5946860381043067632

Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 775801

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

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

Review

Capillaherbal

RobertoDepartment of

a r t i c l

Article history:Received 18 OReceived in re22 NovemberAccepted 26 NAvailable onlin

Keywords:PhytochemicaElectromigratiOptimizationHerbal drugs

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7762. Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 776

2.1. Tropane alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7802.2. Phenylethylamine alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7812.3.2.4.2.5.2.6.2.7.2.8.2.9.2.10.

3. Polyp3.1.3.2.3.3.3.4.

4. Carbo5. Lipids6. Terpe

Tel.: +39 0E-mail add

0731-7085/$ doi:10.1016/j.Aconitine alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783Quinolizidine alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783Isoquinoline alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784Pyrrolizidine alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785Indole alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785Methylxanthine alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786Morphinane alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786Miscellaneous alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787

henols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787Phenolic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788Coumarins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788Naphtoquinones and anthraquinones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789Flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7903.4.1. CZE of avonoids in borate buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7903.4.2. CZE of avonoids in phosphate or mixed phosphate-borate buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7903.4.3. CZE of avonoids using additives to BGE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7903.4.4. Electrochemical detection in CZE analysis of avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7913.4.5. EKC in analysis of avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792

hydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795nes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796

51 2099729; fax: +39 051 2099734.ress: [email protected]

see front matter 2010 Elsevier B.V. All rights reserved.jpba.2010.11.041ry electrophoresis of phytochemical substances indrugs and medicinal plants

Gotti

Pharmaceutical Science, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy

e i n f o

ctober 2010vised form2010ovember 2010e 2 December 2010

l substanceson methods

a b s t r a c t

This paper reviews the applications of electromigrationmethods in analysis of phytochemical substancesin herbal drugs and medicinal plants.

A short description of the basic principles of capillary electrophoretic techniques is rstly given, thenthe overview deals with the applications of selected methods published in the period 20052010.

The phytochemical substances have been classied according to their chemical nature (e.g. alkaloids,polyphenols, carbohydrates, lipids, terpenes) and the applied CE approaches, namely CZE, NACE, MEKC,MEEKC and CEC, together with the different detection methods, are critically discussed for each of theconsidered classes of natural compounds.

2010 Elsevier B.V. All rights reserved.

776 R. Gotti / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 775801

6.1. Monoterpenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7966.2. Diterpenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7966.3. Triterpenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7966.4. Sesquiterpenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797

7. Concl . . . . .Refer . . . . . .

1. Introdu

In the laobserved tosons may bfrom plantsources; thechemicals cfrom total ctain an imprfraction (offor biomedMedicine ofof potential

The discchallenge inseparation tnuclearmagmost imporof the new

Beside thmetabolitestitative anacrude plant

The thermultiple coone or moreity. Neverthherbal medthe quantitmost direct

Capillaryquality conseparationallows the amolecularwcations andproducts bymodes oftrophoresisis based onlytes. This aparticular,owing to thless the pHin analysislites and inet al. reviewin analysislar mode ofwhich empvents instehydrophobiwidened thwhich are king soluteimportantly

e diffcultlly suh vo.trokctrok4 byin aersads. Iintr

tionte as a coen psphordulat, butl as bimulnd nmulC u

entedsed offer (ed bis a sed byts [14illaryqueshromandolid ses, per

(in cadriveassutioneen rpreme

drug

aloid

aloidve oxnd arusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ction

st years a renewed and increasing interest has beenwardsbioactivephytochemical substances. Several rea-e invoked to explain the attention towards substanceskingdom: their availability from almost inexhaustiblerelatively low-cost (in some cases the bioactive phyto-

an be affordable if comparedwith compounds obtainedhemical synthesis); individual plant species may con-essivenumber of chemical substances andonly aminorboth plants and phytochemicals) have been studied

ical application; ethnopharmacology and Traditionalfer the opportunity for the investigation of awide rangely active principles.overy of novel chemotypes from nature is an intriguing

which bioactive assay plays a primary role. Further,echniques combinedwithmass spectrometry (MS) andnetic resonance spectrometry (NMR) are applied as thetant tools for identication and structural elucidationmolecules [1].e phytochemical investigations on new bioactive plant, there is an increasing need for qualitative and quan-lytical methods in quality control of herbal drugs andextracts.apeutic efcacy of the phytopreparations results frommponents at multiple targets, thus the quantitation ofactive components does not always represent its qual-eless, in order to provide high efcacy and safe use oficines as well as in ensuring stability of their quality,ation of active components undoubtedly represent theand important issue [2,3].electrophoresis (CE) meets the requirements for the

trol of herbal drugs because of its versatility and highpower. The wide opportunity for selectivity tuningnalysis of molecules with a wide range of polarity andeight [4,5]. Recently,Unger reviewed the current appli-the promising advances of CE in analysis of naturalfocusing on the different approaches and separation

electromigration methods [6]. Capillary zone elec-(CZE) is the simplest CE mode in which the separationdifferences in the charge-to-mass ratio of the ana-

pproach can be effective in a number of applications; inquaternary alkaloids and anthocyansanthocyanidins,e permanent charge, are ideal solutes in CZE, regard-of the running buffer. Furthermore, CZE is also suitableof other important classes of secondary plant metabo-particular alkaloids, phenolic acids and avonoids. Lied the optimisation of CZEmethods specically appliedof phytochemical bioactive compounds [7]. A particu-CZE is non-aqueous capillary electrophoresis (NACE),loy non-aqueous buffer system. Use of organic sol-ad of water rstly helps in increasing the solubility of

notablare difbe ideathe hig[810]

Eleclar elein 198choicemost vmethoSDS) iscentrageneranism ibetweelectrobe motrationas welto be sbasic aMicroeto MEKreprescompoous bustabilizwhichobtainsolven

Captechniwith cMEKCand a sparticlalso bein situphase,(EOF),separahave b

Thegrationherbal

2. Alk

Alknegatidom ac analytes but also improves selectivity. Actually NACEe set of physicochemical characteristics of the solvents,nown to affect the electrophoretic behavior by inuenc-solvent, soluteadditive and ionion interactions. Most, pKa values of basic analytes in organic solvents are

and as planmarked phare often uprotonatedtrolytes are. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797

erent from those in water allowing separations whichto be obtained in water. In addition, NACE appears toited for online couplingwithmass spectrometry due to

latility and low surface tension ofmany organic solvents

inetic chromatography (EKC), and in particular micel-inetic chromatography (MEKC) has been introducedTerabe and proved to be not only the method of

nalysis of neutral compounds but also one of thetile separation approach among the electromigrationn MEKC a surfactant (often sodium dodecyl sulfate,oduced into the background electrolyte (BGE) at con-above the critical micelle concentration in order tomicellar pseudo-stationary phase. Separation mecha-mbination of chromatographic partitioning of soluteseudo-stationary phase and continuous phase and theetic mechanism. In MEKC the separation selectivity caned not only by variation of BGE type, pH and concen-also by prot of the proper selection of the surfactanty optimizing its concentration. Because phytomarkerstaneously analysed in plant materials are often acidic,eutral compounds, MEKC is widely applied [1113].

sion electrokinetic chromatography (MEEKC), similarlytilizes as separation media, pseudo-stationary phasesby microemulsions. Typically the microemulsions aref nanometer-sized oil droplets suspended in an aque-oil-in-water microemulsions, O/W). These systems arey the presence of surfactant i.e. SDS and a co-surfactant,hort-chain alcohol such as butanol. The oil droplets aredispersion of n-octane or other types of hydrophobic].electrochromatography (CEC) as other electrokinetic

combines the electromigration of the charged solutesatographic partition; the latter event, differently to

MEEKC, is established between a liquid mobile phasetationary phase packed in a fused-silica capillary (small3m) xed by frits prepared by silica sintering. CEC canformed in monolithic stationary phases formed by thepillary) polymerizationofmonomers. InCEC themobilen through the stationary phase by electroosmotic ow

mes a at and homogenous prole that leads to highefciency. Specic CEC applications on natural productsecently extensively reviewed [15,16].sent review focuses on the applications of electromi-thods to phytochemical analysis and quality control ofs in the last ve years.

s

s are cyclic organic compounds containing nitrogen in aidation state; they are widely distributed in plant king-e thought to play role in plant protection, germination

t growth stimulants. These compounds usually have

armacological activity and alkaloids-containing plantssed as traditional medicines [17]. Alkaloids are easilyto form water-soluble salts, thus aqueous acidic elec-often used as the BGEs in CZE analysis of plant extracts.

R. Gotti / Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 775801 777

Table 1Application of electromigration techniques to the analysis of alkaloids.

Alkaloids Sample Separation conditions Detection Ref

Tropane alkaloidsCalystegine A3 and B2 Solanaceae 20mM histidine, 20mM

BES, 20% methanolIndirect UV at300 nm

[18]

Atropine Cannabis sativa 20mM ammoinum acetatepH 8.5

CZE-ESI-MS [25]

Tropine, belladonnine,norhyoscyamine, apoatropine,hyoscyamine, 6-hyoscyamine,scopolamine

Atropa belladonna 60mM ammonium acetatepH 8.5, 5% isopropanol

CE-ESI-TOF-MS [23]

Atropine, scopolamine, anisodamine Flos daturae20mM phosphate pH 7.0,4mM CD

ECL [21]

20mM phosphate pH 8.48 ECL [20]NACE: 1M acetic acid,20mM sodium acetate,2.5mMtetrabutylammoniumperchlorate inacetonitrile:2-propanol(4:1, v/v)

Dual ECL/EC [22]

3-Senecioyloxy-7-hydroxytropane

Schizanthus grahamii

NACE: 1M triuoroacetic acid,25mM ammonium formate inmethanol:ethanol (40:60, v/v)or methanol:tetrahydrofuran(80:20, v/v)

UV at 220nm andESI-MS

[24]3-Hydroxy-7-senecioyloxytropane3-Hydroxy-7-angeloyloxytropane3-Hydroxy-7-tigloyloxytropanePhenylethylamine alkaloids()-Ephedrine, (+)-pseudoephedrine Ephedra supplements 25mM phosphate-

triethanolamine, pH 2.5,7.5% highly sulfated CD

UV at 190nm [36]

()-Ephedrine, (+)-pseudoephedrine,()-N-methylephedrine

Ephedra sinica andphytopreparations

20mM Trisphosphate pH2.5, 20mg/mL DM-CD, 5%tetrabutylammoniumchloride

UV at 210nm [39]

()-Ephedrine, (+)-pseudoephedrine Ephedra Herba (MaHuang)

20mM tris-phosphate (pH4.5) containing 2.5mMCu(II)-L-lysine (molar ratio1:2)

UV at 254nm [30]

()-Ephedrine, (+)-pseudoephedrine SRM supplementsBGE: 25mM phosphatebuffer (pH 2.5)

UV at 210nm [38]Method A: 2.8% sulfatedCD + 1.2% DM-CDMethod B: 4% DM-CDMethod C: 4% HP-CD

()-Ephedrine, (+)-pseudoephedrine Ephedra herb phytopreparations NACE: 80mM ammoniumacetate, 3% acetsic acid inmethanol

LIF ex 488, em520

[33]

MEKC: 25mM tetraboratepH 9.7, 20mM SDS

LIF ex 488 em520

[31]

MEKC: 20mM tetraboratepH 9.8, 20mM SDS, 15%acetonitrile

LIF ex 488 em520

[32]

()-Ephedrine; (+)-pseudoephedrine;()-methylephedrine;()-norephedrine;(+)-norpseudoephedrine;(+)-N-methylpseudoephedrine

SRM supplements MEKC: 15mM ammoniumacetate, 35mM polysodiumN-undecenoxycarbonyl-L-leucinate, pH 6.0, 30%acetonitrile

UV and ESI-MS [40,41]

()-Octopamine, ()-synephrine,tyramine, N-methyltyramine,hornedine

Citrus species andrelated genera

70mM phosphate pH 3.1,40mM HP-CD, 9mMCD, 8.2mM DM-CD

UV at 210nm [37]

Aconitine alkaloidsAconitine, mesaconitine, hypaconitine,

benzoylaconine, benzoylmesaconine,benzoylhypaconine

Aconitum roots 200mM Tris150mMperchloric acid, 40%1,4-dioxane (pH 7.8)

UV at 214nm [46]

Aconitine, mesaconitine, hypaconitine Aconitum kusnezofi A.carmichaeli

30mM phosphate, pH 8.4 ECL [45]

35mM 1B-3MI-TFB (ionicliquid) pH 8.5

UV at 254nm [44]

20mM phosphate, 35%acetonitrile pH 9.5

UV at 235nm [43]

Quinolizidine alkaloidsMatrine, sophoridine, sophocarpine,

oxymatrineSophora avescens rootsand phytopreparations

60mM borate pH 8.5 UV at 204nm [48]

Sophocarpine, matrine, lehmanine,sophoranol, oxymatrine,oxysophocarpine, cytisine

Sophora tonkinensis 50mM phosphate pH 2.5,1% HP-CD and 3.3%isopropanol

UV at 200nm [49]


Table 1 (Continued ).


Sophoridine, matrine, oxymatrine Sophora avescens 50mM phosphate, pH 8.4 ECL [50]Matrine, oxymatrine Sophora avescens

roots,phytopreparations

NACE: 70mM ammoniumacetate, 7.0% acetic acid,10% acetonitrile inmethanol

UV at 205nm [51]

Sparteine, lupanine, angustifoline,13-hydroxylupanine

Lupinus species NACE: 100mM ammoniumformate inmethanol/acetonitrile/water70/20/10, 1% acetic acid

UV at 210nmand ESI-MS

[52]

Matrine, sophoridine, oxymatrine,oxysophcarpine, cytisine

Sophora avescens roots MEEKC: 98.2% 1mMtetraborate-2mMphosphate pH 6.5, 21mMsodium cholate, 4mMMg2+, 1.2% 1-butanol and0.6% ethylacetate

UV-FASS at 200and 214nm

[53]

Isoquinoline alkaloidsBerberine, hydrastine Hydrastis Canadensis CZE: 100mM ammonium

acetateacetic acid pH 3.4and methanol (20:80, v/v).

UV at 225nm [58]

Berberine, palmatine, jatrorrhizine,columbamine, epiberberine,coptisine, tetradehydroscoulerine,tetrahydrocheilanthifolinium

Rhizoma coptidis NACE (I): 50mMammonium acetate pH* 6.8in acetonitrilemethanol(20:80, v/v)

UV at 230, 265,350nm

[63]

CZE (II): 50mMammonium acetate pH 6.8in acetonitrilewater(50:50, v/v)

ESI-MS

Berberine, jatrorrhizine, palmatine Rhizoma coptidis, Caulismahoniae, Cortexberberidis, Cortexphellodendri, Herbachelidonii

NACE: 50mM ammoniumacetate, 0.5% acetic acid inacetonitrilemethanol(10:90, v/v)

UV at 214nm [56]

Berberine, jatrorrhzine, palmatine Rhizoma coptidis, Caulismahoniae

NACE: 35mM ammonium acetate,0.25% acetic acid inacetonitrilemethanol (5:95, v/v)

LIF (nativeuorescence)

[59]

em 488nm em520nm

Berberine, coptisine, palmatine,tetrandrine

Coptidis rhizoma MEKC: 3mMborate-10mM phosphatepH 7.3, 50mM sodiumdeoxycholate, 30%acetonitrile

UV at 270nm [64]

Berberine, coptisine, palmatine, Coptidis chinensis MEKC: 100mM phosphoricacid, 15mM SDS, 10%tetrahydrofurane, pH 1.82

UV at 264nmwith sweeping

[65]

Berberine, jatrorrhizine, palmatine Coptidis chinensis CEC: strongcation-exchangemonolithic silica column.Mobile phase: 12.5mMphosphate pH7.4-acetonitrile (40:60, v/v)

UV at 263nm [67]

Sanguinarine, chelerythrine Macleaya cordata,Chelidonium majus

NACE:40 mM ammoniumacetate,acetonitrilemethanol(1:1), acetic acid pH* 5.6

LIF (nativeuorescence)

[60]

Chelerythrine, nitidine Zanthoxylum nitidum NACE: 100mM sodiumacetate (pH 5.0) inmethanol:water (4:1, v/v)

UV at 228nm [55]

Coptisine, palmatine,N-methyllaudanine, allocryptopine,protopine, corycavidine, glaucine,corydine, bulbocapnine, corydaline,corypalmine, tetrahydropalmatine,canadine, thalictrivacine

Corydalis species NACE: 50mM ammoniumacetate, 1M acetic acid and10% methanol inacetonitrile

ESI-MS [62]

Protopine, cryptopine, corydamine,sinactine, (+)-adlumine,()--hydrastine, (+)-corlumine,()-fumarophycine,()-O-methylfumarophycine,(+)-parfumine

Fumaria ofcinalis andphytopreparations

NACE: 60mM ammoniumacetate inacetonitrile-methanol (9:1)and 2.2M acetic acid

ESI-MS [61]

Fangchinoline, tetrandrine Radix Stephaniatetrandrae andphytopreparations

NACE: 50mM ammoniumacetate, 0.5% acetic acid inacetonitrile-methanol(50:50, v/v)

UV at 214nm [57]




Fangchinoline, tetrandrine Radix Stephaniatetrandrae

Flow injection MEKC:15mM acetic acid15mMsodium acetate3% Tween205% methanol at pH 5.5

UV at 254 nm [66]

Pyrrolizidine alkaloidsSenkirkine, senecionine, retrorsine,

seneciphyllineKuan donghua and Qianliguang

MEKC: 20mM borate pH9.1, 30mM SDS and 20%methanol

UV at 220nm [70]

Sencionine, seneciphylline Gynura segetum MEKC: 120mMTris35mM lauric acid (pHnot reported) and 20%methanol

UV at 220nm [71]

Senkirkine, senecionine, retrorsine,seneciphylline

Kuan donghua MEKC: 20mM borate pH9.1, 30mM SDS and 20%methanol; analysis usingdynamic pH junctionsweeping technique

UV at 220nm [69]

Indole alkaloidsStrychnine, brucine S. nux-vomica NACE: 30 mM ammonium

acetate, 1.0% acetic acidand 15% acetonitrile inmethanol

UV at 214nm [73]

Shen jin huo luo wan NACE: 25mM Tris-boricacid (pH 4.0) and 40%methanol in acetonitrile

UV at 214nm [74]

MEKC: 50mM phosphoricacid (pH 2.0), 100mM SDSand 20% acetonitrileanalysis performed usingsweeping technique

UV at 203nm [75]

Mesembrine, mesembrenone,7

mesembrenone, mesembranol,epimesembranol

Sceletium tortuosumtablets

CZE: 50mM phosphate pH1.5

UV at 228nm [77]

Harmaline, tetrahydroharmine,harmine, harmane, harmolnorharmane

Psychotria viridis l CZE: 200mM formic acidand 7 mM ammonia inwater with 10% acetonitrile

LIF (ex 266nm)and CE-MS

[78]

Yohimbine Pausinystalia yohimbe NACE: 30mM ammoniumacetate, 0.5% acetic acid inmethanol

UV at 220 nm [76]

Methylxanthine alkaloidsCaffeine, theophylline, theobromine guarana CZE: 20mM tetraborate pH

9.2UV at 212nm [80]

Caffeine, theophylline, theobromine Beverages (coca cola,tea drinks) roastedcoffee, tea leaves

CZE: 15mM tetraborate(pH 9.2) analysisperformed using anano-valve as sampleinjector

UV at 274nm [81]

Theobromine, caffeine Green tea CD-MEKC: 25mMborate-phosphate, pH 2.5,90mM SDS and 25 mMHP-CD

UV at 200nm [86]

Thebromine, caffeine Theobroma cacao CD-MEKC: 50mMBritton-Robinson buffer atpH 2.5, 90mM SDS and 12mM HP-CD

UV at 220nm [85]

Theobromine, caffeine Tea leaves at differentfermentation degree

MEKC: 10mM phosphate,4mM borate (pH 7.0),45mM SDS and 0.5%ethanol

UV-DAD atdifferentwavelengths inthe range200280nm

[84]

Caffeine Decaffeinated coffee MEKC: 10mM carbonate(pH 11.0), 50mM SDS

UV at 206nm [83]

Caffeine, theobromine Beverages (yerba mate,coffee, cocoa, tea)

MEKC: 90 mM boratebuffer (pH 8.5), 50 mM SDS

UV at 200 nm [82]

Caffeine, theophylline Beverages CEC: organicinorganichybrid silica monolithC18-sulfonic groups usinga mobile phase containing25mM phosphateboratepH 8acetonitrile (40:60,v/v)

UV at 254 nm [87]

Morphinane alkaloidsMorphine, codeine, thebaine,

papaverine, narcotineOpium CZE: 100 mM sodium

acetateacetic acid (pH3.1)and 70% methanol

UV at 224 nm [88]




Morphine, codeine, oripavine, thebaine Industrial liquors (fromextraction processes)

CD-CZE: 100 mMTrisphosphate (pH

mM HP-CD

UV at 214 nm [90]

Morphine, c phosphate buffer(pH 3.0) + 5 mM

UV at 214nm [91]

Morphine, c 5mM borate80MI-TFB, pH 9.18

ECL [92]

Papaverine,thebaine,codeine, m

mM phosphate,DS and 25%l

UV at 200 nm [89]

Morphine, thpapaverin

rophilic/cation-monolith;mobile

mM phosphate pHcetonitrile (v/v).

UV at 224 nm [95]

Sinomeninehydroxyl-methylmo

M phosphate pH ECL [93]

mM ammonium.0% acetic acid,nitrile inl

UV at 262 nm [94]

MiscellaneoGalanthamin M phosphate at ECL [99]

Galanthamin mM ammonium.5% acetic acid,nitrile inl

UV at 280 nm [98]

Verticine, ve mM pB-3MI

Camptothec9-methox

mMM SDS

Pyrido[1,2-aoxystemodidehydrostemocurt

BES: N,N-bis(methylimidazchemical detematerial.

In addition,loids becauof hydrophBased on thsubgroups aTable 1 theber of herbasummarize

2.1. Tropan

Solanaceanisodaminboth in traof the pharcholinergicthe weak Ution may ndrugs and ranalysis oftoes (Solana300nm); th

ElectrocCE separatibased on tgenerated2.8) +30odeine, thebaine Papaver species CD-CZE:

100 mMCD

odeine, thebaine, narcotine Dried poppy samples IL-CZE: 2mM 1E-3

narceine, noscapine,reticuline, oripavine,orphine

Poppy straw samples MEKC: 5080 mM Smethano

ebaine, codeine,e, narcotine

Pericarpium papaveris CEC: hydexchangephase: 54.090% a

: 7,8-didheydro-4-3,7-dimthoxy-17-rphinan-6-one

Sinomenium acutum CZE: 50 m5.0

Sinomenium acutum NACE: 80acetate, 220% acetomethano

us alkaloidse Bulbus Lycoridis

RadiataeCZE: 18 mpH 9.0

e, haemanthamine Narcissus species NACE: 90acetate, 025% acetomethano

rticinone Fritillariae species IL-CZE: 840 mM 1

in,ycamptothecin

Nothapodytes foetida MEKC: 108.6, 90 m

dimethyl sulfox

]azepines: stemofoline,kerrin,stemofoline,isinol

Stemona curtisii, S.Morakot, S. collinsae, S.tuberose

NACE-MS: 50 mammonium aceacetic acid, 10%in acetonitrile

2-hydroxyethyl)-2-aminoethanesulfonic acid; 1B-3MI-TFB: 1-butyl-3-methylimidazololium tetrauoroborate-based ionic liquid; CD: -cyclodextrin; CD: -cyclodextrinction; ECL: electrochemiluminescence detection; FASS: eld amplied sample stacking;

organic solvents are widely used in CE analysis of alka-se non-aqueous media help in increasing the solubilityobic compounds; further they can improve selectivity.eir general structures, alkaloids are divided into severalnd the most important of them are reported in Fig. 1. InCE approaches applied in analysis of alkaloids in a num-l drugs, plant materials and phytopharmaceuticals, ared.

e alkaloids

ae contain mainly tropane alkaloids such as atropine,e and scopolamine; these plants are extensively usedditional medicine and as sources for the extractionmacologically important (parasympatolytic and anti-) alkaloids. The analytical challenge is represented byV absorption and the sensitivity of direct UV detec-ot meet the requirement of quality control of herbalelated extracts. Indirect UV detection was applied innortropane alkaloids calystegines A3 and B3 in pota-ceae) using histidine as the UV marker (detection ate detection limit (LOD) of 3g/mL was achieved [18].hemiluminescence (ECL) is frequently combined toon, as a useful and sensitive detection mode. ECL ishe chemiluminescence emission of reactive speciesby electrochemical reactions. The complex tris(2,2-

bipyridyl)ruand it lls aoxidation ohosphate -TFB

ECL [101]

borate pHand 20%

UV at 369 nm [96]ideMtate, 1Mmethanol

ESI-MS [102]

ium tetrauoroborate-based ionic liquid; 1E-3MI-TFB: 1-ethyl-3-; DM-CD: Heptakis(2,6-di-O-methyl)--cyclodextrin; EC: electro-HP-CD: Hydroxypropyl- -cyclodextrin; SRM: standard reference

thenium, Ru(bpy)32+, is often selected as an ECL labelreservoir of the detection cell; the complex undergoesn the electrode surface at the voltage of +1.3V to gen-

Fig. 1. Backbone structure of the principal alkaloids.


erate the reactive Ru(bpy)33+ [19]. In particular, in CE analysis ofalkaloids, the alkyl amine groups react with the complex to formthe excited state [Ru(bpy)32+]*whichwill decay to the ground stateemitting at 610nm. Gao et al. proposed the application of CE-ECLto the deterThe detectio(a Pt disk wPt wire as atained a solThe CE sepaalkaline contion limitsof 0.313ngbeside scop-cyclodexNACE condi

CE comESI-MS) wabelladonna land hairy rabout 0.32

2.2. Phenyl

MedicinCitrus spectheir effectlipolysis anpeople. Sp(Ma Huang(1S,2S)-(+)-(+)-norpseu(1S,2S)-(+)-effects assoFood and Ddietary supthe Nationastandard realkaloids, seral analytprovide relin ephedrain analysisTable 2. Inof these alunder acidiof Cu(II)-l-hydroxyl ga complexmatic ring cconditionswas achievrming themethod tosamples (Mranged wit(LOD 5.0[30].

Higher sand Laser-InMEKC meth(ACN) as orderivatized(DTAF). Theous conditi

in 30min at 45 C using a molar ratio (derivatization reagentto analyte) of 20:1. The method was applied to real samples ofephedra herb and its preparations (tablets and capsules); ade-quate recovery (89.6106.8%) and very high sensitivity (LOD lower

107-nicapil, aquillarytial, pHerfo

wasn tooutusi. Tha heed wportzol (tionaqueonta0minrivatcondnol)es Fe(Traetha

synthntiomctorsingaptabis is oaceuion oanalyine,. Simdrugate-cthe rdrin

onstiameis(2,

ions woresosencsup

te thon

demompleof sy) [37nneyd theeutrctorandmination of atropine and scopolamine in Flos daturae.n systemwas based on a three-electrode congurationorking electrode, an Ag/AgCl reference electrode and acounter electrode). The ECL detection reservoir con-

ution of 5mM Ru(bpy)32+ and 50mM phosphate buffer.ration of the two alkaloidswas obtained under aqueousditions (pH 8.48); by means of this method the detec-

for atropine and scopolamine were found in the order/mL [20]. Using the same detectionmode, anisodamineolamine and atropinewas analysed in Flos daturaeusingtrin (CD) as additive to the aqueous BGE [21] or undertions [22].bined to electrospray (ESI)-mass spectrometry (CE-s applied in analysis of tropane alkaloids in Atropaeaf extracts [23], steam-barkof Schizantus grahamii [24]oot cultures of Cannabis sativa [25]; detection limit ofg/mL was achieved.

ethylamine alkaloids

al herbs containing phenylethylamine alkaloids (i.e.ies and Ephedra sinica) are widespread used fors on human metabolism in particular by stimulatingd thus promoting the fat mass reduction in obeseecically, ephedra extracts such as Ephedra Herba) contain alkaloids such as: (1R,2S)-()-ephedrine,pseudoephedrine, (1R,2S)-()-norephedrine, (1S,2S)-doephedrine, (1R,2S)-()-N-methylephedrine, andN-methylpseudoephedrine [26]. The possible adverseciated to the use of these products prompted the U.S.rug Administration (FDA) to ban ephedra containingplements depending on the dosage [27]. In addition,l Institute of Standards and Technology (NIST) issuedference materials with certied values for ephedraynephrine and caffeine [28]. As a consequence sev-ical methods have been developed with the aim toiable quantitation of ephedrine and pseudoephedrineextracts and herbs [29]. Details on the CE methodsof ephedra alkaloids in real samples are reported inorder to obtain adequate selectivity in separation

kaloids, Li and co-workers developed a CZE methodc conditions (tris-phosphate pH 4.5) in the presencelysine complex as a ligand-exchange modier. Theroup and nitrogen atom of the alkaloids, can yieldwith Cu(II); in addition the -electrons of the aro-an interact with the protonated l-lysine. Under thesehigh selectivity for ephedrine and pseudoephedrineed. The authors provided spectroscopic data con-hypothesized mechanism and applied the validatedquantitation of ephedra alkaloids in Ephedra Herbaa Huang) extracted using ethanol. Recovery valueshin 95.0104.0% (RSD %


Table 2Details on the CE analysis of ephedra alkaloids.

Sample CE method Sample preparation Alkaloid contenta LOD (g/mL) Ref.

Ephedra dietary supplements Chiral CZE-UV Ultrasonication (water-HCl) ()-E: 2% 0.6 [36](+)-PE: 0.5%

Ephedra fruits Chiral CZE-UV Ultrasonication (methanol) ()-E: 2.33.3% n.r. [39](+)-PE: 1.62.5%()-ME: 0.98%

SRM Ephedra sinica Chiral CZE-UV Ultrasonication (methanol) ()-E: 0.037.6% n.r. [38](+)-PE: 0.0050.92%

SRM Ephedra sinica Chiral MEKC-ESI-MS Ultrasonication (methanol) ()-E: 0.0181.29% 0.06250.125 [41](+)-PE: 0.00120.393%()-Nor-E: 0.00030.039%(+)-Nor-PE: 0.000370.073%()-ME: 0.000930.121%(+)-MPE: 0.0000150.0061%

Ephedra herb MEKC-LIF Ultrasonication (water) E: 0.560.05% 1.41107 [31]Keke capsule Derivatization with DTAFb PE: 0.0162.94%Hei Pan tabletsBai Pan tabletsEphedra herb MEKC-LIF Ultrasonication (water) E: 0.5890.03% 1.64.8103 [32]Keke capsules in-capillary derivatization with NBD-Fc PE: 0.014 3.0%Hei Pan tabletsBai Pan tabletsEphedra herba (Ma Huang) Ligand-exchange CE Reux ethanol E: 0.600.84% 5.0 [30]

PE: 0.190.22%

n.r.: not reported.a Symbols: ()-E, ()-ephedrine; (+)-PE: (+)-pseudoephedrine; ()-ME: ()-methylephedrine; ()-Nor-E: ()-norephedrine; (+)-Nor-PE: (+)-norpseudoephedrine; (+)-

MPE: (+)-N-methylpseudoephedrine.b DTAF, 5-(4,6-dichloro-s-triazin-2-ylamino)uorescein.c NBD-F, 4-uoro-7-nitro-2,1,3-benzoxadiazole.

Fig. 2. Analys[38]). Modiedis of standard reference material (by NIST) using three different chiral CE methods. The mfrom [38].ethod conditions and symbols are reported in Tables 1 and 2 (see Ref.


Fig.

vided theStandard reopment atobtained elsamples, onand (+)-pseto be suitatial in idenHP-CD wpresence osimultaneoN-methylepsinica extra

The comperformedtaneous enN-undecenosurfactantMS detectioof four paiESI-MS det0.06250.12

2.3. Aconiti

AconitumAsia and NcarmichaeliPharmacophypaconitinaconitum palso be usedas anti-inaactivity (anof aconitum

CZE wasmesaconitinbuffer suchaqueousorliquid 1-buttion at 235of real samorder of ngtris(2,2-bipbuffer (50mof the nati

ypicaA. car

m); BGH7.8.mper

aconin

esteenzosed ahowa BGE composed of 200mM Tris buffer containing 150mMoric acid and dioxane 40% at pH 7.8, the simultaneoustion of three diester-diterpene alkaloids (the native aconi-ypaconitine and mesaconitine) and the related degradationts (benzoylaconine, benzoylmesaconine and benzoylhy-ne) was accomplished in about 25min (Fig. 4). Theed detection limit was lower than 45ng/mL. The analy-crude and processed herbal preparations clearly showedhe manufacture process affected the content of aconi-kaloids with a signicant decrease of the native diester-nes [46].

inolizidine alkaloids3. Structure of aconitine, mesaconitine and hypaconitine.

best separation of the two couples of enantiomers.ference materials (SRMs) containing ephedra in devel-NIST, were analysed by each of three CE methods. Theectropherograms are reported in Fig. 2; in the SRMly the naturally occurring enantiomers ()-ephedrineudoephedrinewere found, however themethod provedble in detecting product adulteration by its poten-tication of specic stereoisomers [38]. The neutralas also found to be a useful chiral selector in thef tetrabutylammonium chloride as additive, for theus enantioseparation of ephedrine, pseudoephedrine,hedrine and norephedrine enantiomers in Ephedracts [39].prehensive characterization of ephedra extracts wasby chiral MEKC-ESI-MS, which provided the simul-antioseparation of all ephedra alkaloids. Polysodiumxy-carbonyl-l-leucinate was used as polymeric chiralbecause it was found to be compatible with ESI-n. In the optimised conditions the enantioseparationrs of ephedrine enantiomers was achieved and theection allowed high sensitivity (LOD ranging within5g/mL) [40,41].

ne alkaloids

plants (Ranuncolaceae) are widely distributed acrossorth America and two species of them, namely A.Dexb. and A. kusnezofi are listed in the Chinese

oeia. Aconitine and the congeners mesaconitine ande (Fig. 3) are the main diester-diterpene alkaloids of

Fig. 4. T(B) fromi.d. 5040% at p25kV; tebenzoyl[46].

of thenine, bprocesfacts sUsingperchlseparatine, hproducpaconiobtainsis ofthat ttum alditerpe

2.4. Qu

lants and although they show high toxicity they canat low doses because of pharmacological effects suchmmatory and anti-pain [42]. Owing to the biological

d toxicity), CE analyticalmethods for the quality controlalkaloids in herbal drugs have been considered.applied in analysis of aconitine, hypaconitine and

e in extracts from aconitum plants using alkalineas phosphate at pH 8.59.5 in aqueous or mixed

ganic media [43], and in the presence of the ionicyl-3-methylimidazolium (1B-3MI-TFB) [44]. UV detec-or 254nm allowed the sensitivity required in analysisples, however improved LOD (108 M, thus in the/mL) was achieved by means of ECL detection usingyridil)ruthenium(II), Ru(bpy)32+, 5mM in phosphateM) [45]. Song et al. focused on the poor stability

ve aconitum alkaloids, which undergo to hydrolysis

Radix soused in TCMthat quinolherbal drugand thequaneeds for tpKa valuesmatrine, somatrine, oxTheir analybuffers botacidic condinvolved thlarly for othof tertiary al electropherograms of crude aconite roots (A) and processed rootsmichaeli. Conditions. Fused-silica capillary (39.6 cm effective length,E 200mMTris buffer containing 150mMperchloric acid and dioxane

Detection at 214nm; electrokinetic injection at 10kV for 10 s; Voltageature 25 C. Peaks: aconitine (1); mesaconitine (2); hypaconitine (3);e (4); benzoylmesaconine (5); benzoylhypaconine (6). Modied from

r moiety. Monester-diterpenes, namely benzoylaco-ylmesaconine and benzoylhypaconine, can occur inconitum herbal drugs and, interestingly, these arte-less toxicity compared to the parent compounds.phorae avescentis (Sophora avescens) is frequentlyfor treating acute hepatitis and jaundice; it was found

izidine alkaloids were the main constituents of this. Furthermore, these compounds exhibit adverse effectslity control of thequinolizidine-containingherbal drugshe accurate quantitation of the alkaloids content. Thedetermined by CZE of quinolizidine alkaloids such asphoridine, sophocarpine, lehmannine, sophoranol, oxy-ysophocarpine, were found in the range 5.907.72 [47].sis was performed by CE-UV using aqueous runningh in alkaline (borate and phosphate pH 8.5 [48]) anditions (phosphate pH 2.5 [49]). The sample preparatione extraction of plant materials using chloroform. Simi-er alkaloid types, quinolizidines, owing to the presencemine function, reactwith tris(2,2-bipyridyl)ruthenium


(II) to produce ECL response. This approach was applied to thehighly selective and sensitive analysis of S. avescens extracts.Unexpectedly, ECL response was also observed for oxymatrine(quinolizidine N-oxide), which should not be determined becauseof the lack otions the LOby CE-UV aand oxymahyphenatioalkaloids ina small percof the poteline and 13as surfactan(1.2%) as thfound to being cytisineat low concimprove thallowed thereported lim

2.5. Isoquin

Bis-benzthe importberines areammonium(e.g., RhizoPapaveracetex phellodeare berberiloids displical activitbiosynthesiuncouplingcontributeagainst bac[54].

NACE hacompoundsity of a mix4:1 v/v, conin ngerprilum nitidumand the ismatine andcoptidis, CoCaulis mahocomposed10% ACN iof about 0benylisoquiwere determpreparationby injectingsample plu[57].

Mixed opH 3.4 within separatiHydrastis canicant impby prot ofat 520nm)of NACE w

to the analysis of isoquinoline alkaloids in Fumaria ofcinalis. Amixture ACNmethanol (9:1, v/v) containing 60mM ammoniumacetate and 2.2M acetic acid was used as the running buffer.Under these conditions the migration order was found to fol-

e pKion mtion uCE-Misoqapeume Aampharaiguot anallowine, ce bioed m, proalues bumaslkalobamidroced foamerrieda cosolvme

KC anide soactias s

d palve cn) anon, cramsamporteto

hizomherbt byultrand dialidaere ind tere

) waandsepathece thtionresis

y fasts ofmon

g sulf tertiary amino function. Under these detection condi-D values were 1000-fold compared to those achieved

t 200nm [50]. NACE was applied in analysis of matrinetrine in S. avescens extracts by UV detection [51];n with ESI-MS was reported in analysis of quinolizidineLupinus species. The BGE was modied by addition ofentage of water (10%) in order to obtain the separationntially toxic alkaloids sparteine, lupanine, angustifo--hydroxylupanine [52]. MEEKC using sodium cholatet at pH 6.5 (boratephosphate electrolyte), 1-butanole co-surfactant and ethylacetate (0.6%) as the oil, wasuseful in analysis of the quinolizidine alkaloids (includ-) of S. avescens. Interestingly, Mg2+ was supplementedentration (4mM) as an EOF modier; it was found toe separation selectivity. The developed MEEKC systemapplication of eld amplied injection technique; theit of detection was 0.1ng/mL [53].

oline alkaloids

ylisoquinoline alkaloids have attracted attention forant pharmacological effects; in particular protober-

a structural class of organic cations (quaternaryalkaloids) mainly distributed in Ranuncolaceae

ma coptidis), Berberidaceae (e.g. Cortex berberdis),ae (e.g. Herba chelidoni) and Rutaceae (e.g. Cor-ndri). The most considered protoberberine alkaloidsne, palmatine and jatrorrhizine. Protoberberine alka-ay a great variety of biological and pharmacolog-ies, including inhibition of DNA synthesis, proteins, inhibition of membrane permeability, and theof oxidative phosphorylation. These processes likelyto the allelochemical and toxic effects observedteria, fungi, other plants, insects, and vertebrates

s shown to be a useful technique in analysis of thesein herbal drugs and preparations. The high selectiv-ed organic-aqueous running buffer (methanol:water,taining sodium acetate 100mM, pH* 5.0) was appliednt analysis of the Chinese herbal medicine Zanthoxy-containing the protoberberine alkaloids chelerythrine

omer nitidine [55]. Quantitation of berberine, pal-jatrorrhizine, in complex samples such as Rhizoma

rtex berberdis, Herba chelidoni, Cortex phellodendri andniae herbal drugs, was performed using a NACE bufferof 50mM ammonium acetate, 0.5% acetic acid andn methanol. UV detection at 214nm allowed LOD.3g/mL [56]. Under similar NACE conditions, bis-noline alkaloids, namely fangchinoline and tetrandrineined in Radix Stephaniae tetrandrae and its medicinal

s; high detection sensitivity (0.3ng/mL) was obtaineda short plug of ethanol before the injection of the

g, in order to achieve a eld amplied stacking effect

rganic-aqueous BGE system (ammonium acetate atacetic acid in watermethanol, 1:5 v/v) was applied

on of berberine and hydrastine in root powder ofnadensis; detection was performed at 225nm [58]. Sig-rovement of sensitivity (at ng/mL level) was achievedthe native uorescence of the compounds (emission

under laser excitation at 488nm [59,60]. Hyphenationith electrospray ion trap mass detector was applied

low thionizatmentaThe NAmajortotherThe saof 79 swere cunambponenbasis, aprotopputativproposmatine(LOD vaqueouFlight)berof acolumtetrahyvalidatloids nwas caRhizomeratedCE-MS[63].

MEto proving bi50mMine, anbioactibaicaliphyscipheroga realare repappliedRhei Rthreeried ouv/v) inuum awas vues wn=9) aloids wng/mLtisineMEKCusingenhanseparatropho[66].

Verextracboth abearina of the compounds. Furthermore the ESI (positiveode) and MS/MS experiments, provided typical frag-seful to the unambiguous identication of 10 alkaloids.S method was applied in quantitative analysis of the

uinoline alkaloids in F. ofcinalis herba and its phy-tic tablets containing protopine and cryptopine [61].uthors applied NACE-MS conditions to analyze a setles belonging to Corydalis species; 39 analytical peakscterized by the MS spectra and a number of them wereusly identied as isoquinoline alkaloids. Principal com-lysis (PCA) performed using peak areas as the dataed to select 8 compounds (among them allocryptopine,orycavidine, bulbocapnine and terahydropalmatine) asmarkers for discrimination of the Corydalis species. Theethod provided also the sensitive quantitation of pal-topine, bulbocapnine, corydaline, tetrahydropalmatines in the range 0.78.3g/mL) [62]. A mixed organic-ffer was also used in CE coupled to ESI-TOF (Time ofs spectrometry in analysis of Rhizoma coptidis. A num-idswere identied:berberine, palmatine, jatrorrhizine,ne, epiberberine, coptisine, tetradehydroscoulerine andheilanthifolinium. In addition, the CE-MS method wasr linearity, sensitivity and precision on selected alka-

ly berberine, palmatine and jatrorrhizine. The analysisout on real samples represented by dried roots of

ptidis; the sample preparation was performed by accel-ent extraction using ethanol (80% in diluted HCl). Thethod proved to be suitable also in routine analysis

alysis of benzylisoquinoline alkaloids is appliedmainlyeparation of complexmixtures in plant samples includ-ve neutral components. Using sodium deoxycholateurfactant, the MEKC separation of coptisine, berber-matine was achieved simultaneously to that of neutralompounds such as avonoids (wogonin, baicalenin,d anthraquinones (sennoside A and B, emodin, rhein,hrysophanol and aloe-emodin). In Fig. 5 the electro-s of a standard mixture of the considered analytes andle (TCM preparation San-huang-xie-xin-tang extract),d. The method showed excellent selectivity and wasthe analysis of Coptidis Rhizoma, Scutellariae Radix,a (rhubarb) and phytopreparations containing thementioned above. The sample preparation was car-exhaustive extraction using methanolwater (70:30,sonic bath. The extracts were concentrated under vac-ssolved in acetonitrilewater (70:30, v/v). The methodted for linearity, precision, accuracy (recovery val-n the range 98.12107.58%, RSD % inter-assay


Fig. 5. (A) Elec in thephyscion (6), w 2), rheof a real samp sodiuacetonitrile. Fu ed fro

tions (MS-Swas compo60% ACN asmonolithiction selecti[67].

2.6. Pyrroli

Pyrrolizispecies groto the Coera. Someused in TCKuan dongchronic brever, besidof pyrrolizof attentionHealth Bure[68].

Analytichave beenpyrrolizidinsenecioninetain the 4-seneciphyllC13-C23 douthese strictSDS [70] ananalysis ofplant mateaddition ofthe requirejunctionswbased MEKextract wasinto the capThe positivplug towarthe high pare convertpartitioninghigher than

dole

ole aph

nt acchnosic, he, art risehe qin peingE wdiads wcetassolvnd balysisCM)hano0 inpliedre v

g/mL78tropherogram of standard mixture of coptisine (1), berberine (2) and palmatine (3)ogonin (7) baicalin (8), baicalein (9), sennoside B (10), sennoside A (11), emodin (1

le (San-huang-xie-xin-tang extract). Conditions: 3mM sodium tetraborate10mMsed silica capillary (49 cm effective length, i.d. 50m); detection at 270nm. Modi

CX). The mobile phase used in both the applicationssed of phosphate buffer pH 7.4 in the presence oforganic modier. The two methods (conventional CECcolumn and MS-SCX column) showed similar separa-vity (separation prole and migration/retention time)

zidine alkaloids

dine alkaloids are found in a variety of plantwing wideworld such as Gynura segetum that belongsmpositae family and Senecio and Tussilago gen-of these plants and phytopreparations are ofciallyM with the vernacular name of Qian liguang andhua for the treatment of pharyngeal infection andonchitis, asthma and inuenza, respectively. How-e the pharmacological effect, due to the presenceidine alkaloids, these TCM remedies are also focus

of health control Authorities (e.g. German Federalau) for their potential genotoxicity and carcinogenicity

al separation methods involving HPLC and CEproposed for the quantitation of these toxic

es. The most important pyrrolizidine alkaloids, seneciphylline, retrorsine and senkirkine, con-azabicyclo[3.3.0]octane system with senecionine and

2.7. In

Indof themdiffere

Stryanalgebrucinnican[72]. Twell asindex b

NACous mealkaloinium aacid dinine athe anning (Tin metpH* 4.the apods we0.02and0.5ine differing only for the presence in the latter of theble bond. Owing to the same pKa values (5.44) [69],

ly related compounds can be separated only by MEKC.d lauric acid [71] as surfactants were used in MEKCQian liguang and Kuan donghua and Gynura segetumrial, respectively. Using both the MEKC systems themethanol (20%) to the BGE was necessary to achieved selectivity. Yu and Li introduced a dynamic pHeeping technique to improve the sensitivity of the SDS

C method; in particular, the pH of the sample matrixadjusted to acidic value and injected as a large volumeillary lled with the SDS separation solution (pH 9.1).ely charged pyrrolizidines migrated into the sampleds the boundary with the separation solution. Due toH value of the MEKC BGE the cationic pyrrolizidineed to the neutral free base forms and are focused byinto SDSmicelles. The achieved sensitivitywas 90-foldthat obtained using conventional injection [69].

observed uresponse.

MEKC wtechnique.phoric acidof the EOF was a large scould be paity (LODs wrespectivelycompared t

The mobioactive coin tropicalyohimbe webased on amThe samplegas chromapresence of neutral compounds: aloe-emodine (4), chrysophanol (5),in (13). IS: tetrandrine (the internal standard). (B) Electropherogram

m dihydrogen phosphate (pH 7.3), 50mM sodium deoxycholate, 30%m [64].

alkaloids

lkaloids constitute awide class of natural productsmostarmacologically important and characterized by verytivities.s nux-vomica is used in TCM as anti-inammatory andowever the major indole alkaloids, strychnine and

e well-known for their toxic effects consisting in sig-of blood pressure and induction of violent convulsions

uantitation of these compounds in plant materials ashytopreparations is thus of importance, the therapeuticvery low.

as useful in reducing the peak tailing observed in aque-and caused by interaction of the cationic hydrophobicith the SiOH groups of the inner capillary wall. Ammo-te was the selected electrolyte in the presence of aceticed in ACNmethanolmixture. Fast separation of strych-rucine was obtained and the method was applied toof Strychnos nux-vomica plant materials and Yaotont-capsules, after extraction using ammonium hydroxidel [73]. Alternatively a BGE composed of Tris-borate atmethanolACN (60:40, v/v), was used [74]. Althoughelectrophoretic conditions of the two described meth-ery similar, the limit of detections for strychnine wasusing ammoniumacetate as supporting electrolyte [73]g/mLusingTris-boric acid [74]; the sharperpeak shapes

sing ammonium acetate could explain the better UV

as used in combination with sweeping concentrationIn particular, using acidic running buffer (50mM phos-at pH 2.0) containing SDS 100mM, a strong suppressionas obtained. The acidied sample matrix was injected

ample volume and the hydrophobic cationic alkaloidsrtitioned into the SDS micelles. The achieved sensitiv-ere 0.05 and 0.07g/mL for strychnine and brucine,using UV detection at 203nm) was 100-fold higher

o that obtained using conventional injection [75].noterpenoid indole alkaloid yohimbine is the typicalmpound of Pausinystala yohimbe whose barks are usedWest Africa as an aphrodisiac. Methanol extracts of P.re directly injected in an optimized NACE-UV methodmonium acetateacetic acid as the BGE in methanol.

s were simultaneously analysed in NACE-UV and intographymass spectrometry (GCMS), used as a refer-


ence method. As expected, GCMS demonstrated to be superior interms of efciency and opportunities in identication of analytes;however the results obtained in quantitation of yohimbine in realsamples using NACE and GCMS were not signicantly different. Inparticular tof the activ

Hydroin(mesembrestereoisomspecies usebased on pthe separatquantitatioat 228nm;tablets wer

Psychotrica as psycalkaloids thdevelopedof -carbolLIF and ESIbers alloweasy the co200mM forthe baselinharmane wsimultaneoobserved thnal aroundof the nativexception ocence quanbe about 5-

2.8. Methyl

Methylxphylline anplant prodguarana). Bnervous sysused as phying on thei.e. the U.S.energy drinFurther, natthus coffeeout being calso used as

Analyticand derivedful tools in t

CZE in athe analysisdissociationH of the immigrating aUnder simithat howevinjector fordrinks, tea l

MEKC w(yerba mateas surfactancarbonate ppeak shape

tautomerism of methylxanthines [82]. The levels of caffeine foundin different beverages showed high variations depending on thetype; i.e. some of the analysed decaffeinated coffee samples werefound to contain levels of caffeine above the limit set by authori-

razilito bhylxapart

(CD-. The] ore peasm wins, ped [8usin

is of myllinto be

orph

rphine, thetiveseirsionsiond illintern, co

tractistrawmethundse alka verkinglkaloby

codes fro

to beis maf thediffesfullys wa) anondirove.14modvityrumlvenses olkalo) su(5m(LOanting tplicae sohe analysed samples showed to contain about 1.11.2%e alkaloid [76].dole alkaloids such as mesembrine and congenersnone, 7 mesembrenone, mesembranol and itser epimesembranol) have been isolated from Sceletiumd for the psychoactive effects. A simple CZE methodhosphate buffer at pH 1.5 was successfully applied toion of the alkaloids of Sceletium phytopreparations. Then was performed only on mesembrine by UV detectionthe LOD was 1.5g/mL and the commercially availablee found to contain mesembrine at 0.03% level [77].ia viridis, a plant usedby the Shamans fromSouthAmer-hotropic and sacramental drug, contains -carbolineat can be included among indole alkaloids. Huhn et al.a mixed aqueous-organic CE method for the analysisines in extracts of P. viridis. A dual detection system,-MS, was developed; in particular, the use of opticaled the LIF detection to be set close to ESI source makingmparison of the LIF and MS electropherograms. Usingmic acid and 7mM ammonia in water with 10% ACN,e separation of norharmane, harmane and tetrahydro-as accomplished. In addition, the method provided theus separation of harmine, harmaline and harmol. It wasat a solution of concentration 40nM, provided aMS sig-the detection limit. LIF detection was applied by prote uorescence of -carbolines (ex = 266nm); with thef tetrahydroharmane, which possesses a low uores-tum yield, the sensitivity of LIF detection was found tofold higher than ESI-MS [78].

xanthine alkaloids

anthine alkaloids (purine backbone) caffeine, theo-d theobromine are present in a variety of plants anducts used for preparation of beverages (coffee, tea,ecause of their stimulant effects on cardiovascular andtem, methylxanthine-containing plants are sometimestopharmaceuticals and dietary supplements. Depend-country, caffeine level in beverage could be limited;FDA set the limit of 0.02% [79], however the so-calledks when classied as supplements may contain more.ural caffeine levels are exempt from these regulations,based drinks may have more caffeine than 0.02% with-lassied as a supplement. Methylxanthines content isan index of the quality of coffee, tea and cocoa.

almethods for the analysis ofmethylxanthines inplantsproducts have been developed in order to provide use-hequality control of suchawidevarietyof preparations.lkaline conditions (tetraborate, pH 9.2) was applied toof guarana (Paullinia cupana) phytopreparations; theof acidic hydrogen of theobromine (H-1) and that of N-

idazol-moiety of theophylline, made these compoundss anions whereas caffeine occurred with the EOF [80].lar conditions this effect was not observed by Li et al.,er focused their study on the development of a samplehigh precision analysis of methylxanthines containingeaves and roasted coffee extracts [81].as developed for methylxanthines analysis in drinks, coffee, cocoa, tea, decaffeinated coffee, etc.) using SDSt under alkaline conditions (tetraborate, pH 8.5 [82] orH 11.0 [83]). However alkaline buffers provided poors and detrimental peak splitting due to the keto-enol

ties (Bfoundof metples. InMEKCtuningtral [84also thtomericatechobtain

CECanalystheophfound[87].

2.9. M

Mocodeinderivaties; thdepresdeprestive anstrict iniferumfor expoppylyticalcompophinanthat isin checnane aappliedbaine,samplefoundanalysbasis oto thesuccessample(pH 2.5these cto imp0.050

CD-selectiPapaverich soprocesnane a2.83.0(CD)sitivitysigniction usthis apalkalinan legislation set the limit of 0.1%) [82,83]. MEKC wase useful also in simultaneous separation and analysisnthines and catechins in tea and Theobroma cacao sam-icular the use of cyclodextrins in cyclodextrin-modiedMEKC)modeoffered the opportunity for high selectivitypoor stability of catechins at pH>8 led to choose neu-acidic running buffers [85,86]; under these conditionsk splitting of methylxanthine due to keto-enolate tau-as circumvented and fast analysis of methylxanthines,rocyanidins, phenolic acids, vitaminCand theavinwas486].g hybrid silica monolith columns allowed a very fastethylxanthines in tea beverages; in analysed samples,

e was not detected whereas the caffeine content wasmore than 10-fold lower compared to the tea infusions

inane alkaloids

ane alkaloids (opium alkaloids) such as morphine,baine, papaverine andnarcotine belong to isoquinolineand show a broad range of pharmacological activi-

major application is in analgesia, sedation and cough. They also exert a number of side effects as respiratory. In addition morphinane alkaloids are strongly addic-citly used as narcotic drugs; for this reason are underational control. Opium, the exudates from Papaver som-ntains more than 30 alkaloids and is the raw materialon; also the dried heads of P. somniferum, so-called, is used as a source of morphine and thebaine. Ana-ods are of importance for the determination of thesein plant materials because the content of specic mor-aloids could be used to differentiate the origin of opiumy important issue to help the law enforcement agenciesthe illicit production of narcotic drugs. CZE of morphi-ids (100mM sodium acetateacetic acid, pH 3.1), wasSashidhar and co-workers to the determination of the-ine, morphine, papaverine and narcotine in 124 opiumm different regions of India. The alkaloids prole wasdependent on sample type. Application of discriminantde it possible to cluster the analysed samples on their geographical origin [88]. A chemometric approach

rentiation of opium and poppy straw samples was alsoapplied by Reid and co-workers. The analysis of the

s carried out by MEKC using 50mM phosphate bufferd 80mM SDS in the presence of 25% methanol. Undertions a sweeping concentration technique was appliedsensitivity; the obtained LOD values were in the rangeg/mL (UV detection at 200nm) [89].ied CZE was applied to improve the separationin the analysis of morphinane alkaloids in differentspecies and in the liquors (rich extracts, spent extractst liquors) produced during the industrial manufacturef alkaloids extraction. Fast separation of the morphi-ids was achieved by using acidic running buffer (pHpplemented with HP-CD (30mM) or -cyclodextrinM) [90,91]. The UV detection allowed for adequate sen-D values were in the order of 0.1g/mL). However aimprovement of sensitivity was obtained by ECL detec-ris(2,2-bipyridyl)ruthenium(II) in the detection cell. Intion the separation buffer was based on 25mM boratelution supplemented with the ionic liquid 1-ethyl-3-


methylimidazolium tetrauoroborate (1E-3MI-TFB). Borate bufferwas useful in improving the separation selectivity by complexa-tion of borate anions with the hydroxyl groups of alkaloids. Onthe other hand, the ionic liquid, because of the higher conductivitycompared tprovided a(LOD of 1in CZE anadimethoxy-found in Sireported tothan that ob

CEC wasine, thebainmonolith coary phase.groups whieffected asconcentratiinteractionseparation

2.10. Misce

CamptotCamptothecand Tabernthese compment of colobtained frodownstreamoping analyin plant maalkaloids froide; subseqof camptothcompatibilisolvent, a Mdimethyl suborate (pHtive in incrby promotilate as a conto the charsamples wacould be ascation (3.76[96].

Amaryllline derivaGalanthus amacologicais consideramaryllidacspecic comlycoramineapplied asNarcissus pformed usinmethanolAwas compaof plant mof galanthaextractswaical performwere simila

of ECL detection after CZE separation (18mM aqueous phosphatebuffer, pH9.0)was applied to the analysis of galanthamine inBulbusLycoridis Radiatae, widely used in TCM. The ECL detection based ontris(2,2-bipyridyl)ruthenium(II) allowed LOD of 0.25ng/mL with a

antnthawasoidaed bycycliundsi.e. Bse, TThe B, antriae soni

es waion. Iatepthesertics shs (FrF. usg wir levrgii aona

medrepatussirallyplanned

ion hloidsof Ming cppliedros

yphe

demiundsrdio05].een is mananeixturalitytativing

s oximeth091liter

moreticnal pcatiolic aidsyanio that of phosphate buffer present in the detection cell,eld amplied effect, resulting in improved sensitivity106 to 1109 M) [92]. ECL detection was appliedlysis of sinomenine (7,8-didehydro-4-hydroxyl-3,7-17-methylmorphinan-6-one), themorphinanealkaloidnomenium acutum. The achieved detection limit wasbe 2103g/mL [93], thus about 1000-fold lowertained using a conventional NACE-UV method [94].applied to the determination of narcotine, papaver-e, codeine, morphine in Pericarpum papaveris using ansistingofhydrophilic/cation-exchangemixed station-The cation-exchanger function was based on sulfonicch also afforded for the EOF, whereas the diol groupshydrophilic interaction sites. It was shown that highon of organicmodier (90%ACN) promoted hydrophilicof the analytes with the stationary phase improving[95].

llaneous alkaloids

hecines are valuable quinoline alkaloids present ina acuminata, Nothapodytes foetida, Ophiorrhiza pumilaaemontana heyneana. The increasing interest towardsounds is due to the demonstrated activity in the treat-orectal and ovarian cancer. Camptothecin is currentlym natural sources both for the clinical use and for thesynthesis of the derivatives. The main issue in devel-

tical methods for the determination of camptothecinterials is the poor water solubility. Extraction of activemplantmaterialwas optimizedusing dimethyl sulfox-uently a CE method was developed for the separationecin and 9-methoxycamptothecin. In order to achievety of the CE separation medium with the extractionEKC running buffer containing 90mM SDS and 20%

lfoxide in 10mM borate buffer was used. Interestingly,> 8) as supporting electrolyte was found to be effec-easing the solubility of camptothecin and derivativesng the equilibrium of the lactone forms to the carboxy-sequence of the complexation with borate ions. Owingacteristic UV spectrum, the selective detection in reals performed at the wavelength of 369nm and the LODsessed as about 2g/mL thus adequate to this appli-mg/g of camptothecin in N. foetida plant materials)

idaceae alkaloids are an important class of isoquino-tives; among them galanthamine, that is found innd Narcissus species, has been approved for the phar-l treatment of Alzheimers disease [97]. GCMS analysised the method of choice for the characterization ofeae alkaloids; by means of this approach a number ofpounds have been identied in Narcissus species (i.e.

, narwedine, haemanthamine, tazettine). NACE-UV wasa conrmatory and orthogonal method in analysis ofseudonarcissus and Narcissus jonquilla. NACE was per-g 90mM ammonium acetate and acetic acid (0.5%) inCN mixture (75/25, v/v). The organic running buffertible with the real samples obtained by extractionaterial (crushed bulbs) using methanol. The amountmine and haemanthamine in the analysed Narcissuss found tobe2.2and0.32mg/g, respectively. Theanalyt-ances of NACE-UV in terms of sensitivity and accuracyr to those of the GCMS reference method [98]. The use

signicof galarations

Steracteriza hexacompofamilyJapane[100].tussiveFritillaof ammresidudetectphosphUndersis of vsampleextractbergii,ranginat lowethunbe

Stemtionalphytoptis, perstructuin thismentiodetectof alkameansN-bearfully adidehy

3. Pol

Epicompocer, ca[1031have bpoundand ligplex mand ququanticontainsuch aration[106,1recent

Thetrophomediciclassi(phenoavonoanthocgain compared to the UV detection; however the levelmine found in the Bulbus Lycoridis Radiatae phytoprepa-0.13mg/g thus affordable also by UV detection [99].l alkaloids such as verticine and verticinone are char-cholestane carbon skeleton (isosteroid alkaloids) with

c benzo[7,8]uoreno[2,1-b]quinolizine nucleus. Thesehave been isolated from plants belonging to Liliaceaeulbus fritillariae used as a traditional herb remedy inurkish, Pakistani and south-east Asian folk medicines. fritillariae-based phytopreparations are used as anti-i-asthmatics and expectorants. Extracts from differentpecies were obtained with chloroform in the presencea to afford the free base of the alkaloids. The organics dissolved in methanol and analysed by CZE using ECLn particular the running buffer was constituted of 8mMH8.0 in thepresenceof40mM1B-3MI-TFB ionic liquid.

e conditions a very low LOD (about 50pg/mL) in analy-ine and verticinone was achieved. The analysis of realowed that verticine was found in any of the analyseditillariae hupehensis, F. waluajewii, F. cirrhosa, F. thun-suriensis, F. pallidiora and F. przewalski) at amountsthin 5.4012.35mg/L, whereas verticinone was foundels (5.668.07mg/L) only in F. hupehensis, F. cirrhosa, F.nd F. ussuriensis [101]., from the family of Stemonaceae, is known in tradi-icine of south-east Asia, China and Japan because itsrations (primary the roots) are used to treat bronchi-s and tubercolosis. Interestingly a number of alkaloids,dened as pyrido[1,2-a]azepines, have been identiedt species and are considered to be responsible for thepharmacological activity. NACE coupled with ESI-MSas been developed to characterize the highest numberin S. curtisii, S. collinsae, S. tuberosa. In particular by

Sn experiments at least 40 and 50 alkaloids or chargedompounds, were detected. The method was success-d to the quantitation of stemofoline, oxystemokerrin,temofoline and stemocurtisinol [102].

nols

ological studies have shown that natural phenolicplay an important role against biomarkers for can-

vascular diseases and other degenerative diseasesSeveral thousand molecules with polyphenol structuredentied in higher plants and edible plants; these com-y be classied as phenolic acids, avonoids, stilbenes. In natural sources, polyphenols are contained as com-es and their composition is highly variable in amount; as a consequence the analytical prole (quali- ande) is very important in standardization of herbal drugspolyphenols as well as in monitoring the food changesdation. Detailed reviews dealing with advanced sepa-ods [106108], including electromigration techniques12] for analysis of polyphenols, are available in theature.st important aspects involved in capillary elec-analysis of phenolic compounds in herbal drugs andlants, will be dealt below by following the chemicaln based on the constitutive phenolic skeleton: phenolscids and coumarins); quinones and naphtoquinones;(avones, isoavones, avanones, avonols, avanols,ns). General structures of avonoids are reported in


Flavan

Anthocy

Flavo

O+

2

5

6

7

2 4

5O

A

B

Fig. 6. Thestwo aromacarbon atomcation is bof B ring; tand /or 5

C glycosid

3.1. Phenol

Phenolicycinnamiccontent in phydroxycinferulic acid)bound formdeterminatcan be useddrugs, howeerally incluwith othermethods spcompounds

CZE usinanalysis ofstrong UVfound to becinnamic, clic, caffeic aobtained inorate in theSoxhlet ext

sequent Solid Phase Extraction (SPE) procedure (reversed-phasesorbent) before injection. The amounts of phenolic acids in realsamples ranged within 2.84.3g/g of the dried plant material

ecovery varying from 96 to 101% and RSD


tively high levels (>85mg/g, recovery of 102%) of sophoricoside, aninhibitor of interleukins, were found in Fructus sophorae japonicae[117].

CEC and pCEC (pressurized CEC) have been widely applied inanalysis of cillary (i.d. 1the presencable EOF. Coseparationhydrate, bya mixtureas the mobthe pressurcoumarinsIn this applthan that a(LOD


the continuous automated sampling without physical movementof the CE capillary. The method was applied to the analysis ofRhubarb and different herbal drugs and preparations. However,rhein, one of the most characteristic anthraquinones in Rhubarb,was not conconditionspropyl alcoto the analtime of abtime (analy(35mM, pHwas introdity of the cof rhubarbextraction imajor numSoxhlet extchrysophan-d-glucosid-glucosidedetected ustaining CDabout 30mshowed anwas thus netary methoof the 11 cohowever thA and B waHPLC resultfound to btime in theemodin anddeoxycholathe presencing for therhein) in lesis of Cassiaof the analyCZE, makinanalysis ofadvantagesmonolithiciso-butyl macid in thev/v) as porofrom Rhuba different[127].

3.4. Flavon

Flavonoibioactive pochromane ation of shikdifferent suas O- or C-gand are alsovegetables.

3.4.1. CZE oCapillary

analysis ofbecause ofand/or sacc

mobile ve-membered-ring complexes (with 1,2-diols) andsix-membered ring complexes (with 1,3-diols) thus increasingthe selectivity of separation [128]. A typical ion-dipole or ion-induced dipole interaction between avonoids and borate was also

ed bide, isociainteiophcircuin thundrateitexits frordiotmiladin Aed frpimeavo-glu-p-cneraopyrrhamtradinat

n-7-gis ofyricermornamets wate 40

CZE o

edsep

ts ofest

ms.pHrimeydroyranerceysis oonalral stornate-of

in) from/v)of c

tin [

CZE oide torgarformclodeffesidered in this study [122]. Actually, when similar CZEinvolving 50mM borate (pH 8.2) in the presence of iso-hol and 25% ACN, as the running buffer, were appliedysis of powdered Rhubarb, rhein showed a migrationout 40min [123]. A signicant reduction of analysissis time


enantiomers of the avanone eriodictyol in Balanophora involu-crata Hook. f. [140]. Jc et al. proposed a novel electrophoreticbuffer containing tungstate as complex-forming reagent for theanalysis of avonoids. In the presence of tungstate, the solutesbearing viciplexes withof 50mM Nat pH 7.4, smethanol,hyperosideforatum ext

Also ionbuffer in sstances wittheir use asYanes andionic liquiddazolium canodic EOFhydrophobitionswith twith imidaz interacation of ioseparationstudies 1-buto be suitaquercetin, istopharmacearteanoavtrimethoxyfurther add

The mafew exceptnin, the bioradix. Hu anACNmethaAdditionofthe separatarteanoavin different

3.4.4. ElectrThe app

of UV deteous and nontwo majorband at 300namoyl systthe absorptits reportedgeneral depinner capillto the relatplant extracern; e.g. thrangewithi[131]; rutinratum leave6.77927.8

Electroction approaupon CE seoxidized atcan be veryminimum p

teins, carbohydrates and lipids, normally do not engender signalinterference. The design of the CE-ED detector is based on the end-column approach in which the working electrode (a carbon-diskelectrode with 300500m diameter) is simply placed at the out-

he sffer rent

de, as satentind tion canalyvoltast oponhenoth tng ine poinatre;d pranaits

0.02ion.ate rnoid

tionady mce oapplres

atetiond nonsitiderinaodex(pHnd eferold thes [16roveof aworktentbor

n abde blectrer otionextritivie ai-woroactid in

exedlysislkalisponnal hydroxy groups can form negatively charged com-W(VI) as the central ion. A running buffer composed

-(2-hydroxyethyl)piperazine-2-(2-ethansulfonic acid)upplemented with tungstate 2.2mM and 25% (v/v) ofwas effective in the separation of apigenin, luteolin,, quercetin, rutin and phenolic acids in Hypericum per-racts [141].ic liquids were explored as additive to borate runningeparation of avonoids. Ionic liquids are ionic sub-h melting points at or close to room temperature andadditive to improve the CE separation is increasing.

co-workers used 1-alkyl-3-methylimidazolium-basedas additive to aqueous running electrolyte. The imi-ations cover the inner capillary wall generating anand they are also supposed to be able in establishingc, hydrogen bonding, ion-dipole/ion-induced interac-he analytes. In particular, the association of polyphenolsolium ions was hypothesized to be occurring through

ctions [142]. Two independent studies involving appli-nic liquid as additives to borate running buffer in

of avonoids have been recently reported. In both thetyl-3-methylimidazolium tetrauoroboratewas foundble in separation of avonoids, namely kaempferol,orhamnetin inHippophae rhamnoidesextracts andphy-utical preparations (tablets) [143]. The separation ofone, eupatilin hispidulin and 5,7,4-trihydroxy-6,3,5-avone was obtained only in the presence of CD as aitive [144].jority of the avonoids are freely soluble in water;ions are represented by puerarin, daidzein and wogo-active components of Puerariae radix and Scutellariaed co-workers applied advantageouslyNACE (mixture ofnol) to improve the solubility of these analytes [145].sodiumcholate toNACEbufferwasnecessary to achieveion of fraxin, esculin and esculetin [146], eupatilin,one, 5,7,4-trihydroxy-6,35-trimethoxyavone [147]medicinal plants extracts.

ochemical detection in CZE analysis of avonoidslications described above were performed by meansction which was conveniently applied both in aque--aqueous buffers. The UV spectra of avonoids exhibitabsorption bands in the region of 240400nm; the380nm is associated with the absorption of the cin-em,whereas thebandat240280nmis associatedwithion of the benzoyl system [148]. The detection lim-in the cited literature varied within 0.016g/mL in

ending on the avonoid type, the selected wavelength,ary section and running buffer nature and pH. Owingively high content of bioactive avonoids in medicinalcts, the detection sensitivity is not a challenging con-e total avonoids extract in Epimedium is reported ton39.0172.1mg/gdependingon the considered speciesis found at a level of about 9.2mg/g inHypericum perfo-s [141] and esculin levels in Cortex fraxini rangedwithin9mg/g [146].hemical detection (ED) is a useful alternative detec-ch, widely applied in the determination of avonoidsparation. Since polyphenols can be electrochemicallya relatively moderate potential, the CE-ED detectionselective also when real samples are analysed after aretreatment. In fact, matrix constituents such as pro-

let of tthe buinstrumelectro(such athe potivity adetectof thenamicfor mothe resever, wSCE), bresultitions thdetermliteratucost anof CZEtion limwithindetect

Borof avocentraAs alrepresenCE-ED100mMphosphseparashowetion seand unformedof cyclbufferechin akaemption anextract

Impmeanspositeoverpo50mMrated ielectroence ethe ordconvenysis ofof senswith thand colyze bioxidizecompltrocatausing arent reeparation capillary and the detection is carried out ineservoir that contains the ground electrode of the CE. The detection cell contains, together with the workingplatinum auxiliary electrode and a reference electrodeturated calomel electrode, SCE, or Ag/AgCl). In CE-ED,al applied to the working electrode affects the sensi-he stability of the system. The investigation of the bestonditions involves the evaluation of the peak currentte when increasing potential is applied. The hydrody-mmograms (peak current versus potential) obtained

f the avonoids, show that a progressive increase ofse is achieved when the potential is increased. How-the applied potential is higher than +950mV (e.g. vshe baseline noise and the background current increase,unstable baseline. In most of the published applica-

tential of +950mVwas used. Several examples of CE-EDions of avonoids in plant extracts are available in thethe assay by means of this approach is rapid and lowovides satisfying sensitivity. In Table 3 recent exampleslysis of avonoids by CE-ED are reported. The detec-reported for most of the bioactive polyphenols ranged6g/mL, thus comparable to those obtained using UV

unning buffer resulted to be suitable in CZE separations with ED detection; in general tetraborate in the con-range 50100mM (pH 8.459.0) was used [149155].entioned, the separation selectivity is improved in the

f relatively high concentrations of borate; however inications, the use of concentration values higher thanulted in baseline noise and sensitivity loss. Addition ofto borate buffer was found to be benecial in the CZEof avonoids using ED detection; Na2B4O7-NaH2PO4t only good resolution but also allowed for high detec-vity. The optimum pH values ranged within 7.58.8these conditions the analysis of avonoids was per-varietyofherbaldrugs [156160].Additionof low levelstrins (in particular 3mM CD) to boratephosphate8.8) was effective in improving the separation of cat-picatechin beside avonoids such as apigenin, luteolin,, quercetin. The additive did not affect the EC detec-methodwas applied in the analysis of chrysanthemum1].d sensitivity in CE-ED of avonoids was achieved bycarbon nanotube/poly(ethylene-co-vinyl acetate) com-ing electrode that showed to signicantly reduce the

ial. Under conventional separation conditions using aate buffer (pH 9.2), esculine and esculetin were sepa-out 8min and detected by CE-ED using the modiedy applying a potential of +0.8V (versus Ag/AgCl refer-ode). The method showed an improved sensitivity (inf 30-fold) compared to the CE-ED method employingal carbonworking electrodes and it was applied in anal-acts of Cortex fraxinii [162]. Beside the improvementty, modied working electrodes have been developedm to enhance the range of detectable compounds. Hekers applied a copperdisc working electrode to ana-ve components of oolong tea infusions. Copper can beto its higher transitional state Cu(II) and Cu(III), whichwith the polyhydroxyl compounds and resulted in elec-oxidation. By means of this system, upon CE separationne buffers, sugars and amino acids produced good cur-se. A phosphate-borate (30mM40mM, pH 8.5) BGE


Table 3CE-ED determination of avonoids in herbal drugs.

Sample Flavonoids Buffer/electrode Ref.

Ilex purpurea Hassk Kaempferol, quercetin, Borate/c.-d. [149]

Ricinus commFrucus auran

Ipomea bataSophora japo

Cynomorium

Perilla frutesAgrimonia pi

Portulaca ole

Lonicera conHouttuynia c

chinensis (Leonurus het

Chrysanthen

Cortex fraxinOolong tea i

c.-d.: carbond

allowed thel-glutaminecatechin ga

3.4.5. EKC iMEKC is

because ofof selectivitin analysis

The CZEone of theAsian counof the matsome constmized by cbaicalin, bagin. Baicalianti-tumorin Scutellarilevel of baicnin withinallowed fordifferent orpossesing pused to conchemical paanalysis itwhigh simila

Catechintive avonobeen includies includinshown the

ce oatechsupas mphenolic acidsunis Rutin, quercetintii Naringin, naringenin,

hesperidintas Rutin, quercetinnica L. Genistin, genistein,

quercetin, kaempferol,rutin

songaricum Epicatechin, catechin,luteolin, quercetin,naringenin, rutin,phloridzin

cens L. Catechin, apigenin, luteolinlosa Ledeb Catechin, quercetin, rutin,

quercitrin, hyperosideracea L. Kaempferol, apigenin,

myricetin, quercetin,luteolin

fusa DC. Luteolin, phenolic acidsordata Thub. SaururusLour) Bail

Quercetin, hyperoside,rutin, quercitrin

erophyllus Sweet Kaempferol, quercetin,hyperoside rutin,quercitrin

um Catechin, epicatechin,apigenin, luteolin,kaempferol quercetin

ii Esculine, esculetinnfusions L-theanine, L-glutamine,

sucrose, glucose, fructose,ascorbic acid,epigallocatechin gallate

isk electrode.

simultaneous separation and analysis of l-theanine,, sucrose, glucose, fructose, ascorbic acid and epigallo-llate [163].

incidensis of cdietaryappliedn analysis of avonoidswidely applied in analysis of polyphenols mainly

its high resolution power and for the wide opportunityy tuning. Examples of applications of MEKC and MEEKCof herbal drugs and plant extracts are given in Table 4.analysis of extracts from Scutellaria baicalensis Georgi,most used herbal medicine in China and other Easttries, was unsuccessful because of the interferencerix components and due to the neutral character ofituents of the raw material. A MEKC system was opti-entral composite design to provide the separation oficalein and wogonin in real samples from different ori-n, reported to be an anti-inammatory, anti-HIV andcompound, was found the most represented avonea baicalensis (amount within 24.74143.56mg/g); thealein ranged within 1.5315.12mg/g and that of wogo-0.364.14mg/g. The high specicity of the methoda comparative analysis of ngerprint of samples fromigin. In particular, the relative area values of eight co-eaks in each of the obtained electropherograms, werestruct an eight-dimensional vector to characterize thettern of the ngerprint. By means of this comparativeas found that the samples from the sameorigin showed

rity [164].s represent one of the most studied classes of bioac-ids; they are mainly found in green tea, which hased among the herbal drugs. Actually, a variety of stud-g epidemiological and nutritional investigations havepossible relationships between tea consumption and

A simpleof catechinsEGC), ()-(()-EC) ananthine cafdietary supof green te(PDMS) mic[168]. MEKsional analyin green tematographgradient eltions wereshowed fulrst chromaunder the ccatechins, detected [1

Catechinthus acidicpreparationin catechinin their anain the stronSDS micelleas reversedinteractionanodic deteBorate/c.-d. [150]Borate/c.-d. [151]

Borate/c.-d. [152]Borate/c.-d. [153]

Borate/c.-d. [154]

Borate/c.-d. [155]Borate-phosphate/c.-d. [156]

Borate-phosphate/c.-d [157]

Borate-phosphate/c.-d [158]Borate-phosphate/c.-d [159]

Borate-phosphate/c.-d [160]

Borate-phosphate + 3 mM CD/c.-d. [161]

Borate/carbon nanotube [162]Borate-phosphate/copper [163]

f cancer and cardiovascular diseases [165,166]. Analy-ins content in green tea, tea beverages and tea extractplements is thus very important and MEKC has beenethod of choice.

MEKC method allowed for the separation of a number, namely (+)-catechin ((+)-C), ()-epigallocatechin (()-epigallocatechin gallate (()-EGCG), ()-epicatechind ()-epicatechin gallate, (()-ECG) and the methylx-feine and it was applied in analysis of green tea extractplements [167]. Under similar conditions the same seta catechins were analysed in a polydimethylsiloxanerochip platform using pulsed amperometric detection

C was also off-line coupled to LC separation in bidimen-sis of complex mixtures of phenolic acids and avonesa extracts. The samples were analysed by liquid chro-yonaPEGcolumnusingammoniumacetateACNunderution (rst dimension of separation). The eluted frac-collected and successively analysed by MEKC, whichl compatibility with the fractions collected from thetographic dimension. Green tea sampleswere analysed

omprehensive 2Dmethod; besides the typical green teaavones such as naringin, biochanin A, hesperetin were69].s undergo rapid oxidation under alkaline conditions,conditions should be preferred not only in samplebut also during separation [170]. The lack of charge

s molecules at low pH values makes MEKC mandatorylysis. The use of acidic running buffers (pH 2.5) resultsg suppression of the EOF and the negatively chargeds show anodic migration. Using this approach, denedow MEKC (RF-MEKC), the analytes showing strongerwith the micelle are the faster migrating towards thection end.Additionof cyclodextrins to theMEKCsystem


Table 4MEKC and MEEKC in analysis of avonoids.

Sample Flavonoids Buffer Ref.

Scutellaria baicalensis Georgi Baicalin, baicalein, wogonin 15mM SDS, 15mM borate40 [164]

Green tea di

Green tea sa sA,

Theobroma cin

Green tea sa

Green tea be

Azadirachtain

Alpinia katsu

tea leaves, tec,

silymarin ca hin,

Radix Astrag

ACN:, Acetonit

gives rise tointo

5946860381043067632

Documents

Transcript of 5946860381043067632