Applications of mass spectrometry to plant phenols

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Applications of mass spectrometry to plantphenolsDanielle Ryan, Kevin Robards*, Paul Prenzler, Michael AntolovichSchool of Science and Technology, P.O. Box 588, Wagga Wagga 2678, Australia

Plant phenols embrace a considerable rangeof compounds and are de¢ned as those sub-stances derived from the shikimate acid path-way and phenylpropanoid metabolism. Thepresent article examines the application ofmass spectrometry to the analysis of thesecompounds and traces the chronologicaldevelopment of analyte ionisation meth-ods. z1999 Elsevier Science B.V. All rightsreserved.

Keywords: Soft ionization; Phenols, Plants

1. Introduction

Terminology relating to the plant phenols is oftenconfusing and a brief overview is presented beforeconsidering aspects of their mass spectrometry. Theplant phenols (Table 1) are a diverse group of second-ary metabolites possessing an aromatic ring bearingone or more hydroxy substituents. However, thisde¢nition is not entirely satisfactory since it inevitablyincludes compounds such as oestrone, the female sexhormone which is principally terpenoid in origin. Forthis reason, a de¢nition based on metabolic origin ispreferable, the plant phenols being regarded as thosesubstances derived from the shikimate pathway andphenylpropanoid metabolism (Fig. 1 ). Some mem-bers are characterised as `polyphenols', an unfortunateterm since not all are polyhydroxy derivatives. In par-ticular, a number of compounds, for example, cin-namic acid, elenolic acid, shikimic acid and quinicacid, are treated in the present discussion as phenolicsbecause of metabolic considerations although theylack a phenolic group or even an aromatic ring. Plantphenols (Table 2) have been classi¢ed into majorgroupings distinguished by the number of constitutivecarbon atoms in conjunction with the structure of thebasic phenolic skeleton. The most widespread and

diverse of the phenolics are the £avonoids which arebuilt upon a C6-C3-C6 £avone skeleton in which thethree-carbon bridge between the phenyl groups iscommonly cyclised with oxygen. Several classes of£avonoid (Fig. 2 ) are differentiated on the degree ofunsaturation and degree of oxidation of the three-car-bon segment. Within the various classes, further dif-ferentiation is possible based on the number and natureof substituent groups attached to the rings. The rangeof known phenolics is thus vast and also includes pol-ymeric lignins and condensed tannins although thesespecies are not considered in the present review.

Additional structural complexity is introduced bythe common occurrence of certain phenolics as theO-glycosides (or, less commonly, as C-glycosides )in which one or more of the phenolic hydroxyl groupsis bound to a sugar or sugars by an acid-labile hemi-acetal bond. Glucose is the most commonly encoun-tered sugar with rhamnose and the disaccharide,rutinose (6-O-K-L-rhamnosyl-D-glucose ), is alsoencountered. Anthocyanins are intensely colouredplant pigments in which an anthocyanidin aglyconeis glycosidically linked to a sugar( s ). Acylation ofthe glycosides in which one or more of the sugarhydroxyls is derivatised with an acid such as aceticor ferulic acid is occasionally observed. Althoughthe number of identi¢ed phenols is increasing expo-nentially, the phenolic content of most plants consti-tutes a complex mixture, the complete chemical natureof which has not, as yet, been elucidated for any spe-cies. For example, there are many phenolics present inlow concentrations which remain unidenti¢ed butwhose signi¢cance may far outweigh their concentra-tion level. Isolation and structure elucidation of thesecompounds are the initial steps to understanding theirsigni¢cance and action. Information on their biosyn-thesis is essential to understanding the interactionbetween plants and the environment. It will also facil-itate genetic manipulation ( both qualitative and quan-titative ) of the phenolic content of plants for dietarymodulation of disease and to provide an environmen-tally sustainable feedstock for the petrochemicalindustry.

0165-9936/99/$ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 5 - 9 9 3 6 ( 9 8 ) 0 0 1 1 8 - 6

*Corresponding author.E-mail: [email protected]

362 trends in analytical chemistry, vol. 18, no. 5, 1999

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Methods of characterisation and identi¢cation ofplant phenols follow those in general use for naturalsubstances. Hence, preparation of an extract, biolog-ical screening, bioguided fractionation, isolation andstructure elucidation is the usual approach. For thelatter, physical methods based on spectral character-istics feature prominently although older chemical andbiochemical approaches should be considered partic-ularly as adjuncts to spectral analysis. Of these tech-niques, none has been more useful in providing struc-tural information than mass spectrometry (MS).However, its use has not been restricted to this rolefor mass spectrometry is increasingly viewed as amass-speci¢c detector [ 1 ] for both qualitative and

quantitative applications in high resolution chroma-tography. Tandem mass spectrometry (MS^MS) isparticularly attractive for trace analysis where highselectivity is essential [ 2 ]. In another role, MS of phe-nolic compounds meets the multivariate criteria for¢ngerprinting food samples in adulteration testing[ 3 ]. Representative applications of MS in the analysisof plant phenols are presented in Table 3.

2. Classical ionisation techniques

The traditional mode of MS involves electronimpact ionisation (EI ) in which the neutral samplemolecule is impacted in the gas phase with an electronbeam of energy 70^100 eV. EI mass spectrometry(EI^MS) is used extensively in structural studies ofa number of plant phenols including various £avo-noids. EI produces a positive radical ion M�c whichif detected provides information on the molecularmass of the analyte. Comprehensive informationabout the mass spectra of £avonoids has been pub-lished [ 26,27 ]. In general, the EI mass spectra of£avonoid aglycones are characterised [ 24,25 ] byintense molecular ion peaks plus signi¢cant fragmentsfrom both A and B rings. The fragmentations oftenprovide suf¢cient information to determine molecularmass, elemental formula, substitution patterns in the Aand B rings, and the class of £avonoid. For example,the EI mass spectrum of substituted £avones shows thetypical fragmentation pattern to yield two fragmentscharacteristic of the A and B rings. In the case of adisubstituted £avone there are three possibilities,either both substituents are located on the A or Bring or one is located on each ring and the mass spec-trum will provide unambiguous information on thesubstitution pattern. In less favourable situations

Table 1De¢nition of terms used

Plant phenols Aromatic substances bearing one or morehydroxy groups on an aromatic ring andderived from the shikimate pathway or phen-ylpropanoid metabolism

Flavonoids A diverse class of plant phenols based on aC6^C3^C6 £avone skeleton in which thethree-carbon bridge between the phenylgroups is commonly cyclised with oxygen

Glycoside In the current context, a molecule formed bylinking a phenol and a sugar(s ) by an acid-labile hemiacetal bond between the phenolichydroxyl group(s) and the sugar(s )

Aglycone The phenolic portion of a glycosideAPI Atmospheric pressure ionisation (generic

term)APCI Atmospheric pressure ionisation by chemi-

cal ionisationElectrosprayionisation

Ion formation from samples in solution bydispersion of liquids into an electricallycharged aerosol due to the action of an elec-tric ¢eld

Table 2Classi¢cation of plant phenols

Class Basestructure

Examples Comments

Simple phenols C6 catechol, resorcinolPhenolic acids C6C1 p-hydroxybenzoic acid, salicylic acid,

gallic acidHydroxycinnamicacids

C6C3 caffeic acid, ferulic acid among the most common of all phenolic compounds

Lignans (C6C3 )2 nordihydroguaiaretic acidCoumarins C6C3 umbelliferone some coumarins are common and often found in

glycosidic formChromones C6C3 eugenin more common than coumarin analoguesFlavonoids C6C3C6 quercitin, cyanidin most common of all phenolic compounds

trends in analytical chemistry, vol. 18, no. 5, 1999 363

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where there are many substituents, the mass spectrumwill assist in structural elucidation although othertechniques such as nuclear magnetic resonance willbe required for a de¢nitive assignment of the substitu-tion.

EI more commonly produces an internally excitedparent ion, M�c, which fragments so extensively thatthe parent radical cation is not observed. This is thesituation with glycosidically linked phenols where theEI (and chemical ionisation, CI ) mass spectra [ 28 ] aredominated by the same ions as for the correspondingaglycones [ 2,28 ], the protonated aglycone invariablybeing the base peak in the mass spectrum. A distinc-tion with the mass spectra of the aglycones is the rel-atively weak fragments from ¢ssion of the A and Brings. However, EI is generally unsuitable for glyco-sidically bound phenols because these compounds arepolar and thermolabile. Similarly, CI [ 29 ] has limitedapplication for the broader range of plant phenols

because vaporisation of the analyte prior to ionisationis once again a prerequisite.

Chemical derivatisation overcomes the limitationsof restricted volatility and thermal stability of polarphenols but presents further dif¢culties by increasingthe molecular mass of the analyte possibly beyond therange of the mass analyser. This is a major consider-ation for glycosidic species with numerous hydroxylgroups although permethylation or perdeuteromethy-lation may be suitable. However, these methods oftenproduce mixtures of partially derivatised compoundsand, in some cases, permethylation can produce arte-facts, e.g., when ester groups are present. Usually onlyweak molecular ion signals are observed when per-methylated compounds are studied under EI condi-tions. The application of chemical derivatisation isextended in the discussion of coupled techniques.

The development of desorption ionisation tech-niques in which ionisation of thermolabile molecules

Table 3Mass spectrometric techniques used in the analysis of phenolic compounds in plants and related samples

Sample Phenolic compounds Technique Ref.

Wines phenolic acids, £avonoids GC^MS [ 3 ]Food review LC^MS [ 4 ]Olive oil GC^MS [ 5 ]Krameria triandra roots low to intermediate molecular weight

polyphenol constituents with radicalscavenging activity

LC^MS (PB, EI ); FAB^MS (negativeion mode) and FAB^MS^MS

[ 6 ]

Olive leaf oleuropein, ligstroside ESI-LC^MS [ 7 ]Oat groats and hulls phenolic acids GC^MS [ 8 ]Spruce shoots and galls 47 phenolics and phenolic glucosides GC^MS (TMS derivatives) [ 9 ]Not applied 15 benzoic and cinnamic acid derivatives PB-LC^MS (EI ) [ 10 ]Olive mill wastewater 15 phenolic compounds LC^APCI-MS (negative ion) [ 11]White and silver birch gallic and chlorogenic acids [ 12 ]

iso£avones CE^ESI-MS (negative ion) [ 13 ]Onions £avonoids [ 14 ]Juice of Sedum telephium leaves 6 £avonol glycosides FAB^MS (negative ion) [ 15 ]Plant extracts £avonoid precursors (cis trans isomers ) LC^MS [ 16 ]Epilobium species 19 £avonol glycosides Thermospray LC^MS [ 17 ]Lemon peel coumarins, phenyl propanoid glycosides,

£avone-C-glucosides, £avonols, £avone-O-glycosides and £avonones

Thermospray LC^MS [ 18 ]

Vitis vinifera L. anthocyanins: 3-glucosides, the 3-acetylgluco-sides, and the 3-p-coumaroylglucosides ofdelphinidin, cyanidin, petunidin, peonidin, andmalvidin; also 3,5-diglucosides

API ion spray LC^MS [ 19 ]

Passionfruit swertisin [ 20 ]Black tea £avanols, £avonol glycosides, chlorogenic acid Plasmaspray^LC^MS [ 21]Sorghum bicolor anthocyanins and anthocyanidins Plasma desorption MS [ 22 ]Soy foods iso£avone glycosides APCI-LC^MS (positive ion) [ 23 ](Orange oil ) 39 polymethoxylated £avones EI-GC^MS [ 24 ]Not applied 43 £avones and £avonols, 7 iso£avones,

18 £avanones and dihydro£avonols, and11 chalcones and dihydrochalcones

EI and CI-MS [ 25 ]


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occurs directly from the condensed phase provides asolution to the problem of limited analyte volatilityand stability. Field desorption (FD) was the ¢rst tech-nique employed for direct analysis of polar and ther-molabile phenolic species. FD has been applied to theanalysis of £avonoid glycosides [ 30 ] and providesmolecular mass data but usually little structural infor-mation and is `̀ notorious for the transient nature of thespectra'' [ 31]. Desorption chemical ionisation (DCI)uses a probe consisting of an electrically heated tung-sten wire introduced into the CI source. DCI providesrapid heating of the analyte and overcomes the prob-lem of thermal decomposition inherent in conven-tional CI but it was the development of particleinduced desorption or sputtering techniques thattruly extended the range of analyte polarities andsizes. Fast atom bombardment (FAB) [ 32 ] is themost successful and in this technique the analyte issolubilised in a non-volatile polar matrix (e.g., glyc-erol, thioglycerol ) and deposited on a copper targetwhich is bombarded with fast neutral energised par-ticles such as xenon or argon thereby inducing thedesorption and ionisation. The value of FAB-MS isdemonstrated by the molecular mass and structuralinformation provided for large polar glycosides inboth positive and negative ion modes (e.g., [ 33 ]).One disadvantage is that the nature of the spectra are

very dependent on the choice of the matrix. Further-more, background matrix signals ( from glycerol orthioglycerol ) can complicate interpretation of thespectra. Nevertheless, when combined with collision-ally induced dissociation of positive ions and tandemmass spectrometric techniques, FAB-MS can provideinformation on the aglycone moiety, the carbohydratesequence and the glycosylation position of glycosides[ 34 ].

3. Coupled techniques

The on-line coupling of methods is of enormouspotential because the selectivity can then be tuned inan optimal way, which in turn can be translated toeither a faster analysis or an improved determinationlimit. Coupled gas chromatography-mass spectrome-try (GC^MS) is now well established as a routinetechnique carried out with either EI or CI sources,since these are appropriate for the introduction of vol-atile compounds. Thus, Berahia et al. [ 24 ] analysed39 polymethoxylated £avones by GC^MS. In additionto common behaviour of £avones under electronimpact, such as a retro-Diels^Alder reaction whichgives a characteristic fragment from the phenylgroup of the £avone skeleton, new fragmentation

Fig. 1. Biosynthetic pathways leading to the formation of phenolic substances.


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Fig. 2. Chemical structures of the various classes of £avonoids.


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pathways were identi¢ed and proposed. Ions charac-teristic of various substitution patterns were also iden-ti¢ed. Improvements in GC column technology haveincreased the range of £avonoids amenable to GC^MSas the underivatised compounds. For instance,Schmidt et al. [ 35 ] analysed 49 £avones, £avonols,£avanones and chalcones without derivatisation byGC^MS in EI mode. Compared with direct inletmass spectra, the GC^MS data exhibited the sametypical fragmentation patterns but with slight differ-ences in intensities.

However, because of limited volatility, analysis ofphenolic compounds and their glycosides, in particu-lar, by GC^MS has not generally found favour. Twoapproaches have been used, namely, hydrolysis of theglycosides to their corresponding aglycones and /orchemical derivatisation. Angerosa et al. [ 36 ] haveshown GC^MS with CI to be an effective tool forphenol identi¢cation after extraction from olive oilwith methanol and derivatisation with bis( trimethyl-silyl )tri£uoracetamide. Peaks in the mass spectra atm / z 192 or at m / z 280 were useful for assigning thephenolic nature to minor components. The advantagesof CI with ammonia for providing molecular masses ofthe aglycones were also demonstrated. One of theproblems associated with derivatisation, namely, theformation of several derivatives from a single analyte,was highlighted by this work. Derivatisation has alsobeen applied to the analysis of the phenolic compo-nents of wine which were extracted and separated [ 3 ]as trimethylsilyl derivatives on a DB-5HT capillarycolumn using MS detection with one target and twoqualifying ions for each compound in a total run timeof 26 min. Excellent resolution of 15 phenolic com-pounds was achieved. Flavonoid glycosides in fruitjuices have been characterised [ 37 ] by GC^MS asthe corresponding aglycones following extraction,hydrolysis and derivatisation to TMS ethers. In thecase of anthocyanins, derivatisation is an essentialstep for GC^MS [ 19 ].

The on-line coupling of LC and MS [ 4,38^40 ] is ofenormous potential as demonstrated in Fig. 3. Thefundamental limitations relate to the problem of inter-facing a high pressure LC system with a mass spec-trometer ion source located inside a high-vacuumenvelope and of producing gas-phase ions, particu-larly intact molecular ion species, without the appli-cation of heat. First-generation LC^MS instrumentssuch as the moving wire or belt interface [ 31] over-came incompatibility between high vacuum and theintroduction of solvent by removing the liquid. Themoving belt system enabled EI, CI and FAB ionisation

but here also the classical mass spectrometric gas-phase ionisation techniques were of limited applica-tion to more polar compounds such as the plant phen-ols and these systems never achieved widespreadacceptance. A recent report [ 10 ] however comparedparticle beam (PB) EI-MS with ultraviolet and electro-chemical detection of phenolic acids. In the secondgeneration, for example, continuous £ow FAB (ordynamic FAB), soft ionisation techniques werecoupled with liquid introduction. When introduced,continuous £ow FAB-MS rapidly superseded allother ionisation methods for £avonoids and, in par-ticular, anthocyanin studies as it provided an idealtechnique for the analysis of highly polar com-pounds, without the need for derivatisation. It hadthe advantage of producing a molecular ion plusvarious fragmentation ions which provided structur-al information. Nowadays, interfacing and ionisationhave merged in third-generation instruments suchas the thermospray and electrospray mass spectrom-eters.

4. Development of newer soft ionisationtechniques

Thermospray ionisation (TSI ) [ 41] was the ¢rstmethod to combine true LC^MS compatibility withthe ability to determine non-volatile thermally labilecompounds. In this approach, the chromatographiceluate passes through a resistively heated stainlesssteel capillary tube located in the thermospray probe.A supersonic jet of vapour is created by adjusting thetemperature of the capillary to a level where the sol-vent is partially vaporised and expansion of the result-ing vapour provides the gas dynamic forces needed foratomisation of the remaining liquid. The vapour jetcontains an entrained `mist' of small, statistically gen-erated electrically charged droplets that ¢nally as aresult of mechanisms detailed later give rise to theanalyte ions. There is no conventional externalmeans of ionisation which is accomplished duringthe solvent vaporisation and desolvation of the smalldroplets. The analyte ions leave the thermospraysource through an ori¢ce in a sampling cone. Theprocess is greatly enhanced if the analyte is itselfionic or by the presence of a volatile electrolyte.Ammonium acetate is the best general-purpose elec-trolyte for ionising neutral analytes although othervolatile salts, acids, bases or no electrolyte at all maybe preferred in certain applications. Reverse-phaseeluents containing a high percentage of water are pre-


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ferred for good sensitivity at conventional LC £owrates, conditions well suited to the analysis of polarphenolic species [ 42 ]. However, TSI is not without itsdif¢culties, in particular, the ef¢ciency of ion produc-

tion varies widely with compound type and the £owrate and temperature of the inlet tube must be opti-mised for each different compound class. Moreover,each class of compound requires different conditions


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for optimal ionisation and this is further complicatedby gradient elution.

TSI continues to ¢nd applications despite its limi-tations [ 42,43 ] but will probably be gradually phasedout by atmospheric pressure chemical ionisation(APCI) and electrospray (see Fig. 1 in [ 44 ]). In themean time, TSI LC^MS^MS has provided [ 43 ] char-acterisation of catechins and £avonoids from their col-lision induced dissociation spectra of the quasi-molec-ular ion. Flavonoids exhibited three types of ringcleavage in the pyran ring and differentiation among£avanone, £avone and £avonol was possible.

In TSI, the fundamental problem of coupling aliquid chromatograph and a mass spectrometer,namely, that of pressure reduction, is addressed byconnecting an additional pumping line directly to theion source. The majority of the vaporised solvent andmist goes to this auxiliary vacuum pump after it tra-verses a skimmer aperture to the mass spectrometer. Amore elegant solution which obviates the problementirely is to eliminate the vacuum altogether. Inatmospheric pressure ionisation (API ) [ 38,39,45 ],pumping at the ion source is eliminated and atmos-pheric pressure operation of the ion source facilitateseasier installation, ¢tting and cleaning.

In APCI, a combination of a heated capillary and acorona discharge is used to promote the formation ofions from the nebulised sample. Ionisation occurs inthe gas phase by ion molecule reactions and followsthe sequence

Sample in solution!sample vapour!sample ions

In coupled mode, the eluant from the HPLC isevaporated completely and the mixture of solventand sample vapour is then ionised by chemical ionisa-tion involving proton transfer, adduct formation andcharge exchange reactions in positive ion mode orproton abstraction, anion attachment and electron cap-ture reactions in the negative mode. APCI is compat-ible with 100% aqueous or 100% organic mobilephases at £ow rates of up to 2 ml / min [ 23 ] and is

therefore ideal for normal or reverse-phase operationwith conventional HPLC columns. In this fashion ithas been used to determine various iso£avones. Neg-ative ion mode provided quality mass spectra whichgave not only the molecular mass of the iso£avones,but also their molecular structures. Deuterium oxidewas used to induce peak shifts in the mass spectra todetermine the number of exchangeable hydrogenatoms in each molecule. Aramend|èa et al. [ 11]reported the LC^APCI^MS of phenolics in olivemill wastewater. Analytes were separated on a C18phase by gradient elution with methanol^water con-taining formic acid. Mass spectral conditions wereoptimised by direct infusion of standards in £ow injec-tion mode into the APCI source. The study wasrestricted to negative ion mode with detection limitsin total ion current mode ranging from 0.5 ng to 500ng. These detection limits were about 20 times betterwhen working in selected ion monitoring mode andmonitoring the [M-H ]3 ion. Mass spectra wererecorded with soft (315 V) and strong (350 V) vol-tages applied at the ion source of the mass spectrom-eter. With the smaller voltages, deprotonated molec-ular species [M-H ]3 were the major ions observed inthe mass spectra with the appearance of very few frag-ment ions which were all of low intensity. The pres-ence of substantial fragmentation from collisionallyinduced dissociation processes which became evidenton increasing the voltage applied at the source (extrac-tion and cone) voltages, gave structural informationabout the molecules. Structures were assigned tomajor eluent cluster ions from methanol^water^for-mic acid mixtures occurring at m / z 91, 113, 137,159, 181 and 183.

APCI still has a major drawback for polar thermo-labile plant phenols: volatilisation of sample mustoccur before ionisation. The newer soft ionisationmethods overcome lack of analyte volatility by directformation or emission of ions from the surface of acondensed phase and sample ions are collected fromthe condensed phase inside the ion source and trans-ferred to the mass analyser. Hence, they eliminate the

Fig. 3. Chromatograms comparing mass spectrometric detection with ultraviolet detection at 280 nm for the reverse-phasechromatography of phenolics extracted from olive fruit. Separation was achieved on a Waters C18 column (2 mmU15 cm)thermostatted at 35³C with a gradient using water with 0.1% formic acid and methanol with 0.1% formic acid. Mass spectrawere collected on a Quattro II quadrupole mass spectrometer (MS) (Micromass, Altrincham, Cheshire, UK) by electrosprayionisation (ESI). An injection volume of 10 Wl and a constant £ow rate of 0.200 ml / min was used for each analysis with a splitratio of approximately 10:1 (UV detector:MS). Chromatograms are shown for total ion current (TIC) and extracted ionmonitoring at m /z values of 525 and 541 corresponding to detection of ligstroside and oleuropein at 18.9 and 16.5 min,respectively. The peak at 10.3 min in the TIC chromatogram is elenolic acid, a non-phenolic degradation product of oleuropein.6


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need for neutral molecule volatilisation prior to ionisa-tion and generally minimise thermal degradation ofthe molecular species. The process is termed electro-spray [ 46 ] and it is most suited to compounds thateither exist as ions in solution, can be ionised at anappropriate pH or polar neutral molecules that canassociate with small ions such as Na�, ammonium orchloride. It is therefore ideally suited to the plant phe-nols. Electrospray is used as a generic term that alsocovers several variants of the basic technique that dif-fer in the precise manner in which charged droplets ofsample are produced. These techniques collectivelyhave revolutionised the ¢eld of mass spectrometry[ 47 ].

Electrospray ion production requires two steps: dis-persal of highly charged droplets at near atmosphericpressure, followed by conditions resulting in dropletevaporation. An electrospray is generally produced byinjection of a solution of the analyte through a metalcapillary maintained at a potential of several kV rela-tive to the surrounding chamber walls. There are twowidely debated mechanisms for the formation of gas-phase ions from solute species in charged droplets. Inthe charged residue model [ 47^50 ], a strong electric¢eld generated by the potential difference causes theeluate to be expelled from the capillary as a plume ofcharged droplets. As solvent evaporates from thesmall droplets, a critical size ^ the Rayleigh limit ^ isreached where Coulomb repulsion between thecharged entities in the droplet ( mobile phase, electro-lyte and sample ions ) becomes greater than the surfacetension forces. At this point the droplet breaks intoseveral smaller droplets and the process is repeateduntil the droplets become so small that they containonly one analyte molecule. This molecule retainssome of the droplet charge when the last solvent mol-ecules evaporate. The ionised species enter the massanalyser through a skimmer cone. The same sequenceof evaporation and Coulomb ¢ssion steps is envisaged

in the ion desorption (evaporation) model [ 47,51,52 ].However, at some intermediate stage before the drop-lets are so small that they contain only one analytemolecule, the surface ¢eld is suf¢ciently intense tocause expulsion of a charged analyte ion from thedroplet surface. Thus, the fundamental differencebetween theories is in how the analyte ion becomesseparated from other species in the droplet. The proc-ess of ESI appears reminiscent of what occurs in TSI.The difference is that in ESI most or all of the neb-ulisation work is done by electrostatic forces whilethat work is performed by gas dynamic forces inTSI.

Electrospray ideally operates with £ow rates at lessthan 10 Wl / min using microbore columns or a conven-tional column equipped with an ef£uent splitter. How-ever, conventional columns and £ow rates are compat-ible with ESI without the need for splitting by use ofpneumatic nebulisers or thermal input. The latterenhance the spray process and increase the rate ofsolvent evaporation from the highly charged dropletsand hence counter to some degree the decreasing ion-isation ef¢ciency at higher £ows. ESI spectra of gly-cosidic compounds typically show a pseudomolecularion (e.g., [M+H ]�), aglycone ion and ions associatedwith the solvent although fragmentation can often beinduced by raising the cone voltage. Acid (acetic orformic ) is often added to mobile phases in positive ionelectrospray as a source of protons to assist ionisation.Sensitivity is improved when the organic content inthe mobile phase exceeds 20%.

The type of information required in an analysis(e.g., structural elucidation ^ molecular mass versusfragmentation data; quantitative determination) andthe nature of the analyte, and more speci¢cally itspolarity, are the prime factors in the choice of theLC^MS interface (Table 4). Nonetheless, electrosprayis undoubtedly the fastest developing approach [ 44 ].The power of electrospray as an alternative, highly

Table 4Application areas of various mass spectrometric methods with respect to analyte polarity and molecular mass

Thermospray Particle beam Electrospray APCI

Sample polarity medium^high low^medium medium^high low^mediumMolecular massrange

100^2000 (polarity ) 100^2000(volatility )

100^ca 100 000 ( multiple charges) 100^1500 ( thermallability )

HPLC compatability medium good good goodEase of use low medium medium mediumSensitivity medium low high highStructural information medium

(with fragmentor )high (EI and CI ) medium (with CID) medium (with CID)


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sensitive soft ionisation technique for investigation ofpolar, non-volatile and thermolabile molecules such asanthocyanins has been demonstrated [ 53 ]. Ion sprayionisation modi¢cation [ 54 ] incorporated pneumaticnebulisation with true electrospray to improve theef¢ciency of the spray generation process. Thischange enabled operation with higher £ow rates of2^200 Wl / min [ 54 ] when used with conventional(e.g., 2U100 mm) reverse-phase columns as illus-trated by the structure determination of anthocyanins[ 53 ] and the identi¢cation [ 7 ] of the phenolic gluco-side content of olive leaves. In one application, ananthocyanic extract of blueberry was applied [ 19 ] toa solid-phase cartridge and lipophilic substances (e.g.,chlorophyll and carotenoids ) were removed with hex-ane following which £avonoids and phenolic acidswere eluted with ethyl acetate. Hydrophilic speciesincluding anthocyanins, sugars and organic acidswere then desorbed with methanol and after furtherclean-up the anthocyanin fraction was analysed inthe £avylium cationic form by ion spray MS. Strong(quasi-)molecular glycoside ions were observed andthe API ion spray interface enabled the use of the sameconditions as in conventional HPLC with ultravioletdetection.

On rare occasions, MS can provide data suf¢cientfor full structure analysis but more generally it is usedto determine molecular mass and to establish the dis-tribution of substituents on the phenolic ring(s ). Thissituation will improve with new developments. Tan-dem mass spectrometry (MS^MS) has been appliedsuccessfully to problems involving trace analysis ofcitrus £avanones and metabolite identi¢cation [ 2 ].Positive CI MS^MS was superior to EI MS^MS forthe detection of a common daughter ion for £avanonesat m / z 153. Using this approach, the £avanones nar-ingenin and hesperitin were detected in human urineafter citrus ingestion. Glycosides were labile under theexperimental conditions, probably during ionisation.MS^MS particularly in combination with LC and softionisation techniques [ 43 ] can be expected to improvesigni¢cantly separations of complex samples. Theinformation available from such methods can beexpected to increase with the development of newertechnologies including collisionally induced dissoci-ation spectra [ 43 ]. For the latter, alternating low andhigh ori¢ce voltages are used in which no fragmenta-tion occurs at low voltage and fragmentation isinduced at the high ori¢ce voltage. This permits simul-taneous measurement of molecular mass and struc-tural characterisation.

5. Future directions

An increasing application of multistage ion analy-sers coupled with various MS ionisation methods willfacilitate the identi¢cation of an increasing range ofplant phenolics. In routine applications, mass-selec-tive detection will provide rapid trace analysis of phe-nolics and the selectivity of MS^MS and collision-induced dissociation spectra will be used to advantageto facilitate dif¢cult separations.

Developments still in their infancy, at least in appli-cations to phenolic analysis, include the coupling ofcapillary electrophoresis with MS (CE^MS). In com-bination with the inherent sensitivity and selectivity ofmass spectrometry, CE^MS [ 38,39 ] becomes a verypowerful technique. The correspondence between CEand electrospray ionisation £ow rates provides thebasis for an extremely attractive technique. Hence,various iso£avones were separated [ 13 ] on anuncoated fused-silica column using 25 mM ammo-nium acetate buffer and negative ion electrospray ion-isation mass spectrometric detection. This approachpermitted the determination of molecular mass of theiso£avones as well as the presence of various func-tional groups according to observed losses from the[M-H ]3 ion during collision-induced dissociationeffected by adjusting MS parameters.

References

[ 1 ] F. Angerosa, N. d'Alessandro, P. Konstantinou, L. DiGiacinto, J. Agric. Food Chem. 43 (1995) 1802.

[ 2 ] R.A. Weintraub, B. Ameer, J.V. Johnson, R.A. Yost,J. Agric. Food Chem. 43 (1995) 1966.

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Danielle Ryan is currently in the ¢rst year of her Ph.D. atCharles Sturt University Riverina. Her Ph.D. projectconcerns phenolic compounds in olive fruits. Sheobtained her undergraduate degree in analytical chem-istry and her Honours project concerned the role of phe-nolic compounds in olive fruits as a function ofphysiological development.

Kevin Robards is Associate Professor of Chemistry atCharles Sturt University Riverina. He obtained his Ph.D. inanalytical chemistry from the University of New SouthWales in 1979. His research interests are focused on theapplication of analytical chemistry to food science and inparticular the identi¢cation and role of naturally occurringphenolic compounds in fruits.

Paul Prenzler is a lecturer in Chemistry at Charles SturtUniversity Riverina. He received his Ph.D. in 1992 fromthe University of Queensland and has held postdoctoralappointments with Hitachi Ltd in Japan and the ResearchSchool of Chemistry, Australian National University. Priorto joining CSU he was a Research Fellow with Prof. TonyWedd at the University of Melbourne. His current researchinterests include applications of electrospray massspectrometry to metal complexes, particularly those withphenolic compounds.

Michael Antolovich is a lecturer at Charles SturtUniversity Riverina. He completed his Ph.D. in 1989 atthe University of New South Wales, followed by apostdoctoral position at Princeton University working onmetalloporphyrins. He returned to Australia on a ResearchFellowship. His current interests are in computationalchemistry and its application to food and environmentalsciences.


Applications of mass spectrometry to plant phenols

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