G.N. Zhang, F. Zhang, Z.J. Zhang, Z.T. Wang, L.S. Xu, Biomed. Chromatogr. 21 (2007) 687. M. Brolis, B. Gabetta, N. Fuzzati, R. Pace, F. Panzeri, F. Peterlongo, J. Chromatogr. A 825 (1998) 9. E.C. Tatsis, S. Boeren, V. Exarchou, A.N. Troganis, J. Vervoort, I.P. Gerothanassis, Phytochemistry 68 (2007) 383. A. Tolonen, J. Uusitalo, A. Hohtola, J. Jalonen, Rapid Commun. Mass Spectrom. 16 (2002) 396. A. Tolonen, J. Uusitalo, Rapid Commun. Mass Spectrom. 18 (2004) 3113. A. Petsalo, J. Jalonen, A. Tolonen, J. Chromatogr. A 1112 (2006) 224. M. Eeva, J.P. Rauha, P. Vuorela, H. Vuorela, Phytochem. Anal. 15 (2004) 167. J.K. Prasain, A. Reppert, K. Jones, D.R. Moore II, S. Barnes, M.A. Lila, Phytochem. Anal. 18 (2007) 50. N. Fuzzati, B. Gabetta, K. Jayakar, R. Pace, F. Peterlongo, J. Chromatogr. A 854 (1999) 69. G.C. Kite, M.R. Howes, C.J. Leon, M.S.J. Simmonds, Rapid Commun. Mass Spectrom. 17 (2003) 238. K.S. Leung, K. Chan, A. Bensoussan, M.J. Munroe, Phytochem. Anal. 18 (2007) 146. L. Li, R. Tsao, J.P. Dou, F.R. Song, Z.Q. Liu, S.Y. Liu, Anal. Chim. Acta 536 (2005) 21. E.C.Y. Chan, S.L. Yap, A.J. Lau, P.C. Leow, D.F. Toh, H.L. Koh, Rapid Commun. Mass Spectrom. 21 (2007) 519. M.Q. Guo, F.R. Song, Z.Q. Liu, S.Y. Liu, Anal. Chim. Acta 557 (2006) 198. B. Li, Z. Abliz, M.J. Tang, G.M. Fu, S.S. Yu, J. Chromatogr. A 1101 (2006) 53. Q. Xu, X.Q. Ma, X.M. Liang, Phytochem. Anal. 18 (2007) 419. Z.G. Zheng, W.D. Zhang, L.Y. Kong, M.J. Liang, H.L. Li, M. Lin, R.H. Liu, C. Zhang, Rapid Commun. Mass Spectrom. 21 (2007) 279.

2062

M. Yang et al. / J. Chromatogr. A 1216 (2009) 2045–2062 [71] M. Yang, X.M. Wang, S.H. Guan, J.M. Xia, J.H. Sun, H. Guo, D.A. Guo, J. Am. Soc. Mass Spectrom. 18 (2007) 927. [72] M. Ye, D.A. Guo, Rapid Commun. Mass Spectrom. 19 (2005) 1881. [73] S.J. Xu, L. Yang, X. Zeng, M. Zhang, Z.T. Wang, Rapid Commun. Mass Spectrom. 20 (2006) 3275. [74] Y.F. Zhang, Q.R. Shi, P.Y. Shi, W.D. Zhang, Y.Y. Cheng, Rapid Commun. Mass Spectrom. 20 (2006) 2328. [75] K. He, G.F. Pauli, B.L. Zheng, H.K. Wang, N.S. Bai, T.S. Peng, M. Roller, Q.Y. Zheng, J. Chromatogr. A 1112 (2006) 241. ˜ [76] M. Herrero, M.J. Vicente, A. Cifuentes, E. Ibánez, Rapid Commun. Mass Spectrom. 21 (2007) 1729. [77] L. Zhao, Z.Y. Lou, Z.Y. Zhu, H. Zhang, G.Q. Zhang, Y.F. Chai, Biomed. Chromatogr. 22 (2007) 64. [78] Y.L. Wang, Y.Z. Liang, B.M. Chen, Phytochem. Anal. 18 (2007) 265. [79] X.G. Chen, L. Kong, X.Y. Su, H.J. Fu, J.Y. Ni, R.H. Zhao, H.F. Zou, J. Chromatogr. A 1040 (2004) 169. [80] Y. Zhou, H. Dou, S.L. Peng, X.R. Zhang, L.S. Ding, Rapid Commun. Mass Spectrom. 18 (2004) 1172. [81] J. Han, M. Ye, H. Guo, M. Yang, B.R. Wang, D.A. Guo, J. Pharm. Biomed. Anal. 44 (2007) 430. [82] Q. He, X.J. Hu, Y.Y. Cheng, J. Pharm. Biomed. Anal. 41 (2006) 485. [83] H.Y. Zhang, P. Hu, G.A. Luo, Q.L. Liang, Y.L. Wang, S.K. Yan, Y.M. Wang, Anal. Chim. Acta 577 (2006) 190. [84] H.J. Zhang, Y.Y. Cheng, J. Pharm. Biomed. Anal. 40 (2006) 429. [85] H.J. Zhang, P. Shen, Y.Y. Cheng, J. Pharm. Biomed. Anal. 34 (2004) 705. [86] I.H. Lin, M.C. Lee, W.C. Chuang, J. Sep. Sci. 29 (2006) 172. [87] L. Liu, Y.Y. Cheng, H.J. Zhang, Chem. Pharm. Bull. 52 (2004) 1295. [88] J.L. Zhang, M. Cui, Y. He, H.L. Yu, D.A. Guo, J. Pharm. Biomed. Anal. 36 (2005) 1029. [89] D.A. Román, A.M.G. Caravaca, M.G. Romero, A.S. Carretero, A.F. Gutiérrez, J. Pharm. Biomed. Anal. 41 (2006) 1648. [90] M. Unger, M. Dreyer, S. Specker, S. Laug, M. Pelzing, C. Neus, U. Holzgrabe, G. Bringmann, Phytochem. Anal. 15 (2004) 21. [91] P. Geng, R.P. Zhang, H.A. Aisa, J.M. He, K. Qu, H.B. Zhu, Z. Abliz, Rapid Commun. Mass Spectrom. 21 (2007) 1877. [92] J.H. Sun, M. Yang, X.M. Wang, M. Xu, A.H. Liu, D.A. Guo, J. Pharm. Biomed. Anal. 44 (2007) 564. [93] J.H. Sun, M. Yang, J. Han, B.R. Wang, X.C. Ma, M. Xu, P. Liu, D.A. Guo, Rapid Commun. Mass Spectrom. 21 (2007) 2211. [94] M. Xu, Z.C. Zhang, G. Fu, S.F. Sun, J.H. Sun, M. Yang, A.H. Liu, J. Han, D.A. Guo, J. Chromatogr. B 856 (2007) 100. [95] J.L. Zhang, Y. He, M. Cui, L. Li, H.L. Yu, G.F. Zhang, D.A. Guo, Biomed. Chromatogr. 19 (2005) 51.

[46] Y.P. Tai, X.J. Cao, X.Y. Li, Y.J. Pan, Anal. Chim. Acta 572 (2006) 230. [47] S.H. Lin, D.M. Wang, D.P. Yang, J.H. Yao, Y. Tong, J.P. Chen, Anal. Chim. Acta 599 (2007) 98. [48] Y. Wang, F.R. Song, Q.X. Xu, Z.Q. Liu, S.Y. Liu, J. Mass Spectrom. 38 (2003) 962. [49] Q.X. Xu, H. Yue, Z.Q. Liu, Y. Wand, C.Y. Yan, S.Y. Liu, Chem. Res. Chin. Univ. 22 (2006) 343. [50] Y. Wang, L. Shi, F.R. Song, Z.Q. Liu, S.Y. Liu, Rapid Commun. Mass Spectrom. 17 (2003) 279. [51] H. Yue, Z.F. PI, H.L. Li, F.R. Song, Z.Q. Liu, S.Y. Liu, Phytochem. Anal. 19 (2008) 141. [52] W. Wu, Z.T. Liang, Z.Z. Zhao, Z.W. Cai, J. Mass Spectrom. 42 (2007) 58. [53] M.J. Liang, W.D. Zhang, J. Hu, R.H. Liu, C. Zhang, J. Pharm. Biomed. Anal. 42 (2006) 178. [54] M. Cai, Y. Zhou, X.L. Wang, R. Li, X. Liao, L.S. Ding, Rapid Commun. Mass Spectrom. 21 (2007) 1931. [55] Y. Zhou, R.W. Jiang, P.M. Hon, Y.T. Xu, Y.M. Chan, T.W.D. Chan, H.X. Xu, L.S. Ding, P.P.H. But, P.C. Shaw, Rapid Commun. Mass Spectrom. 20 (2006) 1030. [56] H.L. Li, J. Tang, R.H. Liu, M. Lin, B. Wang, Y.F. Lv, H.Q. Huang, C. Zhang, W.D. Zhang, Rapid Commun. Mass Spectrom. 21 (2007) 199. [57] L.J. Cui, Z. Abliz, M. Xia, L.Y. Zhao, S. Gao, W.Y. He, Y. Xiang, F. Liang, S.S. Yu, Rapid Commun. Mass Spectrom. 18 (2004) 184. [58] I.N. Abreu, P. Mazzafera, M.N. Eberlin, M.A.T. Zullo, A.C.H.F. Sawaya, Rapid Commun. Mass Spectrom. 21 (2007) 1205. [59] H.J. Dong, Z.Q. Liu, F.R. Song, Z. Yu, H.L. Li, S.Y. Liu, Rapid Commun. Mass Spectrom. 21 (2007) 3193. [60] L.L. Ren, X.Y. Xue, F.F. Zhang, Y.C. Wang, Y.F. Liu, C.M. Li, X.M. Liang, Rapid Commun. Mass Spectrom. 21 (2007) 3039. [61] Y. Zhou, X. Zou, X. Liu, S.L. Peng, L.S. Ding, Rapid Commun. Mass Spectrom. 21 (2007) 1375. [62] K. Alipieva, T. Kokubun, R. Taskova, L. Evstatieva, N. Handjieva, Biochem. Syst. Ecol. 35 (2007) 17. [63] M. Ye, D.A. Guo, Rapid Commun. Mass Spectrom. 19 (2005) 818. [64] S.J. Ding, E. Dudley, S. Plummer, J.D. Tang, R.P. Newton, A.G. Brenton, Rapid Commun. Mass Spectrom. 20 (2006) 2753. [65] L.S. Jager, G.A. Perfetti, G.W. Diachenko, J. Pharm. Biomed. Anal. 41 (2006) 1552. [66] A.G. Jensen, K. Ndjoko, J.L. Wolfender, K. Hostettmann, F. Camponovo, F. Soldati, Phytochem. Anal. 13 (2002) 31. [67] M. Yang, A.H. Liu, S.H. Guan, J.H. Sun, M. Xu, D.A. Guo, Rapid Commun. Mass Spectrom. 20 (2006) 1266. [68] E.S. Ong, C.N. Ong, Rapid Commun. Mass Spectrom. 21 (2007) 589. [69] O. Schaaf, A.P. Jarvis, S.A. van der Esch, G. Giagnacovo, N.J. Oldham, J. Chromatogr. A 886 (2000) 89. [70] L.Y. Huang, T.W. Chen, Z. Ye, G.N. Chen, J. Mass Spectrom. 42 (2007) 910.

Sponsor Documents

Or use your account on DocShare.tips

Hide

Forgot your password?

Or register your new account on DocShare.tips

Hide

Lost your password? Please enter your email address. You will receive a link to create a new password.

Back to log-in

Close