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Journal of Pharmaceutical and Biomedical Analysis
journal homepage: www.elsevier.com/locate/jpba
Recent advances on HPLC/MS in medicinal plant analysis
Dirk Steinmann, Markus Ganzera ∗
Institute of Pharmacy, Pharmacognosy, University of Innsbruck, Innrain 52, 6020 Innsbruck, Austria
a r t i c l e
i n f o
a b s t r a c t
With gaining popularity of herbal remedies worldwide, the need of assuring safety and efﬁcacy of these products increases as well. By nature they are complex matrices, comprising a multitude of compounds, which are prone to variation due to environmental factors and manufacturing conditions. Furthermore, many traditional preparations compose of multiple herbs, so that only highly selective, sensitive and versatile analytical techniques will be suitable for quality control purposes. By hyphenating high performance liquid chromatography and mass spectrometry (LC–MS) these high demands are fulﬁlled, providing the user with a multitude of technical options and applications. This review intends to reﬂect the impact of LC–MS for medicinal plant analysis focusing on most relevant reports published within the last ﬁve years. Commenced by introductory remarks to the different MS approaches most commonly used (e.g. ion trap and time of ﬂight mass analyzers, fragmentation and ionization modes), respective LC–MS applications on the analysis of natural products in medicinal plants, commercial products and biological samples are presented. Methodological aspects like stationary and mobile phase selection or MS settings are discussed, and advantages or limitations of the described techniques are highlighted. © 2010 Elsevier B.V. All rights reserved.
Article history: Received 10 September 2010 Received in revised form 10 November 2010 Accepted 11 November 2010 Available online 19 November 2010 Keywords: Liquid chromatography Mass spectrometry LC–MS Medicinal plants Natural products
Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Ionization source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Mass analyzer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Coumarins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Phenols and ﬂavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Quinones and xanthones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Terpenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. Fingerprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 744 745 745 745 746 746 746 747 749 749 751 753 754 755 755 755
1. Introduction The inception of mass spectrometry (MS) can be traced back to the beginning of the last century, when Joseph John Thomson observed the fact that magnetic and electrostatic ﬁelds deﬂect positive rays depending on their mass. Yet, it took many years of
∗ Corresponding author. Fax: +43 512 507 2939. E-mail address: email@example.com (M. Ganzera). 0731-7085/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2010.11.015
intense research by scientists like Alfred Nier (1940s: improvement in vacuum technologies and ion sources) or Wolfgang Paul (1953: quadrupole mass analyzer) till this technique became of practical relevance for the analysis of organic compounds. The following years (1960–1980) were highlighted by a multitude of technical developments like MALDI (matrix-assisted laser desorption), FTMS (Fourier transform mass spectrometry), FAB-MS (fast atom bombardment mass spectrometry) or ion-trap instruments, just to name a few [1,2]. Today, a mass spectrometer is nearly standard equipment in a modern analytical laboratory and indispensable for
and the third as ﬁlter again.g. not permitting the fragmentation of ions (MS/MS). Then the ions can be fragmented in a similar way as described above by collision-induced decomposition (CID). This process can be repeated in sequential manner. A ﬁne aerosol is produced. and special emphasis will be put on applicability of the described procedures on real life samples. It is based on the fact that molecular mass is related to velocity. and considering the fact that many traditional herbal preparations of Chinese or Indian origin contain not one but several medicinal plants.000. high performance liquid chromatography (HPLC) was and still is the preferred separation technique for the analysis of natural products. Different data acquiring modes are possible with this setup. A more recent development is atmospheric pressure photoionization (APPI). Crucial parameters are position of the ion source (orthogonal or linear). and they enter the analyzer due to a potential or pressure difference. evaporative light scattering (ELS) and ﬂuorescence detectors to MS-detection.000) and mass precision (1–2 ppm) . Once a critical limit . usually low abundance and variability even within the same species.000 metabolites occur in the plant kingdom . The opposing rods are connected and an RF voltage is applied between one pair of rods and the other.1.000 to 1. and therefore different exact mass). resolution. also called ion mirror. They increase ﬂight distance and further focus the ions. so that a quadrupolar electric ﬁeld is created. yet it is the only technique which produces signals directly correlating with the amount of analytes in the sample . can be obtained using different ionization techniques. In linear ion traps. the second as collision cell. also known as multiple reaction monitoring (MRM) [13. It is estimated that 100. and ionization is achieved by applying a high electric charge to the sample needle. which are ionization source. This review intends to summarize the most interesting and relevant applications of HPLC–MS for natural products analysis reported within the last ﬁve years (2006–2010). A quadrupole can be considered as a mass ﬁlter. A conﬁnement to certain plants or applications is necessary due to the extent of the topic. and just like ESI it can be operated in positive and negative mode . ESI is a soft ionization technique (little fragmentation but often adducts are observed) for a wide range of compounds. The chapters are grouped by compound classes. This is possible by arranging three quadrupole cells in sequence (triple quadrupole). Therefore. The latter has an inner part for the LC eluent and an outer part for a nebulising gas (e. Both conﬁne ions in cyclotron motion. for example the highly sensitive selected reaction monitoring (SRM). and therefore compounds will reach the detector with increasing m/z values. resulting in high levels of resolution (typically from 100. the latter offers excellent sensitivity and selectivity. argon or nitrogen) and applied energy (collision energy). Steinmann. for which Fourier transform ion cyclotron resonance-MS (FT-ICR-MS) and Orbitrap are examples. analyzer and detector. ions are isolated and accumulated due to a special arrangement of hyperbolic and ring shaped electrodes as well as oscillating electric ﬁelds. most commonly chromatographic or electrophoretic techniques in combination with different detectors are employed for this purpose. M. either by strong magnetic ﬁelds (FT-ICR-MS) or electrostatic forces (Orbitrap). In addition to good sensitivity and resolution the technique allows high scan speeds making it suitable for fast separations  and it can be coupled to quadrupole (QTOF-MS) or ion trap (IT-TOF-MS) mass ﬁl- 2. Compared with the foremost mentioned techniques. Thus. length of the ﬂight tube and the reﬂectron . is accountable for the high resolution power of TOF. ranging from photodiode array (DAD) or UV–vis detectors. GLC–MS reports are not covered as they are outside the scope of this manuscript. Solvents like acetone. a preferentially ionized substance that acts as intermediate between photons and analytes. and gas chromatography (GC) is a powerful tool for volatile constituents only (otherwise derivatization of the analytes is required). The setup of an APCI source is similar but charge is applied via a corona pin. which are easier to manipulate than molecules in neutral stage. An increasingly popular type of mass analyzer is time of ﬂight (TOF). It is considered as alternative for compounds that are poorly ionized by ESI and APCI.g. More complex and elaborate trapping technologies are so-called 3D traps. only highly selective and sensitive methods will be suitable for controlling their composition and quality. Direct current is then superimposed. toluene or anisole have been used for this purpose. Recent developments of sub 2 m stationary phases and pumping devices enabling pressures up to 1300 bar further supported this trend .14]. nitrogen). This technique usually requires the addition of a dopant. the most economic type of mass analyzer used nowadays (Fig. in the next chapter the basic principles of MS are brieﬂy discussed and major differences of the most commonly used types are highlighted. The type of detector used. A different concept is followed in ion trap analyzers. consists of a set of rings.8]. which are especially suitable for multiple fragmentation steps (MSn ) rather than quantitative studies. mostly leading to the formation of positively charged compounds . Like all other analyzers it has to be maintained under vacuum in order to enable transition of the ions. which only ions of a certain masses can pass through . so that mass accuracies of 5 ppm are possible. Ganzera / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 744–757 745 scientiﬁc research on a high level. Fragmentation occurs in the collision cell with the help of a collision gas (e. Due to different instrumental characteristics in ionization. Ions. Instrumentation 2. 2. and is based on the interaction of a photon beam produced by a kypton or xenon lamp with vapors formed by the nebulisation of a liquid solution . is reached (Rayleigh limit) ions are formed by a process called “Coulombic explosion”. and fragmentation MS is a versatile technique with described applications from astronomical studies and geophysics to biology and medicine. scan speed. The analysis of secondary metabolites in plants is a challenging task because of their chemical diversity. This technique is especially suitable for non-polar compounds. In order to familiarize the reader with the topic. Thus. Ionization source Any mass spectrometer comprises three major components. This permits the veriﬁcation of elemental composition and differentiation of isobaric compounds (with the same nominal mass but different elemental compositions. so that valuable structural information is obtained . where the ﬁrst serves as mass ﬁlter.2. Due to extremely small sample volumes capillary electrophoresis (CE) often faces sensitivity problems as well . largely depends on the analytes investigated. it is not surprising that the use of LC–MS for the qualitative and quantitative analysis of constituents in medicinal plants steadily increased over the last years. Sensitivity is the disadvantage of using proton nuclear magnetic resonance analysis (1 H NMR) for this purpose. in which the droplet size continuously decreases due to evaporation of the solvent. methodological features and possible advantages or disadvantages compared to alternative approaches. The latter. 1). which is located at the exit of a heated tube.000–200.D. Mass analyzer Four parallel aligned metal rods are the core of any single quadrupole. combined with the ability to elucidate or conﬁrm chemical structures (depending on the instrument used) [7. with electrospray ionization (ESI) and atmospheric pressure ionization (APCI) being most widely used .
which was recorded in SIM mode at m/z = 359. They also studied the ionization characteristics of aristolochic acids (with ESI multiple products like [M−H2 O+H]+ or [M−NO2 +H]+ were observed) and fully validated the procedure. A more comprehensive investigation of the different aristolochic acids in Aristolochia species (e. It consists of a thin plate perforated by tiny tubes or slots typically 6–10 m in diameter. Steinmann. But despite the fact that this incident happened nearly twenty years ago the problem still persists. as indicated by similar LOD values determined (e. These confusions led to the poisoning of more than 50 women in Belgium (“Chinese herbs nephropathy”).32 g/ml in methanol extract). mollissima) was described by Yuan et al. The methanolic plant extracts were separated on reversed phase material (Luna C18. Reproduced with permission from . A. Acids Good examples for the relevance of quality control of herbal preparations are aristolochic acids.1. with AA I being assignable in positive mode as [M+NH4 ]+ adduct. Due to a similar name in Chinese the non-toxic Stephania tetrandra (“Fangji”) is sometimes wrongly substituted with Aristolochia fangchi (“Guang Fangji”).746 D. investigated the distribution of aristolochic acids in Herba Asari including the safest preparation method . Other LC–MS studies targeting aristolochic acids to a smaller extend are those on the biochemical effects induced by AA I  or a cooperative study using MS for conﬁrmation purposes only . and the resulting current is measured on an anode. both are traditionally used as diuretic and for treating rheumatic conditions. After each cascade the MCP has to be recharged before it can detect another signal.08 g/g compared to 0. 2. In 2006 Koh et al. Applications 3. A. Numerous reports on the analysis of phenolic acids (e. in 2007 . 5 m) using methanol and water as mobile phase. who took an adulterated herbal slimming preparation . For much higher gain not just one but two or three plates. Nevertheless. and quantiﬁed these constituents using diode array detection (DAD) and MS (Micromass ZQ 4000 in positive ESI mode). In modern instruments this is commonly achieved using the micro-channel plate (MCP) technology. corresponding to the same adduct as mentioned above (see Table 1 for further methodological details). AA II. the authors considered the described LC–MS assay suitable for quality control purposes because of its good selectivity. LC–MS was not only used for identiﬁcation but also quantiﬁcation of AA I. Schematic design of a single quadrupole (A) and triple quadrupole (B) mass analyzer. by UV at 254 nm: 15 ng/ml). fangchi or A. high resolution MS/MS or MSn analyses are possible as well . caffeic acid and its derivatives) by LC–MS have been published. For MS studies a Finnigan LCQ ion trap mass spectrometer with APCI source was utilized. Ganzera / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 744–757 Fig. They . In the presence of an electric ﬁeld each channel acts as electron multiplier and whenever hit by an ion or photon a cascade of electrons starts .1. AA C. M. For fragmentation helium was used as collision gas and the relative collision energy was set to 35%. are used in many instruments . 3. Detector MS detectors record either a current or charge produced by the ions. for AA I by MS: 12 ng/ml.g. They concluded if an aqueous roots is used it can be considered a safe remedy. They are a mixture of nephrotoxic and carcinogenic nitrophenantrene carboxylic acids occurring in the genus Aristolochia. arranged in V or Z shape. and in nine of them the presence of aristolochic acid I (AA I) was conﬁrmed by LC–MS/MS . Thus. 1. In another study Zhao et al. contorta.3. AA D.g. analyzed ten samples of “Fangji”. They studied the occurrence of six acids (AA I. 7-OH AA I and aristolochic acid) together with four aristololactams. A comparison of both approaches showed that “aristolochic acids are in nature not provided with high ionization efﬁciency”.g. The electrons then exit the channel on the opposite end. as the amounts of AA I are low (0. ters.
Considering sensitivity divergent observations were made. 2). structural conﬁrmation and the identiﬁcation of additional constituents was achieved on a LTQ Orbitrap hybrid mass spectrometer. MS2 and MS3 spectra were found to be especially helpful in differentiating symviridine N-oxide and echimidine. Comfrey is a traditional remedy for the treatment of arthritis and sprains. Ganzera / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 744–757 Table 1 Selected LC–MS assays for the analysis of acids in herbal medicines.SA S. AA C. Quantitative results were nearly identical by both sources.4-dihydrocarbazole-9-ethyl chloroformate prior to analysis. and pyrrolizidine alkaloids in Delphinium  have been published already. acetonitrile. S.2-benzo3. Its amount was markedly decreased if the samples were extracted under pressure at 60 ◦ C compared to a higher temperature without applying pressure. water (+0. for example the LOD of caftaric acid was 0. Val. water.10 ng/ml). chlorogenic. For qualitative and quantitative studies (only lycopsamine was quantiﬁed. followed by ionization within the negative glow region. After carefully optimizing separation conditions. and acetonitrile Source ESI ESI APCI ESI ESI ESI GD EI ESI ESI Quant. which are based on RP stationary phases and acidic mobile phases. either ESI or APCI were employed as ionization sources.: quantiﬁcation. based on a newly developed LC–ESI-MS/MS assay.8%) and accuracy (recovery rates between 64 and 115%). These compounds are also pyrrolizidine alkaloids. and then quantiﬁed them in positive ESI mode on a triple quadrupole MS (Fig. Symphytum ofﬁcinale (comfrey) and Tussilago farfara (coltsfoot).1 M with acceptable precision ( rel interday ≤8. without method validation) a LCQ-Duo ion trap mass spectrometer from Thermo Finnigan was used. propolis ). Eucommia ulmoides . Otherwise similar analyt- . the authors considered their assay superior to previously reported ones. Vinca alkaloids . The latter are also found in two other important medicinal plants. Eucommia ulmoides Salvia miltiorrhiza Human plasma Human plasma and urine Echinacea product Barley Gentiana dahurica LC-conditions Alltech C18 (5 m). for which the authors also discussed the fragmentation characteristics in detail. 3: accuracy. methanol and acetonitrile Inertsil ODS-2 (5 m). SA: sample. MS DAD + MS – MS MS – MS MS FD Val. AA D) Chlorogenic acids and geniposidic acid Caffeic.2% formic acid.SA S.g. lycopsamine. water and methanol ˚ (5 m). but in respect to repeatability EI was advantageous (GD showed relative standard deviations of up to 16. Additionally. was used for identiﬁcation and/or quantiﬁcation purposes. As can be seen in Table 1. so that in countries like Austria only extracts free of 1. described the direct determination of amino acids in barley .70 ng/ml by GD. S: standard. M.1 and 0. Two methods for the LC–MS determination of amino acids in plant material should be mentioned as well. 1. LC–MS was used for conﬁrmatory purposes only.g. Alkaloids Excellent reviews on the analysis (incl. 0. In GD the molecules collide with a cathodic surface. This observation is in contrast to a similar study of the same group on the determination of senkirkine and senecionine in Tussilago farfara . are released into the gas phase by thermal vaporization. Thiele et al.SA S.SA S. All alkaloids were directly assignable at m/z values corresponding to [M+H]+ in positive ESI mode. 0.2-unsaturated derivatives are commercialized . Two ionization sources. describe their determination in medicinal plants and preparations thereof (e.8%). 5% acetic acid Hypersil BDS-C18 (5 m). Volunteers were asked to drink a green coffee bean extract and. LC–MS) of ergot alkaloids . By using an internal standard (deuterated phenylalanine) and selecting the MRM mode they were able to detect the analytes as low as 0. protocatechuic acid Chlorogenic acids and metabolites Hydroxycinnamic acids Caffeic acids Amino acids Amino acids (derivatized) Matrix Asarum spp.5% acetic acid in methanol Kromasil C18 (5 m). A discrepancy that the authors explained by the instable nature of lycopsamine. the pharmacokinetics of the above mentioned compounds were investigated. 0. 4: precision). Matsui et al. whereas for chlorogenic acid just an opposite trend was observed (4. The former separated 20 amino acids on a strong cation exchange HPLC column. Only recently Castro et al. Analytes Aristolochic acid I Aristolochic acids (AA I. 2 1–4 2 1–4 1–4 2 1–4 1–4 1–4 Appl.: validated (1: sensitivity.SA SA S. 50 mM acetic acid in water and acetonitrile Luna C18 (5 m). 0.SA S.SA S. in food items (e. collision energies and by using a triple quadrupole API-4000 (Applied Biosystems) in MRM mode the method showed excellent sensitivity with LOD values between 0. 3. Aristolochia and Asarum spp. Sun et al.          Quant. identiﬁed previously derivatized compounds in Gentiana dahurica .6 ng/ml. 2: speciﬁcity. 10 mM ammonium acetate. used liquid chromatography particle beam mass spectrometry (LC–PB/MS) to determine caffeic acid derivatives in Echinacea extracts . for example studied nine chlorogenic acids and some of their metabolites (ferulic acid and caffeic acid) in human plasma .85 ng/ml versus 3. coltsfoot decoctions are used against coughs and as an anti-inﬂammatory drug.2% acetic acid in water and methanol Agilent C18. A comparison of both ionization techniques revealed similar MS spectra in terms of fragmentation patterns. 30 mM formic acid. but it required derivatization with 1. Liu et al.2.1% formic acid) XDB C18 (4 m). The particle beam served as a “transport-type” interface enabling continuous introduction of sample solutions into the sources without residual solvent vapors or changing the natural chromatographic characteristics. but the results were comparable with both extraction procedures. ofﬁcinale . in EI the molecules are hit by electrons produced in a wire by thermionic emission and thereby are ionized . methanol. were then alternately employed followed by MS detection on a Thermabeam LC/MS quadrupole mass spectrometer.g. Thus.D. AA II. and in biological matrices like plasma  or urine . Appl. The obtained derivatives were then quantiﬁed by ﬂuorescence detection. Steinmann. By selecting three diagnostic product ions for each of the compounds their unequivocal assignment was possible. published a LC–MS method for the determination of several alkaloids (e. Salvia miltiorrhiza ). the effect of different extraction procedures (pressurized hot water extraction and reﬂuxing) on the content of lycopsamine was studied. 30 mM ammonium Luna SCX 100 A acetate (pH 6).1% formic acid in water and acetonitrile Alltima C18 (5 m).64 ng/ml by EI and 7.: application. MS. Besides desired bioactive constituents both herbs contain liver toxic pyrrolizidine alkaloids. two structurally closely related minor alkaloids. With LOD-values in the low femtomole range the method described by Sun was advantageous in terms of sensitivity. in most reports comparable analytical conditions were described. echimidine and lasiocarpine) in S. glow discharge (GD) or electron impact (EI).SA 747 Ref.
M.g.15% (determined by titration) . The plant is widely distributed in Asia and Northern America and. aconitine. despite of the presence of toxic diterpene alkaloids. LC separations were performed on C-18 phases. Selected ion chromatograms of 20 underivatized amino acids (20 M each) and the internal standard d2 -Phe (10 M) recorded in MRM mode. Nevertheless. either using an ammonium acetate buffer or an ammonium bicarbonate solution with pH 9. with a maximum alkaloid content of 0. Joosten and colleagues studied pyrrolizidine alkaloids in Jacobaea vulgaris. Wang reported . 2. Ganzera / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 744–757 Fig. described the analysis of major Aconitum alkaloids (e.748 D. In the Chinese Pharmacopeia only the use of processed plant material (by soaking in salt water or pressure steaming. the alkaloids need to be carefully monitored because they suppress the inactivation of sodium channels and lead to neurotoxic and cardiotoxic degenerations. its tuber plays an important role in TCM as analgesic and cardiotonic herbal medicine. because Kaneko only used spiked plasma samples for developing and validating the assay. Wang their determination in urine by LC–MS/MS . From practical perspectives reference  is more relevant. Several reports on the analysis of alkaloids in Aconitum carmichaeli (Ranunculaceae) have been published within the last few years. the product is called “Fuzi”) is allowed.5 for elution (see Table 2). Both required a sample cleanup on SPE cartridges (Oasis MCX or BondElut certify HF) prior to analysis. As this plant is not of medicinal interest but responsible for livestock losses and the contamination of milk and honey it only is mentioned brieﬂy here . Reproduced with permission from . ical approaches as mentioned before were utilized. mesaconitine) in human plasma . Kaneko et al. Steinmann.
03% diethylamine Zorbax Eclipse XDB C18 (3. The latter experiments were performed on an Alltima C18 column and water/acetonitrile as mobile phase. UV. Both desirable (e. except for . Rutaceae.5 kV.SA S. water and acetonitrile both with 0.SA   Verticine. and then the resulting solutions assessed by MALDI-TOF and LC-QTOF. spray voltage: 4. the following alkaloid levels in the urine of a patient: 50.005% TFA Source ESI Quant. 6 O-trans-feruloylnodakenin. for mesaconitine ranging from 1. which are commonly found in plants belonging to the Apiaceae. 3. Three marker substances (isoimperatorin. capillary voltage: −13 V/+25 V. Appl.: quantiﬁcation. which readily was ionized in negative ESI mode. With a Diamonsil C18 column as stationary phase. 0. They utilized LC–MS for conﬁrmatory purposes only. respectively.5 m). Concerning the alkaloid levels enormous differences were observed among different samples. Xin et al. [M+Na]+ and [2M+Na]+ ). Because of the low content of bergapten the authors considered it not suitable as a marker compound. In 2007 Qian et al. 2: speciﬁcity. 0. lens voltage: +18 V/−16 V. 0. Analytes Pyrrolizidine alkaloids (lycopsamine.6 ng/ml). as well as MS–MS data. Coumarins are natural benzopyrone derivates. water and ACN both with 0. nodakenetin. Strobilanthes cusica ESI ESI MS DAD 1–4 1–4 S.1% formic acid Shiseido UG80 C18.SA     Quant. and 1. Method development was performed by using HPLC-DAD. the latter using an ESI source in positive mode. berberine type alkaloids in Coscinium fenestratum . Both plants species are listed in the People’s Republic of China Pharmacopeia under the name “Qiang Huo”. 4: precision). Eight different derivatives (nodakenin. Asteraceae and Umbelliferae fami- lies. S. 8.8 m).5 m). 2. echimidine) Aconitum alkaloids Yunaconitine. ACN Varian ODS-3 (5 m).SA S. ACN Zorbax StableBond C18 (1. The approaches and methods described do not differ much from already mentioned ones. 3: accuracy.49].9 to 291. . used turbulent-ﬂow chromatography (TFC) for analysis of three alkaloids (verticine. spasmolytic or anti-coagulant) and toxic (e.g. and a methanol/water gradient all eight coumarins could be well separated and identiﬁed in plant samples. indigo.g. 3. bergaptol. notoptol. Detection was achieved at 255 nm and by using a Perkin-Elmer Sciex API 365 triple-quad MS with ion-spray interface. Coumarins The analysis of coumarins and coumarin glycosides by LC–MS is rarely described in literature. The relevance of different processing procedures on the alkaloid content was studied by Lu et al. Other reports on the analysis of alkaloids by LC–MS describe the determination of steroidal alkaloids in Fritillaria species [48.g. capillary: 350 ◦ C.D. and p-hydroxyphenetylanisate) were determined in Notopterygium incisum and N. and based on correlation coefﬁcients close to 0. the column is ﬂushed (to remove unbound constituents). analgesic. and then the retained compounds are eluted to and separated on a second column (Zorbax StableBond C-18). foresaconitine Aconitine. Positive and negative ionization modes were recorded using the following settings: Sheath gas: N2 (80 units). quantiﬁcation was done by DAD at 240 nm. This technique enabled the quantiﬁcation of the alkaloids in rat plasma in 7 min without sacriﬁcing resolution or sensitivity (LLOQ between 0. 20 mM ammonium acetate solution in ACN XTerra RP18 (3. reported on improved analytical procedures for the above mentioned two plant species by using High Performance Thin Layer Chromatography (HPTLC) and LC–MS–MS . S: standard.SA   ESI ESI ESI APCI MS MS – MS 1–4 1–4 2 1–4 S.1% formic acid in water and acetonitrile Zorbax extend C18 (5 m). Phenols and ﬂavonoids The LC analysis of phenols and ﬂavonoids is well established and many respective reports are found in literature. mesaconitine Aconitum alkaloids Matrix Symphytum ofﬁcinale Human plasma Human urine LC-conditions Luna C18(2) (3 m). 10 mM ammoniuim bicarbonate (pH 9. 2 Appl. indigo and indigorubin in Isatis indigotica and Strobilanthes cusica .9 ng/ml yunaconitine. MS Val. Ganzera / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 744–757 Table 2 Selected LC–MS assays for the analysis of alkaloids in herbal medicines. Steinmann. auxiliary gas: N2 (20 units). Plant material was extracted with acetonitrile. notopterol. for identiﬁcation of analytes an HPLC-DAD–MSn system (ThermoQuest Finnigan LCQDECA system with ESI source) was employed.4. while isoimperatorin and notopterol can be used for quality assessment and differentiation. .: application. phototoxic) effects are reported among their pharmacological properties. e.: validated (1: sensitivity.1% formic acid in water and acetonitrile XTerra RP18 (5 m). notopterol and bergapten) were quantiﬁed in 16 batches of Rhizoma and Radix Notopterygii collected in China. By using an internal standard the amounts of individual compounds were compared.9 mg/kg.SA 749 Ref. crassicauline A.1% formic acid in water and methanol Hypurity-Advance C18 (5 m). LC-QTOF results were alike. In a semi-quantitative matter MALDI-TOF allowed the identiﬁcation of hypaconitine in 10 out of 14 “Fuzi” samples. forbesii. hypaconitine.3. isoimperaorin. An identiﬁcation of analytes was possible by their retention times.2 ng/ml foresaconitine.9 ng/ml crassicauline A. Most of the compounds were assignable via pseudo molecular ion formation in positive mode ([M+H]+ . but at the same time allowing the monitoring of a total of 57 peaks.and MS-spectra. verticinone Steroidal alkaloids Oxoprotoberberine alkaloids Tryptanthrin.SA SA S.5). SA: sample.5 mM ammonium bicarbonate (pH 10). These compounds . Coscinium fenestratum Isatis indigotica. with the exception of bergaptol. indigorubin Aconitum carmichaeli Aconitum carmichaeli (processed) Rat plasma Fritillaria spp. Fabaceae. verticinone and isoverticine) in Fritillaria thunbergii. and collision energy from 35 to 50%. M. An interesting study comparing two different MS approaches for the determination of Aconitum alkaloids was published 2009 . Val. water and ACN both with 0.8 the authors concluded that both MS-approaches are suitable for semi-quantitative proﬁling.1 and 0. and tryptanthrin. aconitine and mesaconitine were found in eight. Carcinogenic aﬂatoxins are also considered as coumarins .  ESI ESI – MS 1–4 1–4 S S. TFC is a column switching technique in which the sample is introduced to a ﬁrst column ﬁlled with coarse packing material (in this case 25 m Oasis HLB). The use of coumarins for the differentiation of herbal material was reported by Li et al.
using ESI voltages of 4.68]. ESIMS spectra were recorded from m/z 160 to 800. Tandem MS spectra were recorded on an API 4000 triple quad MS equipped with a turbo ion spray source in SRM mode. concentration of icariin in the standard solution was 4 ng/ml. Ganzera / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 744–757 Fig.5 kV in negative ionization mode. the separation and pharmacological properties of ﬂavonoids were the topic of many reviews in the past already [55–60]. the determination of icariin in rat plasma  and urine  was described. Among other compounds tentatively identiﬁed by their mass spectra. Furthermore. antimicrobial or estrogenic activities. and hypertension [65. are of special interest because of their enormous structural variability (5000 derivatives are known up to now). plants which are relevant not only for medicinal but also for food purposes . up to tumor. Reproduced with permission from . Using a similar setup.750 D. who investigated phenolic compounds in Indian medicinal plants by LC–ESI-MS. The already mentioned review by Vukics and Guttmann also summarized multi-stage MS applications of ﬂavonoid analysis. diuretic.69. At a ﬂow rate of 0. One of the main bioactive constituents in the plant is the ﬂavonol icariin. which is prepared from dried and minced leaves of Ilex paraguariensis. erectile function. 3. but due to the low amount of icariin in the samples a derivatization step with dansyl chloride was required prior to analysis. cardiovascular failures. Based on the LC–MS analysis of ﬂavonoids the differentiation of closely related plant species or those which are listed under equal names has been described [61–63].6 m) as stationary phase. chlorogenic acid and rutin were then selected for quantitative studies. Steinmann. Typical mass chromatograms of dansyl-icariin (Rt = 4. as it is the ﬁrst step to explain the beneﬁts of traditionally used medicinal plants by scientiﬁc means [67. 4 m) column.1% formic acid in water and methanol. Fig. The mobile phase consisted of 0. But besides this recent publication. which were isolated by high-speed counter-current chromatography . This greatly improved ionization efﬁciency in positive ESI mode. The chromatographic system consisted of a Phenomenex Synergi Max-RP 80A (150 mm × 2 mm.2 ml/min. choleretic. Dugo and his colleagues developed a two-dimensional LC separation system followed by MS detec- . which explains their broad spectrum of pharmacological effects and medicinal uses . Mate is a tea-like beverage especially popular in South America. should be mentioned. the identiﬁcation of (unknown) plant constituents is still of interest [64–66]. AIDS and cancer treatment. applied with a ﬂow rate of 0. As an example for the latter the study of Surveswaran et al.7) and dansyl-d5-E2 (Rt = 12. just to name a few . Thus.  developed a method for the trace analysis of icariin in human serum after oral administration of an Epimedium decoction. They selected twelve species of the Asclepiadoideae and Periplocoideae subfamilies. which were published within the last years . bone health. Epimedium species (Berberidaceae) are widely used in TCM to improve menopausal symptoms. The plant is known for numerous health beneﬁts (e. and a binary mobile phase (water/acetonitrile in the ratio of 95/5 and 5/95. Thus. Because of a complex matrix. Vukics and Guttmann reviewed their biological properties ranging from antioxidant. in this review only a few most recent reports are discussed in detail. In 2007 Gong et al. respectively.0 mm. acidiﬁed) applied in gradient mode. 4. interferences and co-elutions single stage LC-separations are usually not sufﬁcient for an accurate characterization of the respective extracts. 3 shows the mass spectra of both compounds at a concentration of 4 ng/ml.2).5 kV in positive and 3. Based on the obtained results the authors pointed out that the identiﬁed phenolic compounds signiﬁcantly contribute to the antioxidant capacity of these plants. indicating excellent sensitivity of the assay. Typical examples are the identiﬁcation of phenylethanoid glycosides in Cistanche deserticola.70]. hepatoprotective. The obtained ﬁngerprints were useful for authentication and quality control purposes. M. An innovative approach is the use of multi-stage MS coupled to a comprehensive LC system (LC × LC) for the characterization of mate extracts . The speciﬁc transitions for dansyl-icariin and dansyld5-E2 (internal standard) were recorded at m/z 910 → 764 and m/z 511 → 171. or the differentiation of diverse Celastraceae species . Qualitative and quantitative results are reported. and hypocholesterolemic).25 ml/min the required analysis time was only 16 min.g. Methanolic extracts were prepared and separated on a Shimadzu LC–MS-2010EV system using a VP-ODS C18 column (250 mm × 2.
isoeleutherin. Parent ions were acquired from m/z 170 to 200 in positive.1% formic acid in water and in acetonitrile Zorbax C18 (5 m). They are found in many medicinally used plants as biologically active constituents.: validated (1: sensitivity.SA 751 Ref. 0. eleutherin and eleutherol) in methanolic extracts of Eleutherine americana . as diuretic. and MS3 fragments ions from m/z 50 to 200. 0. and this review focuses on their LC–MS determination in relevant species belonging to the Iridaceae . methylxanthines and ﬂavonol glycosides. 1% TFA in water. 3.5 mM ammonium formate in water Symmetry C18 (5 m). reported on the ﬁrst procedure for the analysis of ﬁve naphthopyrone derivates (eleutherinoside A.7 m). The ﬁrst separation was performed on an Ascentis RP-Amide phase (250 mm × 1.1% formic acid and 0.6 mm. 0.1% formic acid in water and ACN Synergi Max-RP 80A (4 m).5 m). 2: speciﬁcity. dry gas: 10 l/min). chinensis.8 m).SA SA   Flavonoids and phenolic acids Flavonoids and phenolic acids Curcumin and derivatives Oxyresveratrol Flavonoids (gossypetin. 10 l. 0. M.D. S. 0. indicating the versatility of LC–MS.1% acetic acid in water and ACN Zorbax SB-C18 (5 m). and from m/z 330 to 680 in negative ionization mode. including hydroxycinnamoyl quinic acids. 5 m. with water and acetonitrile as mobile phase. 2. Gentianaceae  and Liliaceae  families. ACN Spherisorb ODS-2 (10 m).  Flavonoid aglyca and glycosides Zorbax SB-C18 (3. Ganzera / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 744–757 Table 3 Selected LC–MS assays for the analysis of phenols and ﬂavonoids in herbal medicines. Appl.1% formic acid in water and ACN ESI + APCI – 2 S. 2 Appl. 2 2 2 1–4 2 SA SA SA S. 2 2 S. and other spp. Table 3 lists these and other applications including experimental details (phenolic acids were discussed in Section 3.: application. The described approaches range from single stage [61–68. MS Val.1% formic acid in water and in methanol Source ESI Quant. ACN and water with FA Synergi Max-RP 80A (4 m). 0. Chrysanthemum morifolium.SA   ESI ESI ESI ESI MS DAD + MS DAD – 1–4 1–4 2 2 S.0 mm.80] to multi stage MS systems [71–73]. and then re-assayed on an Ascentis Express C18 (30 mm × 4. water and acetonitrile Symmetry C18 (3. Quinones and xanthones Quinones and xanthones are classiﬁed as oxidation products of aromatic compounds.05% TFA in water and methanol ESI ESI DAD + MS – 1–4 1. Steinmann. ﬂow rate 4 ml/min) in the second dimension. 0. The compounds of interest were unambigu- . P.1 already). 3: accuracy. Separation temperature. injection volume and detection wavelength were set to 40 ◦ C.5% formic acid in water. with usually intense color (yellow or red) and smell (quinones).5 m). 2 S. SA: sample.1% formic acid in water. Widely cultivated in China and Thailand.74–78. ilicifolia Ilex paraguariensis Geranium robertianum.7 m.SA S. HPLC separations were performed on a Synergi MAX-RP C12 column.70. phtalides Alltima C18 (5 m). Saussurea laniceps. desmethylicaritin Flavonol-3-O-glycosides ESI APCI MS MS 1–4 2 S.6 m).1% formic acid in water and in acetonitrile Zorbax SB-C18 (1.: quantiﬁcation. phenolic acids Flavonoids (e. 0. 0. lignanoids. phenolic acids Matrix Polygonum capitatum. acacetin.SA S. and 254 nm. The LC system was hyphenated with a Shimadzu ion trap-time of ﬂight (ITTOF) MS equipped with ESI source. With this technique the characterization of 26 polyphenols and xanthines in mate extracts was feasible.1 M formic acid in water and MeOH Synergi Fusion RP80 (4 m).SA  Apigenin. this plant is traditionally used for the treatment of coronary diseases. Val. 0. ACN with 0. respectively. chrysoeriol. liquiritigenin. rutin) and phenolic acids Icariin Flavonoids. medusa Asclepiadoideae and Periplocoideae species Epimedium spp. 0. S. Lamiaceae [82.05% acetic acid in water and ACN Alltima C18 (5 m). Because of technical variability and the amount of information obtained it is obvious that LC–MSn can be considered as state of the art technique for the analysis of phenols and ﬂavonoids in complex matrices such as herbal extracts. Artemisia annua.g. MS/MS fragments were recorded at m/z values from 50 to 400. eluting fractions were trapped.SA S. LC-conditions VP-ODS C18 (5 m). tion. which served for conﬁrmatory purposes only. water and acetonitrile Symmetry Shield C8 (5 m). Epimedium ssp. Uncaria tomentosa Curcuma domestica Smilax china Rhodiola rosea Uraria picta Tanacetum parthenium ESI – 2 SA  ESI ESI – – 1. nebuliser gas: 30 psi. herbacetin glycosides) Rhoifolin Antioxidant phenolics Maytenus aquifolium. were recorded in alternating ESI mode (probe temperature: 350 ◦ C. eleuthoside B. icariside I.SA S. M.SA   ESI ESI ESI ESI ESI MS – – DAD – 1. ﬂow rate 10 l/min). Paramapojn et al. Radix Angelica sinensis Epimedium spp.SA SA      Quant. phenolic acids Liquiritin. Saussurea involucrata Glycyrrhiza uralensis Radix Astragali. ACN YMC ODS (5 m). S: standard. icaritin.83]. 0.5. MS spectra. ACN Ascentis RP-Amide (5 m) and Ascentis Express C18 (2.SA S. 0. emetic and purgative.SA     Icariin Icariin. C.1% FA in water and in methanol Shimadzu C18 RP (5 m). coronarium. Analytes Flavonoids. introducing the LC eluent with a split ratio of 1:3. For LC–MS analysis an Agilent 1100 HPLC was connected to a Bruker Esquire 3000 plus ion trap. By coupling these columns with different separation modes high-resolution separations were possible. respectively. isoononin Isoﬂavonoids. 4: precision).79] and tandem mass spectrometry [69.1% formic acid in water and in acetonitrile VP ODS C18 (4.
“Danshen” (Radix Salviae miltiorrhizae) is listed in the Chinese Pharmacopeia for the treatment of coronary heart diseases and for suppressing tumor necrosis. water and acetonitrile as mobile phase. protocatechuic aldehyde (3).20% eleutherol was found to be the major naphthopyrone in most of the samples analyzed. danshexinkun D (9). miltiorrhiza extract. RP-stationary phase. denaturated and then extracted with 75% ethanol. Ganzera / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 744–757 Fig.1% formic acid) and acetonitrile. ously identiﬁed in positive (isoeleutherin. which possess anti-bacterial. With a concentration ranging from 0. . Steinmann. sweroside and swertiamarin) . The following compounds could clearly be identiﬁed (see Fig.39 V. eleutherol and eleutherinoside A) or negative mode (eleuthoside B). but recorded them in MRM mode on . HPLC chromatograms of different cell types incubated with an S. For MS analysis an Agilent G1969 LC/MSD TOF system with ESI source was used. anthelmintic. salvianolic acid B (7). Suryawanshi et al. dihydrotanshinone I (10). allowed the identiﬁcation of unknown metabolites of tanshinone I and dihydrotanshinone I. An interesting approach for the prediction of possible bioactive compounds in the same species was published by Qu et al. tonic and laxative properties. including xanthone (mangiferin) and secoiridoid glycosides (amarogentin. In their concluding remarks the authors stated that their proposed strategy was in coincidence with the holistic characteristics. Liu et al. Due to selectivity and speciﬁcity of tandem MS the authors did not attempt to separate all compounds by chromatographic means. 4. lithospermic acid (6).g. rosmarinic acid (5).752 D. extract of myocardial cells (D). anti-inﬂammatory and anti-coagulative properties [82. Major bioactive compounds in the plant are diterpenequinones like tanshinone I. M. Method validation and quantiﬁcation of the ﬁve marker compounds in plant specimens were accomplished by HPLC-UV. danshexinkun B (11). and cryptotanshinone. and extract of red blood cells (F). Based on the compounds found in these extracts (for this purpose LC–MS was used) the authors attempted to ﬁnd possible bioactive constituents. myocardial or endothelial cells). assessed the bioactive constituents in the plant by LC–ESI-MS/MS. extract of endothelial cells (C).33 and 0. The described analytical conditions are comparable to the previously mentioned ones (e. and they permitted the postulation of respective metabolic pathways. extract of blood platelets (E). Mixture of reference compounds (A). and added it to different types of target cells (e.11 to 0. Once again optimum separations were obtained on RP-material (Zorbax ODS C18) and the mobile phase comprised water (with 0. methylene tanshinone (14). tanshinone IIA. dihydrotanshinone I. Reproduced with permission from .83]. tanshinone II (15) and miltirone (16). ion trap MS in alternating mode).g. They prepared an aqueous ethanolic extract of S. 4): danshensu (1). eleutherin. MS/MS spectra. cryptotanshinone (12). tanshinone I (13). After incubation the cells were washed. miltiorrhiza. salvianolic acid D (4). studied the metabolism of these compounds in rats by LC–MS . which were recorded at collision energies between 0. Swertia chirata (Gentianaceae) is used in traditional Ayurvedic medicine because of its febrifuge. known constituents and acting sites of “Danshen”. extract of Radix Salviae miltiorrhizae (B). amaroswerin.
quinquefolius (American ginseng).05% formic acid in water and ACN Source ESI Quant. 0. three of their metabolites (glucuronide and methyl conjugates of magniferin). Rb1.: quantiﬁcation.0 mm. By LC–MS/MS it was possible to identify the glucuronides of (20S)protopanaxatriol and Rg1 as major metabolites in urine. The authors also evaluated the use of GC–MS2 for this purpose. Additionally. Rf) Ginsenosides (e.5 min only. An APCI interface in negative mode enabled ionization. ACN ESI MS 2 S. For comparison purposes the authors also analyzed the samples on a “normal” HPLC column (Symmetry Shield RP18. the samples were puriﬁed on C18 SPE cartridges and then assayed by LC–MS2 . Val. 5 m).  Ginsenosides (e. in 2008 . japonicus Panax ginseng Euphorbia kansui Centella asiatica Melia toosendan Callilepis laureola LC-conditions ACQUITY BEH C18 (1. notoginseng.7 m). Rb1. The described setup included a Waters ACQUITY UPLC system equipped with an ACQUITY BEH C18 column (100 mm × 2. In this respect it has to be mentioned that different ginseng varieties are commercially available. Analytes Ginsenosides (e. but only after enzymatic hydrolysis and derivatization both aglyca were found in spiked urine samples. 250 mm × 4.0 m). Zhang et al. F1. which often are not differentiated. An UPLC–QTOF/MS method for the differentiation of those species was published by Xie et al. japonicus. Rg1.SA S. water and methanol Eclipse C18 (5 m). Rd. Rf. The roots of this plant are utilized to treat many ailments like cancer. 10 mM ammonium acetate in water and methanol Apollo C18 (5. 0. 3 m). Further in vitro (Rb1 and Rg1 were incubated with horse liver microsomes) and in vivo experiments (1 g of Rg1 was orally administered to two horses) investigated the metabolism of ginsenosides. 4: precision). japonicus and P. After oral administration of an aqueous root decoction. the so-called ginsenosides. yet. Rg1.1 mm. P. F2) Matrix Panax ginseng. and six steroidal saponins were identiﬁed in the samples based on their UV spectra and MS fragmentation patterns. This study is of relevance because these compounds are prohibited feed additives for racing horses and therefore controlled by anti-doping agencies. Indeed. S.D. Rc. it was possible to clearly distinguish among the ﬁve species. Eleven compounds including two xanthones.SA  ESI ESI ESI ESI ESI – – DAD MS MS 2 2 1–4 1–4 1–4 S. M. 1. water. 5 m) column using a linear gradient of acetonitrile and water containing 0. Investigations were focused on the analysis of seven analytes belonging to two different aglyca subtypes. notoginseng.g.g.05% formic acid in water and ACN Supelcosil LC-8-DB (3 m). which were introduced by a speciﬁc interface. Rg1and Rh1). while iridoidO-glycosides revealed typical fragments due to cleavage of the glycoside linkage and retro-Diels-Alder (RDA) reaction within the iridoid moiety. quinquefolium Panax ginseng. LC–MS2 and LC–MS3 on the other hand allowed the direct determination of all seven intact ginsenosides in the same matrix (LOD values from 5 to 100 ng/ml in EI mode). and a Micromass Q-TOF Premier TM.: application.SA S. the obtained fractions were tested for their inﬂammatory potential in . They are Panax ginseng (Chinese or Korean ginseng). Steinmann. 2 Appl.5 m). madecassoside Toosendanin Atractyloside ACQUITY BEH C18 (1. known as “Kansui” in Chinese.1% acetic acid in water and acetonitrile Zorbax SB-C18 (53. Another recent report described the analysis of xanthones and saponins from Anemarrhena asphodeloides in rat urine . Rb1. an API 4000 triple quadrupole mass spectrometer. An ethanolic plant extract was ﬁrst separated by column chromatography using silica gel. optimum LC-separations were obtained on a Supelcosil LC-8-DB column (75 mm × 3.SA 753 Ref. Terpenes Among the most popular herbal remedies is ginseng. Therefore.g. Rd. and ginsenoside Rb2 being accountable for distinction.6 mm. water and acetonitrile XTerra Phenyl (5 m). 25 mM ammonium acetate. For respective experiments a Thermo Finnigan LCQ Advantage MS in negative ESI mode was selected. The method was validated (no detailed results are presented) and used for the quantitative analysis of four sample batches. leukemia. As this interesting study focused on differentiation of Panax species no method validation or quantitative results are presented. the required analysis time under isocratic conditions was 3. HPLC separations were performed at 20 ◦ C on a Diamonsil C18 (200 mm × 4. The latter were recorded using the information-dependent acquisition (IDA) method. P. (20S)-protopanaxadiol (ginsenosides Rb1. It is based on the assessment of speciﬁc. the plant is also known for toxic effects (e. For internal MS calibration lock masses were used (leucine-enkephalin). P. Rh1) Diterpenes (kansuinines and ingerols) Asiatic acid. Rd. All triterpenes were tentatively identiﬁed in 70% aqueous methanolic extracts by their high resolution TOF-MS spectra with mass accuracies of 2 ppm and below. with ginsenoside Rf. 3. The obtained UPLC–QTOF/MS raw data was then statistically analyzed (principal component analysis and partial least square-discriminant-analysis) to ﬁnd possible variables. P. Rg3 and Rh2) and (20S)-protopanaxatriol (ginsenosides Re. – Val.: validated (1: sensitivity. asphodeloides was conﬁrmed for the ﬁrst time the authors pointed out that their results are a signiﬁcant contribution in explaining the use of this valued medicinal plant. Ganzera / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 744–757 Table 4 Selected LC–MS methods for terpene analysis in herbal medicines and medicinal products. notoginseng. Thus. As bioavailability of these constituents in A. P.6 mm. malonyl ginsenoside Rb1.SA      Quant. 2: speciﬁcity.g. K. P. 3: accuracy. with corona discharge voltage set to 4000 V and a collision energies ranging from 20 to 60 V. 0. SA: sample.SA S. S: standard. bioactive triterpenes. or pancreatitis. but in addition to prolonged analysis time (20 min versus 80 min) the number of identiﬁed triterpenes was much higher by UPLC (25 versus 11 compounds). and argon was used as collision gas (3 mTorr). 20(S)-pseudoginsenoside F11. reported on an HPLC-DAD–MS–MS assay to investigate the inﬂammatory constituents in the plant . which allows on-column selective product ion MS/MS data acquisition by using nitrogen as collision gas.4% formic acid (analysis time 35 min). Appl. the fragmentation of [M+H]+ and [M+Na]+ ions of the analytes was studied based on their collision-induced dissociation (CID) spectra. Re.7 m). Another important medicinal plant in TCM is Euphorbia kansui. skin irritation).SA S.7 m). the UPLC approach was preferred. The determination of ginsenosides in biological matrix (horse urine after oral administration) by LC–MS was reported by Chung et al. P. Xanthone-C-glycosides showed characteristic fragment ions because of the opening of the C-glycosidic unit. operating in positive ion electrospray mode (see Table 4 for further details). .6. In a second publication the same authors used a similar approach for metabolite proﬁling in three ginseng varieties . asiaticoside.
4E. and valsartan. illegally included synthetic drugs) in herbal medicinal products [98–100]. Steenkamp et al. Under the same chromatographic conditions the performance of two different mass analyzers (Agilent 6320 Ion Trap and Agilent G1969A TOF) was tested. By LC–MS–MS it was found that this fraction mainly contained diterpenes (kansuinines and deoxyingenol derivatives). diazepam. 0. a schematic design of the setup is presented in Fig. investigated the oral bioavailability of alkamides from Echinacea purpurea formulations (EchinaforceTM ) in human plasma . which revealed that dodeca-2E. 3: accuracy. Others The LCMS analysis of analytes not covered in the previous chapters is compiled in Table 5. A few studies emphasized on the identiﬁcation of single terpenoids in plant material by LC–MS. ibuprofen) Adulterants Amlodipine. I. using sinigrin as internal standard. 2 2 2 S. 5). This and the previously mentioned studies clearly indicate that the quality of herbal products is still a major concern for consumers and . All compounds were assignable in positive ESI mode at m/z values corresponding to [M+Na]+ (ion trap) or [M+H]+ (TOF).8 m). and therefore they were considered by the authors as potential anticancer drugs.SA SA S. illegally sold in Japan for its narcotic effects.754 Table 5 Mixed analytes determined by LC–MS. indapamide.g. In 2009 Shen published a method for the analysis of major triterpenes (asiatic acid. ). acetonitrile and water Source ESI Quant. with 81 of them being advertised for “enhancing sexual potency”.5 m).1% formic acid in water and acetonitrile ESI DAD 2 S.g. valsartan ODS-80TM (5 m). examined one herbal product. Out of 50 species initially selected. published a study on adulterants in a TCM preparation called “Gold Nine Soft Capsules”. both Isatis species could be differentiated.: application. Cnidium ofﬁcinale Hypericum perforatum Linum scabrellum. so that from analytical perspectives a mostly routine application is presented. In a similar study Uchiyama et al. Appl. two biological assays. Another study focused on Mexican medicinal plants and the occurrence of podophyllotoxin type lignans therein . However.6%. For example. Ong and Ong quantiﬁed toosendanin in Melia toosendan by ion trap MS (calibration by external standard. 0. one cannabinoid analog and one cannabimimetic indole.SA Ref. Main emphasize of this study was the evaluation of different extraction procedures (microwaveassisted extraction was found to be the best in terms of extraction efﬁciency). Over 200 samples were investigated.2% acetic acid in water and ACN ˚ (5 m). and the selected compounds monitored in MRM on a SCIEX API 3000 triple-quad MS/MS equipped with a Turbo Ion Spray Interface (TIS). Quantitative results were obtained on a micrOTOF ESI-MS from Bruker Daltronics in negative ionization mode. Woelkart et al.3–7. alkylphthalides.025 ng (20 l injection) a LCQ Deca XP Plus ion trap mass spectrometer from Finnigan was ideal for quantitative studies. sildenaﬁl. content of valsartan: 40 mg/capsule) were found. the analysis of adulterants (e.  Hyperforin. With an LOD of 0. In 2006 Liang et al. Steinmann. 0. ACN Zorbax Eclipse Plus C18 (1.: quantiﬁcation.7. Analytes Phenols. Just recently. Angelica sinensis. indigotica Echinacea purpurea products Herbal products LC-conditions Alltima C18 (5 m). 0. alkylphthalides and phthalide dimers in the rhizomes of Ligusticum chuanxiong and three other related umbelliferous medicinal plants .10E/Z-tetraenoic acid isobutylamides are quickly absorbed within 15 min after oral administration. Hyptis suaveolens Isatis tinctoria. TFA in water and methanol ACQUITY UPLC HSS T3 (1. Using a mobile phase comprising methanol. asiaticoside and madecassoside) in Centella asiatica (Apiaceae) . A. 2: speciﬁcity. Charchoglyan et al. SA: sample. M.SA  ESI ESI ESI TIS – MS MS – 2 1. and the authors considered both equally suitable to identify the three markers in plant samples. published an assay for the determination of atractyloside. famotidine. acutiloba. Even it cannot be considered natural products analysis in a narrower sense. Val. S: standard. two (Linum scabrellum and Hyptis suaveolens) were found to contain such compounds.SA     Herbal product Gold Nine Soft Capsules ESI ESI – DAD 2 2 SA S. Eight respective derivatives could be identiﬁed and based on the occurrence of two of them. a “herbal based” medicine intended for treating hypertension . 1–4 Appl. 0. indigotica seeds . method precision for sample analysis 4. Kesting et al. In all of the above mentioned studies LC–MS or LC–MS/MS were used for the identiﬁcation of compounds. and the constituents in the most active (toxic) one were then determined. 10 mM aqueous Aqua C18 125 A ammonium formate (pH 4.: validated (1: sensitivity. 4: precision). Yi et al. phthalide dimers D. S. 3. The authors investigated the product by LC-HRMS (Agilent G1969A LC/MSD TOFMS) and LC–MS-SPE-NMR (Esquire 4000 ion trap with Bruker/Spark Prospect II LC–SPE–NMR interface. used HPLC-DAD–ESI–MS for the determination of phenolic compounds. DAD Val. Mohn and Hamburger used LC–ESI-MS and LC–MS/MS techniques for the qualitative and quantitative analysis of glu- cosinolates in Isatis tinctoria and I.SA S. water and TFA the samples were separated on C-18 material within 5 min. analyzed hyperforin and three polyprenylated acylphloroglucinol derivatives in in vitro cultures of Hypericum perforatum . in 28 of them (35%!) positive results for sildenaﬁl (viagraTM ) were found. captopril and amoxicillin in herbal products purchased from the Chinese market . and polyprenylated derivatives Podophyllotoxin lignans Glucosinolates Alkamides Adulterants (e.8 m). presented a semi quantitative method for the rapid determination of nine most common adulterants like sildenaﬁl.SA   Quant.01% phosphoric acid in water and acetonitrile Prevail RP C18 (5 m). Ganzera / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 744–757 Matrix Ligusticum chuanxiong. for the presence of cannabimimetic compounds .6) and acetonitrile Zorbax SB-C18 (3. indapamide. Three synthetic antihypertensive drugs (amlodipine. another type of applications should be mentioned as well. a toxic diterpene from Callilepis laureola (Asteraceae). By using GC–MS and LC–ESI-MS they found two respective compounds. namely glucoisatisin and epiglucoisatisin.8Z. dead patient: 280 ng/g) . in plant material (content 9 g/g) and human viscera (venomed.1% formic acid in water.2% formic acid in water and ACN Megachem C18 (5 m).
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