t17

Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 744757

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

journa l homepage: www.e lsev ier .co

Review

Recent

Dirk SteiInstitute of Pha

a r t i c l

Article history:Received 10 SeReceived in re10 NovemberAccepted 11 NAvailable onlin

Keywords:Liquid chromaMass spectromLCMSMedicinal planNatural produ

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7442. Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 745

2.1.2.2.2.3.

3. Applic3.1.3.2.3.3.3.4.3.5.3.6.3.7.3.8.

4. ConclRefer

1. Introdu

The inceto the begison observepositive ray

CorresponE-mail add

0731-7085/$ doi:10.1016/j.Ionization source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 745Mass analyzer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 745Detector. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 746ations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 746Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 746Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747Coumarins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 749Phenols and avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 749Quinones and xanthones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751Terpenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754Fingerprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 755

usions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 755ences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 755

ction

ption of mass spectrometry (MS) can be traced backnning of the last century, when Joseph John Thom-d the fact that magnetic and electrostatic elds deects depending on their mass. Yet, it took many years of

ding author. Fax: +43 512 507 2939.ress: [email protected] (M. Ganzera).

intense research by scientists likeAlfredNier (1940s: improvementin vacuum technologies and ion sources) or Wolfgang Paul (1953:quadrupole mass analyzer) till this technique became of practicalrelevance for the analysis of organic compounds. The followingyears (19601980) were highlighted by a multitude of techni-cal developments like MALDI (matrix-assisted laser desorption),FTMS (Fourier transform mass spectrometry), FAB-MS (fast atombombardment mass spectrometry) or ion-trap instruments, just toname a few [1,2]. Today, a mass spectrometer is nearly standardequipment in a modern analytical laboratory and indispensable for

see front matter 2010 Elsevier B.V. All rights reserved.jpba.2010.11.015advances on HPLC/MS in medicinal plant analysis

nmann, Markus Ganzera

rmacy, Pharmacognosy, University of Innsbruck, Innrain 52, 6020 Innsbruck, Austria

e i n f o

ptember 2010vised form2010ovember 2010e 19 November 2010

tographyetry

tscts

a b s t r a c t

With gaining popularity of herbal remedies worldwide, the need of assuring safety and efcacy of theseproducts 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 andversatile analytical techniques will be suitable for quality control purposes. By hyphenating high per-formance liquid chromatography and mass spectrometry (LCMS) these high demands are fullled,providing the user with a multitude of technical options and applications. This review intends to reectthe impact of LCMS for medicinal plant analysis focusing on most relevant reports published withinthe last ve years. Commenced by introductory remarks to the different MS approaches most commonlyused (e.g. ion trap and time of ight mass analyzers, fragmentation and ionization modes), respectiveLCMS applications on the analysis of natural products in medicinal plants, commercial products andbiological samples are presented. Methodological aspects like stationary and mobile phase selection orMS settings are discussed, and advantages or limitations of the described techniques are highlighted.

2010 Elsevier B.V. All rights reserved.m/locate / jpba

D. Steinmann, M. Ganzera / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 744757 745

scientic research on a high level. Due to different instrumentalcharacteristics in ionization, resolution, scan speed, and fragmen-tation MS is a versatile technique with described applications fromastronomical studies and geophysics to biology and medicine.

The analtask becausand variabi100,00020consideringChinese orplants, onlyfor controlladvantage oNMR) for thsignals direple [4]. Thutechniquesfor this purelectrophorgas chromaonly (otherhighperformpreferred seRecent deving devicesthis trend [array (DADand uoresthe analytetechniques,combined wtures (depesurprising ttive analysiover the las

This revrelevant apreported ware not covA connemto the extenclasses, anddescribed pand possiblapproachesthe next chand major dlighted.

2. Instrum

2.1. Ionizat

Any mawhich areare easier tobtained usionization (most widelmentationcompoundstric chargethe LC eluenA ne aerosdecreases d

is reached (Rayleigh limit) ions are formed by a process calledCoulombic explosion, and they enter the analyzer due to a poten-tial or pressure difference. The setup of an APCI source is similarbut charge is applied via a corona pin, which is located at the exit

atedundsve mre phundseracith

his tntias ansed fchar

ass a

r papoleig. 1betperionlynalytranlter,iblepolen celisiond aparelecten moiffereally suantcumapeds calisioned in

is ologieon ronn(FT-IvelsrecisincreIt isy, aning(ort

ron [ringsncreaccurof eundspos

d senmak

d to qysis of secondary metabolites in plants is a challenginge of their chemical diversity, usually low abundancelity even within the same species. It is estimated that0,000 metabolites occur in the plant kingdom [3], andthe fact that many traditional herbal preparations ofIndian origin contain not one but several medicinalhighly selective and sensitive methods will be suitable

ing their composition and quality. Sensitivity is the dis-f using proton nuclearmagnetic resonance analysis (1His purpose, yet it is the only technique which producesctly correlating with the amount of analytes in the sam-s, most commonly chromatographic or electrophoreticin combination with different detectors are employedpose. Due to extremely small sample volumes capillaryesis (CE)often faces sensitivityproblemsaswell [5], andtography (GC) is apowerful tool for volatile constituentswise derivatization of the analytes is required). Thus,ance liquid chromatography (HPLC)wasand still is the

paration technique for the analysis of natural products.elopments of sub 2m stationary phases and pump-enabling pressures up to 1300bar further supported

6]. The type of detector used, ranging from photodiode) or UVvis detectors, evaporative light scattering (ELS)cence detectors to MS-detection, largely depends ons investigated. Compared with the foremost mentionedthe latter offers excellent sensitivity and selectivity,ith the ability to elucidate or conrm chemical struc-

nding on the instrument used) [7,8]. Therefore, it is nothat the use of LCMS for the qualitative and quantita-s of constituents in medicinal plants steadily increasedt years.iew intends to summarize the most interesting andplications of HPLCMS for natural products analysisithin the last ve years (20062010); GLCMS reportsered as they are outside the scope of this manuscript.ent to certain plants or applications is necessary duet of the topic. The chapters are grouped by compoundspecial emphasis will be put on applicability of the

rocedures on real life samples, methodological featurese advantages or disadvantages compared to alternative. In order to familiarize the reader with the topic, inapter the basic principles of MS are briey discussedifferences of the most commonly used types are high-

entation

ion source

ss spectrometer comprises three major components,ionization source, analyzer and detector. Ions, whicho manipulate than molecules in neutral stage, can being different ionization techniques, with electrosprayESI) and atmospheric pressure ionization (APCI) beingy used [9]. ESI is a soft ionization technique (little frag-but often adducts are observed) for a wide range of, and ionization is achieved by applying a high elec-to the sample needle. The latter has an inner part fort and an outer part for a nebulising gas (e.g. nitrogen).ol is produced, in which the droplet size continuouslyue to evaporation of the solvent. Once a critical limit

of a hecomponegatipressucompothe intlamp w[11]. Tpreferephotonbeen uitively

2.2. M

Fouquadrudays (Fappliedthen suwhichother aenablemass is possquadrucollisiothe colgen) anmodestive sereactio

A despecithan qand acring shthe ionby colrepeatmationtechnocyclotrBoth celdshigh lemass p

An(TOF).velocitincreassourcereectset ofThey imass acationcompotal comto goospeedscoupletube. This technique is especially suitable for non-polar, and just like ESI it can be operated in positive andode [10]. A more recent development is atmosphericotoionization (APPI). It is considered as alternative forthat are poorly ionized by ESI and APCI, and is based on

tion of a photon beam produced by a kypton or xenonvapors formed by the nebulisation of a liquid solutionechnique usually requires the addition of a dopant, ally ionized substance that acts as intermediate betweendanalytes. Solvents like acetone, tolueneor anisole haveor this purpose, mostly leading to the formation of pos-ged compounds [12].

nalyzer

rallel aligned metal rods are the core of any single, the most economic type of mass analyzer used nowa-). The opposing rods are connected and an RF voltage isween one pair of rods and the other. Direct current ismposed, so that a quadrupolar electric eld is created,ions of a certain masses can pass through [9]. Like allzers it has to be maintained under vacuum in order tosition of the ions. A quadrupole can be considered as anot permitting the fragmentation of ions (MS/MS). Thisby arranging three quadrupole cells in sequence (triple), where the rst serves as mass lter, the second asll, and the third as lter again. Fragmentation occurs incell with the help of a collision gas (e.g. argon or nitro-

plied energy (collision energy). Different data acquiringpossible with this setup, for example the highly sensi-d reaction monitoring (SRM), also known as multiplenitoring (MRM) [13,14].nt concept is followed in ion trap analyzers, which areuitable for multiple fragmentation steps (MSn) ratheritative studies. In linear ion traps, ions are isolatedulated due to a special arrangement of hyperbolic and

electrodes as well as oscillating electric elds. Thenn be fragmented in a similar way as described above-induced decomposition (CID). This process can besequential manner, so that valuable structural infor-btained [15]. More complex and elaborate trappings are so-called 3D traps, forwhich Fourier transform ionesonance-MS (FT-ICR-MS) and Orbitrap are examples.e ions in cyclotron motion, either by strong magneticCR-MS) or electrostatic forces (Orbitrap), resulting inof resolution (typically from 100,000 to 1,000,000) andion (12ppm) [16].asingly popular type of mass analyzer is time of ightbased on the fact that molecular mass is related tod therefore compounds will reach the detector withm/z values. Crucial parameters are position of the ionhogonal or linear), length of the ight tube and the17]. The latter, also called ion mirror, consists of a, is accountable for the high resolution power of TOF.se ight distance and further focus the ions, so thatacies of 5ppm are possible. This permits the veri-lemental composition and differentiation of isobaric(with the same nominal mass but different elemen-

itions, and therefore different exact mass). In additionsitivity and resolution the technique allows high scaning it suitable for fast separations [18] and it can beuadrupole (QTOF-MS) or ion trap (IT-TOF-MS)mass l-

746 D. Steinmann, M. Ganzera / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 744757

iple qReproduced w

ters. Thus, hwell [8].

2.3. Detecto

MS deteions. In momicro-chanperforatedthe presenctiplier and wstarts [19]. Tand the resucade theMCFor much hin V or Z sh

3. Applicat

3.1. Acids

Good expreparationtoxic andcain the genunon-toxic Ssubstitutedditionally uThese confuBelgium (Cherbal slimmdent happeIn 2006 Koof them theby LCMS/M

ersedas mpectrble iwas

t toon oationFig. 1. Schematic design of a single quadrupole (A) and trith permission from [9].

igh resolution MS/MS or MSn analyses are possible as

r

ctors record either a current or charge produced by thedern instruments this is commonly achieved using thenel plate (MCP) technology. It consists of a thin plate

on revwatermass sassignaheliumwas setributipreparby tiny tubes or slots typically 610m in diameter. Ine of an electric eld each channel acts as electron mul-henever hit by an ion or photon a cascade of electronshe electrons then exit the channel on the opposite end,lting current is measured on an anode. After each cas-Phas tobe rechargedbefore it candetect another signal.igher gain not just one but two or three plates, arrangedape, are used in many instruments [20].

ions

amples for the relevance of quality control of herbals are aristolochic acids. They are a mixture of nephro-rcinogenicnitrophenantrene carboxylic acidsoccurrings Aristolochia. Due to a similar name in Chinese thetephania tetrandra (Fangji) is sometimes wronglywith Aristolochia fangchi (Guang Fangji); both are tra-sed as diuretic and for treating rheumatic conditions.sions led to the poisoning of more than 50 women inhinese herbs nephropathy), who took an adulteratedingpreparation [21]. But despite the fact that this inci-

ned nearly twenty years ago the problem still persists.h et al. analyzed ten samples of Fangji, and in ninepresence of aristolochic acid I (AA I) was conrmedS [22]. The methanolic plant extracts were separated

used it canare low (0.LCMS wasofAA I,whicing to the samethodolog

A moretolochic acor A. mollisstudied theAA I and arquantiedand MS (Mison of botnature notby similar Lby UV at 25the describbecause ofcharacteris[MH2O+Hthe procedua smaller eAA I [25] oronly [26].

Numerofeic acid anuadrupole (B) mass analyzer.

phase material (Luna C18; 5m) using methanol andobile phase. For MS studies a Finnigan LCQ ion trapometer with APCI source was utilized, with AA I beingnpositivemodeas [M+NH4]+ adduct. For fragmentationused as collision gas and the relative collision energy

35%. In another study Zhao et al. investigated the dis-f aristolochic acids in Herba Asari including the safest

method [23]. They concluded if an aqueous roots is

be considered a safe remedy, as the amounts of AA I

08g/g compared to 0.32g/ml in methanol extract).not only used for identication but also quanticationhwas recorded inSIMmodeatm/z=359.1, correspond-me adduct as mentioned above (see Table 1 for furtherical details).comprehensive investigation of the different aris-

ids in Aristolochia species (e.g. A. contorta, A. fangchisima) was described by Yuan et al. in 2007 [24]. Theyoccurrence of six acids (AA I, AA II, AA C, AA D, 7-OH

istolochic acid) together with four aristololactams, andthese constituents using diode array detection (DAD)icromass ZQ 4000 in positive ESI mode). A compar-h approaches showed that aristolochic acids are inprovided with high ionization efciency, as indicatedOD values determined (e.g. for AA I by MS: 12ng/ml;4nm: 15ng/ml). Nevertheless, the authors considereded LCMS assay suitable for quality control purposesits good selectivity. They also studied the ionizationtics of aristolochic acids (with ESImultiple products like]+or [MNO2+H]+ were observed) and fully validatedre. Other LCMS studies targeting aristolochic acids to

xtend are those on the biochemical effects induced bya cooperative study usingMS for conrmation purposes

us reports on the analysis of phenolic acids (e.g. caf-d its derivatives) by LCMS have been published. They


Table 1Selected LCMS assays for the analysis of acids in herbal medicines.

Analytes Matrix LC-conditions Source Quant. Val. Appl. Ref.

Aristolochic acid I Asarum spp. Alltech C18 (5m), 10mM ammonium acetate,formi

ESI MS 1, 2 S,SA [23]

AristolochicAA II, AA C

ater

Chlorogenicgeniposid

anol

Caffeic, chloprotocate

d, me

Chlorogenicmetabolit

acid in

Hydroxycinn water

Caffeic acids anolAmino acids onium

Amino acids mic ac

Quant.: quanti pl.: ap

describe ththereof (e.gfood items ([30] or urinparable anaRP stationaAPCI werecation andstudied ninulic acid anasked to drdeveloped Lmentionedmizing septriple quadrmethod shoand 0.6ng/mthe compouthe authorsones.

Only reccle beam mderivativesas a transpof samplevapors or cTwo ioniza(EI), were tha Thermabemolecules cphase by thnegative gloduced in a[33]. A comMS spectrativity divercaftaric acidchlorogenicversus 3.10both source(GD showed

Two meplant materthe direct didentied p[35]. The f

ge Hde ondartheyableetwran

of seydroed deMSs als

kaloi

elleninca ave bmpossilagatmet couve cods, ssatura LCcopsalitaed,metmethanol, acetonitrile, water (+0.1%acids (AA I,, AA D)

Aristolochia andAsarum spp.

XDB C18 (4m), 0.2% acetic acid in wmethanol

acids andic acid

Eucommia ulmoides Agilent C18, 0.5% acetic acid in meth

rogenic,chuic acid

Salvia miltiorrhiza Kromasil C18 (5m), 0.2% formic aciand acetonitrile

acids andes

Human plasma Inertsil ODS-2 (5m), 50mM aceticwater and acetonitrile

amic acids Human plasma andurine

Luna C18 (5m), 0.1% formic acid inacetonitrile

Echinacea product Alltima C18 (5m), water and methBarley Luna SCX 100 A (5m), 30mM amm

acetate (pH 6), 5% acetic acid(derivatized) Gentiana dahurica Hypersil BDS-C18 (5m), 30mM for

water, and acetonitrile

cation; Val.: validated (1: sensitivity; 2: specicity; 3: accuracy; 4: precision); Ap

eir determination in medicinal plants and preparations. Eucommia ulmoides [27], Salvia miltiorrhiza [28]), ine.g. propolis [29]), and inbiologicalmatrices likeplasmae [31]. As can be seen in Table 1, in most reports com-lytical conditions were described, which are based onry phases and acidic mobile phases. MS, either ESI oremployed as ionization sources, was used for identi-/or quantication purposes. Matsui et al. for examplee chlorogenic acids and some of their metabolites (fer-d caffeic acid) in human plasma [30]. Volunteers wereink a green coffee bean extract and, based on a newlyCESI-MS/MS assay, the pharmacokinetics of the abovecompounds were investigated. After carefully opti-aration conditions, collision energies and by using aupoleAPI-4000 (AppliedBiosystems) inMRMmode thewed excellent sensitivity with LOD values between 0.1l. By selecting three diagnostic product ions for each ofnds their unequivocal assignment was possible. Thus,considered their assay superior to previously reported

ently Castro et al. used liquid chromatography parti-ass spectrometry (LCPB/MS) to determine caffeic acidin Echinacea extracts [32]. The particle beam servedort-type interface enabling continuous introductionsolutions into the sources without residual solventhanging the natural chromatographic characteristics.tion sources, glow discharge (GD) or electron impacten alternately employed followed by MS detection onam LC/MS quadrupole mass spectrometer. In GD theollide with a cathodic surface, are released into the gas

exchanESI monal stamodeacceptrates btomoleterms3,4-dihobtaintion. LCauthor

3.2. Al

Exc[36], V[38] haother iand Tuthe treagainsbioactialkaloi1,2-unlished(e.g. lyFor ququantispectroermal vaporization, followed by ionization within thew region; in EI the molecules are hit by electrons pro-wire by thermionic emission and thereby are ionizedparison of both ionization techniques revealed similarin terms of fragmentation patterns. Considering sensi-gent observations were made, for example the LOD ofwas 0.64ng/ml byEI and7.70ng/ml byGD,whereas foracid just an opposite trend was observed (4.85ng/mlng/ml). Quantitative results were nearly identical bys, but in respect to repeatability EI was advantageousrelative standard deviations of up to 16.8%).

thods for the LCMS determination of amino acids inial should be mentioned as well. Thiele et al. describedetermination of amino acids in barley [34], Sun et al.reviously derivatized compounds in Gentiana dahuricaormer separated 20 amino acids on a strong cation

tion and thon a LTQ Odirectly assitive ESI mohelpful in dstructurallyof differenttion and reamount waunder presout applyinstudy of thsenecioninepyrrolizidinextractionby the instc acid)and ESI DAD+MS 14 S,SA [24]

APCI 2 SA [27]

thanol ESI MS 14 S,SA [28]

ESI MS 14 S,SA [30]

and ESI 2 S,SA [31]

GD EI MS 14 S,SA [32]ESI MS 14 S,SA [34]

id, ESI FD 14 S,SA [35]

plication; S: standard; SA: sample.

PLC column, and then quantied them in positiven a triple quadrupole MS (Fig. 2). By using an inter-d (deuterated phenylalanine) and selecting the MRMwere able to detect the analytes as low as 0.1M withprecision (rel interday 8.8%) and accuracy (recoveryeen 64 and 115%). With LOD-values in the low fem-ge the method described by Sun was advantageous innsitivity, but it required derivatization with 1,2-benzo-carbazole-9-ethyl chloroformate prior to analysis. Therivatives were then quantied by uorescence detec-was used for conrmatory purposes only, forwhich the

o discussed the fragmentation characteristics in detail.

ds

t reviewson the analysis (incl. LCMS) of ergot alkaloidslkaloids [37], and pyrrolizidine alkaloids in Delphiniumeen published already. The latter are also found in twortant medicinal plants, Symphytum ofcinale (comfrey)o farfara (coltsfoot). Comfrey is a traditional remedy fornt of arthritis and sprains, coltsfoot decoctions are usedghs and as an anti-inammatory drug. Besides desirednstituents both herbs contain liver toxic pyrrolizidineo that in countries like Austria only extracts free ofated derivatives are commercialized [39]. Liu et al. pub-MS method for the determination of several alkaloids

amine, echimidine and lasiocarpine) in S. ofcinale [40].tive and quantitative studies (only lycopsamine waswithout method validation) a LCQ-Duo ion trap masser fromThermoFinniganwasused, structural conrma-

e identication of additional constituents was achievedrbitrap hybrid mass spectrometer. All alkaloids wereignable at m/z values corresponding to [M+H]+ in pos-de. MS2 and MS3 spectra were found to be especially

ifferentiating symviridine N-oxide and echimidine, twoclosely related minor alkaloids. Additionally, the effectextraction procedures (pressurized hot water extrac-

uxing) on the content of lycopsamine was studied. Itss markedly decreased if the samples were extractedsure at 60 C compared to a higher temperature with-g pressure. This observation is in contrast to a similare same group on the determination of senkirkine and

in Tussilago farfara [41]. These compounds are alsoe alkaloids, but the results were comparable with bothprocedures. A discrepancy that the authors explainedable nature of lycopsamine. Otherwise similar analyt-


FigReproduced w

ical approacolleaguesthis plant islosses and tbriey here

Severalcarmichaelifew years.America anits tuber pdiotonic heuse of procsure steam. 2. Selected ion chromatograms of 20 underivatized amino acids (20M each) and the iith permission from [34].

ches as mentioned before were utilized. Joosten andstudied pyrrolizidine alkaloids in Jacobaea vulgaris. Asnot of medicinal interest but responsible for livestock

hecontaminationofmilkandhoney it only ismentioned[42].reports on the analysis of alkaloids in Aconitum(Ranunculaceae) have been published within the lastThe plant is widely distributed in Asia and Northernd, despite of the presence of toxic diterpene alkaloids,lays an important role in TCM as analgesic and car-rbal medicine. In the Chinese Pharmacopeia only theessed plant material (by soaking in salt water or pres-ing; the product is called Fuzi) is allowed, with a

maximum aNeverthelethey supprneurotoxicthe analysistine) in huby LCMS/Mtridges (Oaseparationsniumacetat9.5 for eluti[45] is morsamples fornternal standard d2-Phe (10M) recorded in MRM mode.

lkaloid content of 0.15% (determined by titration) [43].ss, the alkaloids need to be carefully monitored becauseess the inactivation of sodium channels and lead toand cardiotoxic degenerations. Kaneko et al. describedof major Aconitum alkaloids (e.g. aconitine, mesaconi-

man plasma [44], Wang their determination in urineS [45]. Both required a sample cleanup on SPE car-

sis MCX or BondElut certify HF) prior to analysis. LCwereperformedonC-18phases, eitherusinganammo-e buffer or an ammoniumbicarbonate solutionwith pHon (see Table 2). From practical perspectives referencee relevant, because Kaneko only used spiked plasmadeveloping and validating the assay. Wang reported


Table 2Selected LCMS assays for the analysis of alkaloids in herbal medicines.


Pyrrolizidine alkaloids(lycopsamechimidin

Symphytum Luna C18(2) (3m), water and acetonitrile both ESI MS 2 S,SA [40]

Aconitum alk um ac

Yunaconitincrassicaulforesacon

niuim

Aconitine, hmesaconi

id in w

Aconitum alk ium

Verticine, ve % form

Steroidal alk d ACN

Oxoprotobealkaloids

1% for

Tryptanthrinindigorub

r and


the followinyunaconitintine. An intfor the dete[46]. Plantresulting sousing an ESter MALDI-Tof 14 Fuzieight. LC-Qthe monitorthe amounton correlatiboth MS-apThe relevancontentwasmatory purConcerningamong diffe291.9mg/k

Other rethe determiberberine tythrin, indigcusica [51].much fromturbulent-(verticine, vis a columnduced to a case 25mconstituentseparated onique enab7min witho0.1 and 0.6

3.3. Couma

The analrarely descrderivates, wApiaceae, F

th dxic (egica

marinuse

portenetis-ferined

s areundedADSI sodes2 (80olta

16Vl C18nt alplanmol

M+Nin nine,e)

ofcinale with 0.1% formic acid

aloids Human plasma Shiseido UG80 C18, 20mM ammonisolution in ACN

e,ine A,itine

Human urine XTerra RP18 (3.5m), 10mM ammobicarbonate (pH 9.5), ACN

ypaconitine,tine

Aconitumcarmichaeli

Varian ODS-3 (5m), 0.1% formic acand acetonitrile

aloids Aconitumcarmichaeli(processed)

XTerra RP18 (5m), 2.5mM ammonbicarbonate (pH 10), ACN

rticinone Rat plasma Zorbax StableBond C18 (1.8m), 0.1acid in water and acetonitrile

aloids Fritillaria spp. Zorbax extend C18 (5m), water anwith 0.03% diethylamine

rberine Cosciniumfenestratum

Zorbax Eclipse XDB C18 (3.5m), 0.acid in water and methanol

, indigo,in

Isatis indigotica,Strobilanthes cusica

Hypurity-Advance C18 (5m), wateboth with 0.005% TFA


g alkaloid levels in the urine of a patient: 50.9ng/mle, 8.9ng/ml crassicauline A, and 1.2ng/ml foresaconi-

eresting study comparing two different MS approachesrmination of Aconitum alkaloids was published 2009

material was extracted with acetonitrile, and then thelutions assessed by MALDI-TOF and LC-QTOF; the latterI source in positive mode. In a semi-quantitative mat-OF allowed the identication of hypaconitine in 10 out

samples, aconitine and mesaconitine were found inTOF results were alike, but at the same time allowinging of a total of 57 peaks. By using an internal standards of individual compounds were compared, and basedon coefcients close to 0.8 the authors concluded thatproaches are suitable for semi-quantitative proling.ce of different processing procedures on the alkaloidstudiedby Lu et al. [47]. Theyutilized LCMS for conr-

poses only; quantication was done by DAD at 240nm.thealkaloid levels enormousdifferenceswereobservedrent samples, e.g. for mesaconitine ranging from 1.9 to

g.ports on the analysis of alkaloids by LCMS describenation of steroidal alkaloids in Fritillaria species [48,49],pe alkaloids inCoscinium fenestratum [50], and tryptan-o and indigorubin in Isatis indigotica and Strobilanthes

lies. Boand tomacoloas cou

Thewas renodakeO-trandetermspeciecopeiaperformHPLC-Dwith Etion mgas: Nspray v+18V/monsigradieed inpseudoand [2ionizedThe approaches and methods described do not differalready mentioned ones, except for [48]. Xin et al. usedowchromatography (TFC) for analysisof threealkaloidserticinone and isoverticine) in Fritillaria thunbergii. TFCswitching technique in which the sample is intro-

rst column lled with coarse packing material (in thisOasis HLB), the column is ushed (to remove unbounds), and then the retained compounds are eluted to andn a second column (Zorbax StableBond C-18). This tech-led the quantication of the alkaloids in rat plasma inut sacricing resolution or sensitivity (LLOQ between

ng/ml).

rins

ysis of coumarins and coumarin glycosides by LCMS isibed in literature. Coumarins are natural benzopyronehich are commonly found in plants belonging to the

abaceae, Rutaceae, Asteraceae and Umbelliferae fami-

In 2007for the abomance ThinThe latter eand water/at 255nm aMS with ionlytes was pas well as Mnotopteroland Radix Ntent of bergcompound,quality asse

3.4. Phenol

The LC amany respeetate ESI 14 S [44]

ESI MS 14 S,SA [45]

ater ESI MS 14 S,SA [46]

ESI DAD 14 S,SA [47]

ic ESI MS 14 S,SA [48]

both ESI MS 14 S,SA [49]

mic ESI 2 SA [50]

ACN APCI MS 14 S,SA [51]


esirable (e.g. analgesic, spasmolytic or anti-coagulant).g. phototoxic) effects are reported among their phar-l properties. Carcinogenic aatoxins are also considereds [52].of coumarins for the differentiation of herbal materialdby Li et al. [53]. Eight different derivatives (nodakenin,n, bergaptol, isoimperaorin, notoptol, notopterol, 6-uloylnodakenin, and p-hydroxyphenetylanisate) werein Notopterygium incisum and N. forbesii. Both plantslisted in the Peoples Republic of China Pharma-

er the name Qiang Huo. Method development wasby using HPLC-DAD, for identication of analytes anMSn system (ThermoQuest Finnigan LCQDECA system

ource) was employed. Positive and negative ioniza-were recorded using the following settings: Sheathunits), auxiliary gas: N2 (20units), capillary: 350 C,

ge: 4.5 kV, capillary voltage: 13V/+25V, lens voltage:, and collision energy from 35 to 50%. With a Dia-column as stationary phase, and a methanol/water

l eight coumarins could be well separated and identi-t samples. Most of the compounds were assignable viaecular ion formation inpositivemode ([M+H]+, [M+Na]+

a]+), with the exception of bergaptol, which readily wasegative ESI mode.

Qian et al. reported on improved analytical proceduresve mentioned two plant species by using High Perfor-Layer Chromatography (HPTLC) and LCMSMS [54].

xperiments were performed on an Alltima C18 columnacetonitrile as mobile phase. Detection was achievednd by using a Perkin-Elmer Sciex API 365 triple-quad-spray interface, respectively. An identication of ana-ossible by their retention times, UV- and MS-spectra,SMS data. Three marker substances (isoimperatorin,

andbergapten)werequantied in16batchesofRhizomaotopterygii collected in China. Because of the low con-apten the authors considered it not suitable as a markerwhile isoimperatorin and notopterol can be used forssment and differentiation.

s and avonoids

nalysis of phenols andavonoids iswell established andctive reports are found in literature. These compounds


Fig. 3 .2); cReproduced w

are of speciaity (5000 dbroad spectVukics anding from antumor, AIDbesides thiscal propertipast alreadreports are

Based onclosely relanames hastion of (unas it is themedicinal pthe latter twho investby LCESI-Mand Periplofor medicinwere prepatem usingstationary pin water anMS spectraof 4.5 kV intively. Amospectra, chltive studiesthat the idethe antioxidwere useful

Epimediuimprovemediovascular

ve cot al. [an sualit

amansyed i

raph50m/acet. Typical mass chromatograms of dansyl-icariin (Rt =4.7) and dansyl-d5-E2 (Rt =12ith permission from [69].

l interest becauseof their enormous structural variabil-erivatives are known up to now), which explains theirrum of pharmacological effects and medicinal uses [5].Guttmann reviewed their biological properties rang-tioxidant, antimicrobial or estrogenic activities, up toS and cancer treatment, just to name a few [55]. Butrecent publication, the separation and pharmacologi-es of avonoids were the topic of many reviews in they [5560]. Thus, in this review only a few most recentdiscussed in detail.

bioactiGong ein humtion. Qthe lowwith dimprovmatog80A (1(waterthe LCMS analysis of avonoids the differentiation ofted plant species or those which are listed under equalbeen described [6163]. Furthermore, the identica-known) plant constituents is still of interest [6466],rst step to explain the benets of traditionally usedlants by scientic means [67,68]. As an example for

he study of Surveswaran et al. should be mentioned,igated phenolic compounds in Indian medicinal plantsS. They selected twelve species of the Asclepiadoideae

coideae subfamilies, plants which are relevant not onlyal but also for food purposes [64]. Methanolic extractsred and separated on a Shimadzu LCMS-2010EV sys-a VP-ODS C18 column (250mm2.0mm, 4.6m) ashase. The mobile phase consisted of 0.1% formic acid

d methanol, applied with a ow rate of 0.2ml/min. ESI-were recorded from m/z 160 to 800, using ESI voltagespositive and 3.5 kV in negative ionizationmode, respec-ngother compounds tentatively identiedby theirmassorogenic acid and rutinwere then selected for quantita-. Based on the obtained results the authors pointed outntied phenolic compounds signicantly contribute toant capacity of these plants. The obtained ngerprintsfor authentication and quality control purposes.m species (Berberidaceae) are widely used in TCM tonopausal symptoms,bonehealth, erectile function, car-failures, and hypertension [65,69,70]. One of the main

in gradientsis time waAPI 4000 trSRM mode.d5-E2 (inte511171.a concentraassay. Usinplasma [65

The alresummarizewhich werples are thedeserticola,chromatogrspecies [72MS coupledacterizationespeciallydried and mfor numerodiuretic, aninterferencusually norespectivetwo-dimenoncentration of icariin in the standard solution was 4ng/ml.

nstituents in the plant is the avonol icariin. In 200769] developed a method for the trace analysis of icariinerum after oral administration of an Epimedium decoc-ative and quantitative results are reported, but due toount of icariin in the samples a derivatization stepl chloride was required prior to analysis. This greatlyonization efciency in positive ESI mode. The chro-ic system consisted of a Phenomenex Synergi Max-RPm2mm, 4m) column, and a binary mobile phaseonitrile in the ratio of 95/5 and 5/95, acidied) applied

mode. At a ow rate of 0.25ml/min the required analy-s only 16min. Tandem MS spectra were recorded on aniple quad MS equipped with a turbo ion spray source inThe specic transitions for dansyl-icariin and dansyl-

rnal standard) were recorded at m/z 910764 and m/zFig. 3 shows the mass spectra of both compounds attion of 4ng/ml, indicating excellent sensitivity of theg a similar setup, the determination of icariin in rat] and urine [70] was described.ady mentioned review by Vukics and Guttmann alsod multi-stage MS applications of avonoid analysis,e published within the last years [55]. Typical exam-identicationof phenylethanoid glycosides inCistanchewhich were isolated by high-speed counter-currentaphy [71], or the differentiation of diverse Celastraceae]. An innovative approach is the use of multi-stageto a comprehensive LC system (LC LC) for the char-of mate extracts [73]. Mate is a tea-like beverage

popular in South America, which is prepared frominced leaves of Ilex paraguariensis. The plant is knownus health benets (e.g. hepatoprotective, choleretic,d hypocholesterolemic). Because of a complex matrix,es and co-elutions single stage LC-separations aret sufcient for an accurate characterization of theextracts. Thus, Dugo and his colleagues developed asional LC separation system followed by MS detec-


Table 3Selected LCMS assays for the analysis of phenols and avonoids in herbal medicines.


Flavonoids, phenolic acids Polygonum VP-ODS C18 (5m), 0.1% formic acid in water ESI MS 2 S,SA [61]

Flavonoid agglycoside

acid in

Apigenin, chacacetin,

n wat

Flavonoids (phenolic a

r and

Icariin acid

Flavonoids, lphenolic a

n wat

Liquiritin, liqisoononin

n wate

Isoavonoid acid in

Icariin wate

Icariin, icarisdesmethy

h 0.1%ormat

Flavonol-3-O id in w

Flavonoids aacids

Flavonoids aacids

Curcumin an

OxyresveratFlavonoids (

herbacetiRhoifolinAntioxidant

Quant.: quanti

tion. The rphase (250tions wereC18 (30mmdimension.modes highwas hypheTOF) MS efrom m/z 1negative iorecorded atfrom m/z 50polyphenoling hydroxyglycosides.

Table 3 ltal details (The describtandem masystems [71technical vaobvious thafor the analas herbal excapitatum, P.chinensis, and otherspp.

and in methanol

lyca ands

Chrysanthemummorifolium, C.coronarium,Artemisia annua,

Zorbax SB-C18 (3.5m), 0.1% formicwater and ACN

rysoeriol,phenolic acids

Saussurea laniceps,S. medusa

Alltima C18 (5m), 0.1% formic acid iand in acetonitrile

e.g. rutin) andcids

Asclepiadoideaeand Periplocoideaespecies

VP ODS C18 (4.6m), 0.1% FA in watemethanol

Epimedium spp. Shimadzu C18 RP (5m), 0.05% aceticwater and ACN

ignanoids,cids

Saussureainvolucrata

Alltima C18 (5m), 0.1% formic acid iand in acetonitrile

uiritigenin, Glycyrrhizauralensis

Zorbax C18 (5m), 0.1% formic acid iin acetonitrile

s, phtalides Radix Astragali,Radix Angelicasinensis


Epimedium spp. Synergi Max-RP 80A (4m), ACN andFA

ide I, icaritin,licaritin

Epimedium ssp. Synergi Max-RP 80A (4m), ACN witformic acid and 0.5mM ammonium fwater

-glycosides Maytenusaquifolium, M.ilicifolia

Symmetry C18 (5m), 0.5% formic acACNnd phenolic Ilex paraguariensis Ascentis RP-Amide (5m) and Ascentis ExpC18 (2.7m), water and acetonitrile

nd phenolic Geraniumrobertianum,Uncaria tomentosa

Symmetry Shield C8 (5m), 0.1M formic awater and MeOH

d derivatives Curcuma domestica Synergi Fusion RP80 (4m), 0.1% acetic aciwater and ACN

rol Smilax china Zorbax SB-C18 (5m), water and acetonitrgossypetin,n glycosides)

Rhodiola rosea Symmetry C18 (3.5m), 0.1% formic acid inwater, ACN

Uraria picta Spherisorb ODS-2 (10m), 1% TFA in waterphenolics Tanacetum

partheniumYMC ODS (5m), 0.05% TFA in water andmethanol

cation; Val.: validated (1: sensitivity; 2: specicity; 3: accuracy; 4: precision); Appl.: ap

st separation was performed on an Ascentis RP-Amidemm1.0mm, 5m; ow rate 10l/min), eluting frac-trapped, and then re-assayed on an Ascentis Express4.6mm, 2.7m; ow rate 4ml/min) in the secondBy coupling these columns with different separation-resolution separations were possible. The LC systemnated with a Shimadzu ion trap-time of ight (IT-quipped with ESI source. Parent ions were acquired70 to 200 in positive, and from m/z 330 to 680 innization mode, respectively. MS/MS fragments werem/z values from 50 to 400, and MS3 fragments ionsto 200. With this technique the characterization of 26

s and xanthines in mate extracts was feasible, includ-cinnamoyl quinic acids, methylxanthines and avonol

ists these and other applications including experimen-phenolic acids were discussed in Section 3.1 already).ed approaches range from single stage [6168,79] andss spectrometry [69,70,7478,80] to multi stage MS73], indicating the versatility of LCMS. Because ofriability and the amount of information obtained it ist LCMSn can be considered as state of the art techniqueysis of phenols and avonoids in complexmatrices suchtracts.

3.5. Quinon

Quinonearomatic coand smell (plants as bon their LCIridaceae [8[85] familie

Paramapof ve naphisoeleutherEleutherinethis plant ieases, as dperformedacetonitrilevolume an254nm, rewas connecing the LCserved for cing ESI modry gas: 10ESI +APCI 2 S,SA [62]

er ESI DAD+MS 14 S,SA [63]

in ESI 1, 2 S,SA [64]

in ESI MS 14 S,SA [65]

er ESI DAD+MS 14 S,SA [66]

r and ESI DAD 2 S,SA [67]

ESI 2 S,SA [68]

r with ESI MS 14 S,SA [69]

e inAPCI MS 2 SA [70]

ater, ESI 2 SA [72]ress ESI 1, 2 S,SA [73]

cid in ESI 2 S,SA [74]

d in ESI MS 1, 2 SA [75]

ile ESI 2 SA [76]ESI 2 SA [77]

, ACN ESI DAD 14 S,SA [79]ESI 2 SA [80]


es and xanthones

s and xanthones are classied as oxidation products ofmpounds, with usually intense color (yellow or red)quinones). They are found in many medicinally usediologically active constituents, and this review focusesMS determination in relevant species belonging to the1], Lamiaceae [82,83], Gentianaceae [84] and Liliaceaes.ojn et al. reported on therst procedure for the analysisthopyrone derivates (eleutherinoside A, eleuthoside B,in, eleutherin and eleutherol) in methanolic extracts ofamericana [81].Widely cultivated inChinaandThailand,s traditionally used for the treatment of coronary dis-iuretic, emetic and purgative. HPLC separations wereon a Synergi MAX-RP C12 column, with water andas mobile phase. Separation temperature, injection

d detection wavelength were set to 40 C, 10l, andspectively. For LCMS analysis an Agilent 1100 HPLCted to a Bruker Esquire 3000 plus ion trap, introduc-eluent with a split ratio of 1:3. MS spectra, whichonrmatory purposes only, were recorded in alternat-de (probe temperature: 350 C, nebuliser gas: 30psi,l/min). The compounds of interest were unambigu-


Fig. 4. HPLC cextract of endoReproduced w

ously identand eleuthevalidation aspecimensranging fronaphthopyr

DanshePharmacopfor suppresthe plant ashinone I, tanti-bacteri[82,83]. Liuby LCMS [8to the previand acetoniMS/MS spec0.33 and 0.lites of tansthe postulaapproach fosame speciaqueous ethent types ofhromatograms of different cell types incubated with an S. miltiorrhiza extract. Mixture othelial cells (C), extract of myocardial cells (D), extract of blood platelets (E), and extractith permission from [83].

ied in positive (isoeleutherin, eleutherin, eleutherolrinoside A) or negative mode (eleuthoside B). Methodndquanticationof thevemarker compounds inplantwere accomplished by HPLC-UV. With a concentrationm 0.11 to 0.20% eleutherol was found to be the majorone in most of the samples analyzed.n (Radix Salviae miltiorrhizae) is listed in the Chineseeia for the treatment of coronary heart diseases andsing tumor necrosis. Major bioactive compounds inre diterpenequinones like tanshinone I, dihydrotan-anshinone IIA, and cryptotanshinone, which possessal, anti-inammatory and anti-coagulative propertieset al. studied themetabolismof these compounds in rats2]. The described analytical conditions are comparableously mentioned ones (e.g. RP-stationary phase, watertrile as mobile phase, ion trap MS in alternating mode).tra, which were recorded at collision energies between39V, allowed the identication of unknown metabo-hinone I and dihydrotanshinone I, and they permittedtion of respective metabolic pathways. An interestingr the prediction of possible bioactive compounds in thees was published by Qu et al. [83]. They prepared ananolic extract of S. miltiorrhiza, and added it to differ-target cells (e.g. myocardial or endothelial cells). After

incubationwith 75% et(for this pupossible biowere obtainphase compMS analysiswas used. TFig. 4): dansD (4), rosm(7), danshe(11), cryptonone (14), tremarks thecidence witacting sites

Swertiamedicine bproperties.in the planand secoiridand swertiadem MS thby chromatf reference compounds (A), extract of Radix Salviae miltiorrhizae (B),of red blood cells (F).

the cells were washed, denaturated and then extractedhanol. Based on the compounds found in these extractsrpose LCMS was used) the authors attempted to ndactive constituents. Once again optimum separationsed on RP-material (Zorbax ODS C18) and the mobilerisedwater (with 0.1% formic acid) and acetonitrile. Foran Agilent G1969 LC/MSD TOF system with ESI source

he following compounds could clearly be identied (seehensu (1), protocatechuic aldehyde (3), salvianolic acidarinic acid (5), lithospermic acid (6), salvianolic acid Bxinkun D (9), dihydrotanshinone I (10), danshexinkun Btanshinone (12), tanshinone I (13), methylene tanshi-anshinone II (15) andmiltirone (16). In their concludingauthors stated that theirproposedstrategywas incoin-h the holistic characteristics, known constituents andof Danshen.chirata (Gentianaceae) is used in traditional Ayurvedicecause of its febrifuge, anthelmintic, tonic and laxativeSuryawanshi et al. assessed the bioactive constituentst by LCESI-MS/MS, including xanthone (mangiferin)oid glycosides (amarogentin, amaroswerin, swerosidemarin) [84]. Due to selectivity and specicity of tan-e authors did not attempt to separate all compoundsographic means, but recorded them in MRM mode on


Table 4Selected LCMS methods for terpene analysis in herbal medicines and medicinal products.


Ginsenosides (e.g. Rb1, Rf,K, F1, F2)

Panax ginseng, P. ACQUITY BEH C18 (1.7m), 0.05% formic acid in ESI 2 S,SA [86]

GinsenosideRd, Rg1, R

rmic

GinsenosideRd, Rg1, R

mmon

Diterpenes (and inger

hanol

Asiatic acid,madecass

in wat

Toosendanin acetoAtractylosid nium


an API 4000required anThe methodused for theally, the frawas studiespectra. Theacquisitionuct ion MSgas. Xanthobecause ofO-glycosideglycoside liiridoid moi

Anothersaponins froral adminwere puriHPLC separ(200mmtonitrile an35min). Anargon wasvoltage set60V. Elevenmetabolitesand six stertheir UV spity of theserst time thcontributio

3.6. Terpen

Amongrespect it hcommerciaPanax ginseicus and P.method forXie et al. inbioactive trsetup incluan ACQUITa Micromastrospray mpurposes th

olumaddimbersusAll t

nolicccurassebya sen stleas

Indees, wiinsention.speci. In ach fodetefter8]. Tited-dopevenrotorotoaluaaticed ud thesamepectnotoginseng, P.japonicus, P.quinquefolium

water and ACN

s (e.g. Rb1, Rc,e, Rf)

Panax ginseng, P.notoginseng, P.japonicus

ACQUITY BEH C18 (1.7m), 0.05% fowater and ACN

s (e.g. Rb1,h1)

Panax ginseng Supelcosil LC-8-DB (3m), 10mM aacetate in water and methanol

kansuininesols)

Euphorbia kansui Apollo C18 (5.0m), water and met

asiaticoside,oside

Centella asiatica Eclipse C18 (5m), 0.1% acetic acidacetonitrile

Melia toosendan Zorbax SB-C18 (53.5m), water ande Callilepis laureola XTerra Phenyl (5m), 25mM ammo

acetate, water, ACN


triple quadrupole mass spectrometer. Therefore, thealysis time under isocratic conditions was 3.5min only.was validated (no detailed results are presented) andquantitative analysis of four sample batches. Addition-

gmentation of [M+H]+ and [M+Na]+ ions of the analytesd based on their collision-induced dissociation (CID)latterwere recorded using the information-dependent(IDA) method, which allows on-column selective prod-/MS data acquisition by using nitrogen as collisionne-C-glycosides showed characteristic fragment ionsthe opening of the C-glycosidic unit, while iridoid-s revealed typical fragments due to cleavage of thenkage and retro-Diels-Alder (RDA) reaction within theety.recent report described the analysis of xanthones andom Anemarrhena asphodeloides in rat urine [85]. Afteristration of an aqueous root decoction, the samplesed on C18 SPE cartridges and then assayed by LCMS2.ations were performed at 20 C on a Diamonsil C184.6mm, 5m) column using a linear gradient of ace-d water containing 0.4% formic acid (analysis timeAPCI interface innegativemodeenabled ionization, andused as collision gas (3mTorr), with corona dischargeto 4000V and a collision energies ranging from 20 tocompounds including two xanthones, three of their(glucuronide and methyl conjugates of magniferin),

oidal saponins were identied in the samples based onectra and MS fragmentation patterns. As bioavailabil-constituents in A. asphodeloides was conrmed for thee authors pointed out that their results are a signicantn in explaining the use of this valued medicinal plant.

HPLC cbut inthe nu(25 veferred.methamass alock mducedwas thpartialables.specieonyl gdistincPanaxsentedapproa

Theurine aet al. [8prohibby antisis of s(20S)-p(20S)-palso evenzymin spikallowein theFor reses

the most popular herbal remedies is ginseng. In thisas to be mentioned that different ginseng varieties arelly available,whichoftenarenotdifferentiated. Theyareng (Chinese or Korean ginseng), P. notoginseng, P. japon-quinquefolius (American ginseng). An UPLCQTOF/MSthe differentiation of those species was published by2008 [86]. It is based on the assessment of specic,

iterpenes, the so-called ginsenosides. The describedded a Waters ACQUITY UPLC system equipped withY BEH C18 column (100mm2.1mm; 1.7m), ands Q-TOF Premier TM, operating in positive ion elec-ode (see Table 4 for further details). For comparisone authors also analyzed the samples on a normal

in negativeobtained onFurther inmicrosomeistered to twBy LCMS/Mprotopanax

Anotherknown as Ktreat manyplant is alsoreported onmatory conwas rst seobtained fracid in ESI MS 2 S,SA [87]

ium ESI 2 S,SA [88]

ESI 2 S,SA [89]

er and ESI DAD 14 S,SA [90]

nitrile ESI MS 14 S,SA [91]ESI MS 14 S,SA [92]


n (Symmetry Shield RP18; 250mm4.6mm, 5m),tion to prolonged analysis time (20min versus 80min)r of identied triterpenes was much higher by UPLC11 compounds). Thus, the UPLC approach was pre-riterpenes were tentatively identied in 70% aqueousextracts by their high resolution TOF-MS spectra withacies of 2ppm and below. For internal MS calibrations were used (leucine-enkephalin), which were intro-pecic interface. TheobtainedUPLCQTOF/MSrawdataatistically analyzed (principal component analysis andt square-discriminant-analysis) to nd possible vari-d, it was possible to clearly distinguish among the veth ginsenoside Rf, 20(S)-pseudoginsenoside F11, mal-oside Rb1, and ginsenoside Rb2 being accountable forAs this interesting study focused on differentiation ofes no method validation or quantitative results are pre-second publication the same authors used a similar

r metabolite proling in three ginseng varieties [87].rmination of ginsenosides in biological matrix (horseoral administration) by LCMS was reported by Chunghis study is of relevance because these compounds arefeed additives for racing horses and therefore controlleding agencies. Investigations were focused on the analy-analytes belonging to two different aglyca subtypes,

panaxadiol (ginsenosides Rb1, Rd, Rg3 and Rh2) andpanaxatriol (ginsenosides Re, Rg1andRh1). The authorsted the use of GCMS2 for this purpose, but only afterhydrolysis and derivatization both aglyca were foundrine samples. LCMS2 and LCMS3 on the other handdirect determination of all seven intact ginsenosidesmatrix (LOD values from 5 to 100ng/ml in EI mode).

ive experiments a Thermo Finnigan LCQ Advantage MS

ESI mode was selected, optimum LC-separations werea Supelcosil LC-8-DB column (75mm3.0mm; 3m).vitro (Rb1 and Rg1 were incubated with horse livers) and in vivo experiments (1 g of Rg1was orally admin-o horses) investigated themetabolismof ginsenosides.S it was possible to identify the glucuronides of (20S)-

atriol and Rg1 as major metabolites in urine.important medicinal plant in TCM is Euphorbia kansui,ansui in Chinese. The roots of this plant are utilized toailments like cancer, leukemia, or pancreatitis; yet, theknown for toxic effects (e.g. skin irritation). Zhang et al.an HPLC-DADMSMS assay to investigate the inam-stituents in the plant [89]. An ethanolic plant extractparated by column chromatography using silica gel, theactions were tested for their inammatory potential in


Table 5Mixed analytes determined by LCMS.


Phenols, alkylphthalides,phthalide

Ligusticum Alltima C18 (5m), acetonitrile and water ESI DAD 14 S,SA [93]

Hyperforin,polyprenyderivative

c acid

Podophyllot id in w

Glucosinolat ousetonit

Alkamides acid

Adulterantssildenal,ibuprofen

and

Adulterants form

Amlodipine,valsartan

% for


twobiologione were thfraction magenolderivaof major triin Centellawas the evaassisted extefciency),applicationditions the6320 Ion Trconsideredplant samplat m/z value

A few stpenoids inquantied ttion by ext4.37.6%; [9mination of(Asteraceae(venomed,

3.7. Others

The LCMters is comthe determphthalide dthree otherchoglyan eacylphlorogperforatumplants and t[95]. Out oand Hyptisand therefoanticanceror LCMS/MHowever, Mtechniques

latestive dof twatis sed onizat

nvestpurpith ap madies

obutyistratn it csenshe anin hted anineril ane maand Tdimers chuanxiong,Angelica sinensis, A.acutiloba, Cnidiumofcinale

andlateds

Hypericumperforatum

ODS-80TM (5m), 0.01% phosphoriwater and acetonitrile

oxin lignans Linum scabrellum,Hyptis suaveolens

Prevail RP C18 (5m), 0.2% acetic acand ACN

es Isatis tinctoria, I.indigotica

Aqua C18 125 A (5m), 10mM aqueammonium formate (pH 4.6) and ac

Echinacea purpureaproducts


(e.g.famotidine,)

Herbal products Megachem C18 (5m), TFA in watermethanol

Herbal product ACQUITY UPLC HSS T3 (1.8m), 0.1%in water, ACN

indapamide, Gold Nine SoftCapsules

Zorbax Eclipse Plus C18 (1.8m), 0.1acid in water and acetonitrile


cal assays, and the constituents in themost active (toxic)en determined. By LCMSMS it was found that thisinly contained diterpenes (kansuinines and deoxyin-tives). In2009Shenpublishedamethod for theanalysisterpenes (asiatic acid, asiaticoside and madecassoside)asiatica (Apiaceae) [90]. Main emphasize of this studyluation of different extraction procedures (microwave-raction was found to be the best in terms of extractionso that from analytical perspectives a mostly routineis presented. Under the same chromatographic con-performance of two different mass analyzers (Agilentap andAgilent G1969A TOF)was tested, and the authorsboth equally suitable to identify the three markers ines. All compoundswere assignable in positive ESImodes corresponding to [M+Na]+ (ion trap) or [M+H]+ (TOF).udies emphasized on the identication of single ter-plant material by LCMS. For example, Ong and Ongoosendanin in Melia toosendan by ion trap MS (calibra-ernal standard, method precision for sample analysis1]). Steenkamp et al. published an assay for the deter-atractyloside, a toxic diterpene from Callilepis laureola), in plant material (content 9g/g) and human viscera

cosinorespecrenceboth Isobtaintive ionet al. inacea[97]. Wion trative stuacid isadmin

Everowerwell, tdrugs)presention ofcaptopChineswaterdead patient: 280ng/g) [92].

S analysis of analytes not covered in the previous chap-piled in Table 5. Yi et al. used HPLC-DADESIMS forination of phenolic compounds, alkylphthalides andimers in the rhizomes of Ligusticum chuanxiong andrelated umbelliferous medicinal plants [93]; Char-

t al. analyzed hyperforin and three polyprenylatedlucinol derivatives in in vitro cultures of Hypericum[94]. Another study focused on Mexican medicinalhe occurrence of podophyllotoxin type lignans thereinf 50 species initially selected, two (Linum scabrellumsuaveolens) were found to contain such compounds,re they were considered by the authors as potentialdrugs. In all of the above mentioned studies LCMSS were used for the identication of compounds.ohn and Hamburger used LCESI-MS and LCMS/MSfor the qualitative and quantitative analysis of glu-

5min, and tAPI 3000 tInterface (Tthem beingthem (35%!In a similarillegally solcannabimimthey foundand one calished a stuNine Soft Cing hyperteLC-HRMS (A(Esquire 40interface; aThree synthand valsartThis and thequality of hin ESI DAD 2 S,SA [94]

ater ESI 2 S,SA [95]

rileESI MS 1, 2 SA [96]

in ESI MS 2 S,SA [97]

TIS 2 S,SA [98]

ic acid ESI 2 SA [99]

mic ESI DAD 2 S,SA [100]


in Isatis tinctoria and I. indigotica seeds [96]. Eighterivatives could be identied and based on the occur-o of them, namely glucoisatisin and epiglucoisatisin,pecies couldbedifferentiated.Quantitative resultswere

a micrOTOF ESI-MS from Bruker Daltronics in nega-ion mode, using sinigrin as internal standard. Woelkartigated the oral bioavailability of alkamides from Echi-urea formulations (EchinaforceTM) in human plasman LOD of 0.025ng (20l injection) a LCQ Deca XP Plusss spectrometer from Finnigan was ideal for quantita-, which revealed that dodeca-2E,4E,8Z,10E/Z-tetraenoiclamides are quickly absorbed within 15min after oralion.annot be considered natural products analysis in a nar-e, another type of applications should be mentioned asalysis of adulterants (e.g. illegally included synthetic

erbal medicinal products [98100]. In 2006 Liang et al.semi quantitative method for the rapid determina-most common adulterants like sildenal, diazepam,d amoxicillin in herbal products purchased from therket [98]. Using a mobile phase comprising methanol,FA the sampleswere separated on C-18material within

he selected compounds monitored in MRM on a SCIEXriple-quad MS/MS equipped with a Turbo Ion SprayIS). Over 200 samples were investigated, with 81 ofadvertised for enhancing sexual potency; in 28 of

) positive results for sildenal (viagraTM) were found.study Uchiyama et al. examined one herbal product,d in Japan for its narcotic effects, for the presence ofetic compounds [99]. By using GCMS and LCESI-MStwo respective compounds, one cannabinoid analog

nnabimimetic indole. Just recently, Kesting et al. pub-dy on adulterants in a TCM preparation called Goldapsules, a herbal based medicine intended for treat-nsion [100]. The authors investigated the product bygilent G1969A LC/MSD TOFMS) and LCMS-SPE-NMR

00 ion trap with Bruker/Spark Prospect II LCSPENMRschematic design of the setup is presented in Fig. 5).etic antihypertensive drugs (amlodipine, indapamide,an; content of valsartan: 40mg/capsule) were found.previously mentioned studies clearly indicate that the

erbal products is still amajor concern for consumers and


Fig. 5. SchemReproduced w

regulatory apreparationto be of natthetic adultalso are res

3.8. Fingerp

Chromatnese Pharmthe qualityMany tradiso that thetheir overawhole chrorecorded anefcacy). Bsimilarity erence of coindicate thehas been deand similarpounds is aanalysis eitsingle herbifolius and[103], just tstudiedby therbs used(ten herbalglutionsa an(Radix Ginsapproachesor LCESI-Mones.

4. Conclus

Thismanysis of natuinmedicinaon the currthe methodresearch. Wby high-resstances byamounts byuse of a mof establishniques furthdetectable a

In respeous as well(consequenimproves seolution. As

effects, ion suppression, a diminished linear range compared toother detectors like UV, and the high acquisition costs have to bementioned. Apart from that the desirable features certainly prevail,so that the role of LCMS as analytical key technique will further

ngth

nces

. Dompothe. Bud,Franc.M. Okew wa004). Lew.R. Suetaboa, Ana. Gans: pot4893.J. Ko

f multromar qual.J. Graroma

erbalm1103. Zhroma

216 (2. All

ectrochem. Solerometr008).A. Haressur004)Marchuplin009)Kopkalantbi. Lu,rgeted.C. Hoentiid chrhroma. MakaorningectroHejaznizatime-of1812. Guilometr. Budziley-V

. Richaroteom. Van. Jado. Vanoman41 (19.L. Kohtrandroma

iomedatic design of the LCMS-SPE/NMR system described by Kesting et al.ith permission from [100].

uthorities. Especially, as the use of TCM and Ayurvedics from questionable sources is still popular. Pretendingural origin they sometimes contain a mixture of syn-erants, which explain their (unexpected) potencies butponsible for side effects of unknown reason.

rinting

ographic ngerprinting is recommended by the Chi-acopeia as a potential and reliable strategy forcontrol of complex mixtures like herbal medicines.

tional preparations are composed of multiple herbs,analysis of selected constituents might not reect

ll quality or efcacy. Therefore, in ngerprinting thematographic/electrophoretic/spectroscopic pattern isd compared to a reference (e.g. sample with proven

y using specic software like CASES (computer aidedvaluation system) this can be done based on the occur-mmon peaks, and obtained correlation coefcientsdegree of similarity. The successful use of this approachscribed for authentication, quality assurance, stabilityity studies. Most commonly the identication of com-chieved by LCMS or LCMSn, for actual ngerprinther UV or MS-traces can be used. Typical examples forngerprinting are Euonymus alatus [101], Echinops lat-E. grijsii [102], or Dysosma versipellis and D. pleianthao name a few. Multi herb preparations that have beenhis technique include BaZhenYiMu (amixtureofninefor regulating blood circulation; [104]), Gan Lu Yinconstituents, including Artemisia capillaris, Rhemanniad Eryobotrya japonica; [105]), or Shen Mai injectioneng rubra and Radix Ophiopogonis [106]). The LCMSutilized are manifold (e.g. LCESI-TOFMS, LCAPCI-MSS), but they do not differ much from already described

ions

uscript reviews recent LCMSapplications for the anal-ral products (alkaloids, avonoids, terpenes and others)l plants, commercial products or biological uids. Basedently available literature this approach appears to beof choice for diverse analytical issues in this eld ofhether it is the identication of unknown compoundsolution MS or MSn, the conrmation of known sub-single quadrupole systems, the determination of trace

triple quadrupole or time of ight systems, or theass spectrometer for ngerprinting studies. A varietyed (ESI, APCI) and innovative (APPI) ionization tech-er broadens the spectrum of possible applications and

be stre

Refere

[1] AA

[2] R&

[3] Kn(2

[4] I.AMmtr

[5] Msi3

[6] Wochfo

[7] Mchh9

[8] J.Lch1

[9] J.Wspto

[10] Ctr(2

[11] Kp(2

[12] I.co(2

[13] J.p

[14] Wta

[15] AiduC

[16] AHsp

[17] L.ioti2

[18] Mtr

[19] HW

[20] PP

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[22] HtechBnalytes.ct to chromatographic separations no cessation is obvi-. The increasing use of sub 2m stationary phases andtly) of highly efcient pumps (e.g. UPLC) dramaticallyparation speed and sensitivity without sacricing res-disadvantages of LCMS sometimes observed matrix

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