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Applications of mass spectrometry to plant phenols
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Applications of mass spectrometry to plant phenols Danielle Ryan, Kevin Robards*, Paul Prenzler, Michael Antolovich
School of Science and Technology, P.O. Box 588, Wagga Wagga 2678, Australia Plant phenols embrace a considerable range of compounds and are de¢ned as those substances derived from the shikimate acid pathway and phenylpropanoid metabolism. The present article examines the application of mass spectrometry to the analysis of these compounds and traces the chronological development of analyte ionisation methods. z1999 Elsevier Science B.V. All rights reserved. Keywords: Soft ionization; Phenols, Plants
1. Introduction Terminology relating to the plant phenols is often confusing and a brief overview is presented before considering aspects of their mass spectrometry. The plant phenols (Table 1 ) are a diverse group of secondary metabolites possessing an aromatic ring bearing one or more hydroxy substituents. However, this de¢nition is not entirely satisfactory since it inevitably includes compounds such as oestrone, the female sex hormone which is principally terpenoid in origin. For this reason, a de¢nition based on metabolic origin is preferable, the plant phenols being regarded as those substances derived from the shikimate pathway and phenylpropanoid metabolism ( Fig. 1 ). Some members are characterised as `polyphenols', an unfortunate term since not all are polyhydroxy derivatives. In particular, a number of compounds, for example, cinnamic acid, elenolic acid, shikimic acid and quinic acid, are treated in the present discussion as phenolics because of metabolic considerations although they lack a phenolic group or even an aromatic ring. Plant phenols (Table 2 ) have been classi¢ed into major groupings distinguished by the number of constitutive carbon atoms in conjunction with the structure of the basic phenolic skeleton. The most widespread and *Corresponding author. E-mail: [email protected]
diverse of the phenolics are the £avonoids which are built upon a C6 -C3 -C6 £avone skeleton in which the three-carbon bridge between the phenyl groups is commonly cyclised with oxygen. Several classes of £avonoid ( Fig. 2 ) are differentiated on the degree of unsaturation and degree of oxidation of the three-carbon segment. Within the various classes, further differentiation is possible based on the number and nature of substituent groups attached to the rings. The range of known phenolics is thus vast and also includes polymeric lignins and condensed tannins although these species are not considered in the present review. Additional structural complexity is introduced by the common occurrence of certain phenolics as the O-glycosides ( or, less commonly, as C-glycosides ) in which one or more of the phenolic hydroxyl groups is bound to a sugar or sugars by an acid-labile hemiacetal bond. Glucose is the most commonly encountered sugar with rhamnose and the disaccharide, rutinose ( 6-O-K-L-rhamnosyl-D-glucose ), is also encountered. Anthocyanins are intensely coloured plant pigments in which an anthocyanidin aglycone is glycosidically linked to a sugar( s ). Acylation of the glycosides in which one or more of the sugar hydroxyls is derivatised with an acid such as acetic or ferulic acid is occasionally observed. Although the number of identi¢ed phenols is increasing exponentially, the phenolic content of most plants constitutes a complex mixture, the complete chemical nature of which has not, as yet, been elucidated for any species. For example, there are many phenolics present in low concentrations which remain unidenti¢ed but whose signi¢cance may far outweigh their concentration level. Isolation and structure elucidation of these compounds are the initial steps to understanding their signi¢cance and action. Information on their biosynthesis is essential to understanding the interaction between plants and the environment. It will also facilitate genetic manipulation ( both qualitative and quantitative ) of the phenolic content of plants for dietary modulation of disease and to provide an environmentally sustainable feedstock for the petrochemical industry.
0165-9936/99/$ ^ see front matter PII: S 0 1 6 5 - 9 9 3 6 ( 9 8 ) 0 0 1 1 8 - 6
ß 1999 Elsevier Science B.V. All rights reserved.
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Table 1 De¢nition of terms used Plant phenols
Flavonoids
Glycoside
Aglycone API APCI Electrospray ionisation
Aromatic substances bearing one or more hydroxy groups on an aromatic ring and derived from the shikimate pathway or phenylpropanoid metabolism A diverse class of plant phenols based on a C6 ^C3 ^C6 £avone skeleton in which the three-carbon bridge between the phenyl groups is commonly cyclised with oxygen In the current context, a molecule formed by linking a phenol and a sugar( s ) by an acidlabile hemiacetal bond between the phenolic hydroxyl group( s ) and the sugar( s ) The phenolic portion of a glycoside Atmospheric pressure ionisation ( generic term ) Atmospheric pressure ionisation by chemical ionisation Ion formation from samples in solution by dispersion of liquids into an electrically charged aerosol due to the action of an electric ¢eld
Methods of characterisation and identi¢cation of plant phenols follow those in general use for natural substances. Hence, preparation of an extract, biological screening, bioguided fractionation, isolation and structure elucidation is the usual approach. For the latter, physical methods based on spectral characteristics feature prominently although older chemical and biochemical approaches should be considered particularly as adjuncts to spectral analysis. Of these techniques, none has been more useful in providing structural information than mass spectrometry (MS ). However, its use has not been restricted to this role for mass spectrometry is increasingly viewed as a mass-speci¢c detector [ 1 ] for both qualitative and
quantitative applications in high resolution chromatography. Tandem mass spectrometry (MS^MS ) is particularly attractive for trace analysis where high selectivity is essential [ 2 ]. In another role, MS of phenolic compounds meets the multivariate criteria for ¢ngerprinting food samples in adulteration testing [ 3 ]. Representative applications of MS in the analysis of plant phenols are presented in Table 3.
2. Classical ionisation techniques The traditional mode of MS involves electron impact ionisation ( EI ) in which the neutral sample molecule is impacted in the gas phase with an electron beam of energy 70^100 eV. EI mass spectrometry ( EI^MS ) is used extensively in structural studies of a number of plant phenols including various £avonoids. EI produces a positive radical ion Mc which if detected provides information on the molecular mass of the analyte. Comprehensive information about the mass spectra of £avonoids has been published [ 26,27 ]. In general, the EI mass spectra of £avonoid aglycones are characterised [ 24,25 ] by intense molecular ion peaks plus signi¢cant fragments from both A and B rings. The fragmentations often provide suf¢cient information to determine molecular mass, elemental formula, substitution patterns in the A and B rings, and the class of £avonoid. For example, the EI mass spectrum of substituted £avones shows the typical fragmentation pattern to yield two fragments characteristic of the A and B rings. In the case of a disubstituted £avone there are three possibilities, either both substituents are located on the A or B ring or one is located on each ring and the mass spectrum will provide unambiguous information on the substitution pattern. In less favourable situations
Table 2 Classi¢cation of plant phenols Class
Base structure
Examples
Comments
Simple phenols Phenolic acids
C6 C6 C1
Hydroxycinnamic acids Lignans Coumarins
C6 C3
catechol, resorcinol p-hydroxybenzoic acid, salicylic acid, gallic acid caffeic acid, ferulic acid
among the most common of all phenolic compounds
( C6 C3 ) 2 C6 C3
nordihydroguaiaretic acid umbelliferone
Chromones Flavonoids
C6 C3 C6 C3 C 6
eugenin quercitin, cyanidin
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Table 3 Mass spectrometric techniques used in the analysis of phenolic compounds in plants and related samples Sample
Phenolic compounds
Technique
Ref.
Wines Food Olive oil Krameria triandra roots
phenolic acids, £avonoids review
GC^MS LC^MS GC^MS LC^MS (PB, EI ); FAB^MS ( negative ion mode ) and FAB^MS^MS
[3] [4] [5] [6]
ESI-LC^MS GC^MS GC^MS (TMS derivatives ) PB-LC^MS ( EI ) LC^APCI-MS ( negative ion )
FAB^MS ( negative ion ) LC^MS Thermospray LC^MS Thermospray LC^MS
[7] [8] [9] [ 10 ] [ 11 ] [ 12 ] [ 13 ] [ 14 ] [ 15 ] [ 16 ] [ 17 ] [ 18 ]
API ion spray LC^MS
[ 19 ]
Plasmaspray^LC^MS Plasma desorption MS APCI-LC^MS ( positive ion ) EI-GC^MS EI and CI-MS
[ 20 ] [ 21 ] [ 22 ] [ 23 ] [ 24 ] [ 25 ]
low to intermediate molecular weight polyphenol constituents with radical scavenging activity Olive leaf oleuropein, ligstroside Oat groats and hulls phenolic acids Spruce shoots and galls 47 phenolics and phenolic glucosides Not applied 15 benzoic and cinnamic acid derivatives Olive mill wastewater 15 phenolic compounds White and silver birch gallic and chlorogenic acids iso£avones Onions £avonoids Juice of Sedum telephium leaves 6 £avonol glycosides Plant extracts £avonoid precursors ( cis trans isomers ) Epilobium species 19 £avonol glycosides Lemon peel coumarins, phenyl propanoid glycosides, £avone-C-glucosides, £avonols, £avone-Oglycosides and £avonones Vitis vinifera L. anthocyanins: 3-glucosides, the 3-acetylglucosides, and the 3-p-coumaroylglucosides of delphinidin, cyanidin, petunidin, peonidin, and malvidin; also 3,5-diglucosides Passionfruit swertisin Black tea £avanols, £avonol glycosides, chlorogenic acid Sorghum bicolor anthocyanins and anthocyanidins Soy foods iso£avone glycosides (Orange oil ) 39 polymethoxylated £avones Not applied 43 £avones and £avonols, 7 iso£avones, 18 £avanones and dihydro£avonols, and 11 chalcones and dihydrochalcones
where there are many substituents, the mass spectrum will assist in structural elucidation although other techniques such as nuclear magnetic resonance will be required for a de¢nitive assignment of the substitution. EI more commonly produces an internally excited parent ion, Mc , which fragments so extensively that the parent radical cation is not observed. This is the situation with glycosidically linked phenols where the EI ( and chemical ionisation, CI ) mass spectra [ 28 ] are dominated by the same ions as for the corresponding aglycones [ 2,28 ], the protonated aglycone invariably being the base peak in the mass spectrum. A distinction with the mass spectra of the aglycones is the relatively weak fragments from ¢ssion of the A and B rings. However, EI is generally unsuitable for glycosidically bound phenols because these compounds are polar and thermolabile. Similarly, CI [ 29 ] has limited application for the broader range of plant phenols
CE^ESI-MS ( negative ion )
because vaporisation of the analyte prior to ionisation is once again a prerequisite. Chemical derivatisation overcomes the limitations of restricted volatility and thermal stability of polar phenols but presents further dif¢culties by increasing the molecular mass of the analyte possibly beyond the range of the mass analyser. This is a major consideration for glycosidic species with numerous hydroxyl groups although permethylation or perdeuteromethylation may be suitable. However, these methods often produce mixtures of partially derivatised compounds and, in some cases, permethylation can produce artefacts, e.g., when ester groups are present. Usually only weak molecular ion signals are observed when permethylated compounds are studied under EI conditions. The application of chemical derivatisation is extended in the discussion of coupled techniques. The development of desorption ionisation techniques in which ionisation of thermolabile molecules
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occurs directly from the condensed phase provides a solution to the problem of limited analyte volatility and stability. Field desorption ( FD ) was the ¢rst technique employed for direct analysis of polar and thermolabile phenolic species. FD has been applied to the analysis of £avonoid glycosides [ 30 ] and provides molecular mass data but usually little structural information and is ``notorious for the transient nature of the spectra'' [ 31 ]. Desorption chemical ionisation ( DCI ) uses a probe consisting of an electrically heated tungsten wire introduced into the CI source. DCI provides rapid heating of the analyte and overcomes the problem of thermal decomposition inherent in conventional CI but it was the development of particle induced desorption or sputtering techniques that truly extended the range of analyte polarities and sizes. Fast atom bombardment ( FAB ) [ 32 ] is the most successful and in this technique the analyte is solubilised in a non-volatile polar matrix ( e.g., glycerol, thioglycerol ) and deposited on a copper target which is bombarded with fast neutral energised particles such as xenon or argon thereby inducing the desorption and ionisation. The value of FAB-MS is demonstrated by the molecular mass and structural information provided for large polar glycosides in both 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. Furthermore, background matrix signals ( from glycerol or thioglycerol ) can complicate interpretation of the spectra. Nevertheless, when combined with collisionally induced dissociation of positive ions and tandem mass spectrometric techniques, FAB-MS can provide information on the aglycone moiety, the carbohydrate sequence and the glycosylation position of glycosides [ 34 ].
3. Coupled techniques The on-line coupling of methods is of enormous potential because the selectivity can then be tuned in an optimal way, which in turn can be translated to either a faster analysis or an improved determination limit. Coupled gas chromatography-mass spectrometry ( GC^MS ) is now well established as a routine technique carried out with either EI or CI sources, since these are appropriate for the introduction of volatile compounds. Thus, Berahia et al. [ 24 ] analysed 39 polymethoxylated £avones by GC^MS. In addition to common behaviour of £avones under electron impact, such as a retro-Diels^Alder reaction which gives a characteristic fragment from the phenyl group 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 characteristic of various substitution patterns were also identi¢ed. Improvements in GC column technology have increased the range of £avonoids amenable to GC^MS as the underivatised compounds. For instance, Schmidt et al. [ 35 ] analysed 49 £avones, £avonols, £avanones and chalcones without derivatisation by GC^MS in EI mode. Compared with direct inlet mass spectra, the GC^MS data exhibited the same typical fragmentation patterns but with slight differences in intensities. However, because of limited volatility, analysis of phenolic compounds and their glycosides, in particular, by GC^MS has not generally found favour. Two approaches have been used, namely, hydrolysis of the glycosides to their corresponding aglycones and / or chemical derivatisation. Angerosa et al. [ 36 ] have shown GC^MS with CI to be an effective tool for phenol identi¢cation after extraction from olive oil with methanol and derivatisation with bis( trimethylsilyl )tri£uoracetamide. Peaks in the mass spectra at m / z 192 or at m / z 280 were useful for assigning the phenolic nature to minor components. The advantages of CI with ammonia for providing molecular masses of the aglycones were also demonstrated. One of the problems associated with derivatisation, namely, the formation of several derivatives from a single analyte, was highlighted by this work. Derivatisation has also been applied to the analysis of the phenolic components of wine which were extracted and separated [ 3 ] as trimethylsilyl derivatives on a DB-5HT capillary column using MS detection with one target and two qualifying ions for each compound in a total run time of 26 min. Excellent resolution of 15 phenolic compounds was achieved. Flavonoid glycosides in fruit juices have been characterised [ 37 ] by GC^MS as the corresponding aglycones following extraction, hydrolysis and derivatisation to TMS ethers. In the case of anthocyanins, derivatisation is an essential step for GC^MS [ 19 ]. The on-line coupling of LC and MS [ 4,38^40 ] is of enormous potential as demonstrated in Fig. 3. The fundamental limitations relate to the problem of interfacing a high pressure LC system with a mass spectrometer ion source located inside a high-vacuum envelope and of producing gas-phase ions, particularly intact molecular ion species, without the application of heat. First-generation LC^MS instruments such as the moving wire or belt interface [ 31 ] overcame incompatibility between high vacuum and the introduction of solvent by removing the liquid. The moving belt system enabled EI, CI and FAB ionisation
but here also the classical mass spectrometric gasphase ionisation techniques were of limited application to more polar compounds such as the plant phenols and these systems never achieved widespread acceptance. A recent report [ 10 ] however compared particle beam (PB ) EI-MS with ultraviolet and electrochemical detection of phenolic acids. In the second generation, for example, continuous £ow FAB ( or dynamic FAB ), soft ionisation techniques were coupled with liquid introduction. When introduced, continuous £ow FAB-MS rapidly superseded all other ionisation methods for £avonoids and, in particular, anthocyanin studies as it provided an ideal technique for the analysis of highly polar compounds, without the need for derivatisation. It had the advantage of producing a molecular ion plus various fragmentation ions which provided structural information. Nowadays, interfacing and ionisation have merged in third-generation instruments such as the thermospray and electrospray mass spectrometers.
4. Development of newer soft ionisation techniques Thermospray ionisation (TSI ) [ 41 ] was the ¢rst method to combine true LC^MS compatibility with the ability to determine non-volatile thermally labile compounds. In this approach, the chromatographic eluate passes through a resistively heated stainless steel capillary tube located in the thermospray probe. A supersonic jet of vapour is created by adjusting the temperature of the capillary to a level where the solvent is partially vaporised and expansion of the resulting vapour provides the gas dynamic forces needed for atomisation of the remaining liquid. The vapour jet contains an entrained `mist' of small, statistically generated electrically charged droplets that ¢nally as a result of mechanisms detailed later give rise to the analyte ions. There is no conventional external means of ionisation which is accomplished during the solvent vaporisation and desolvation of the small droplets. The analyte ions leave the thermospray source through an ori¢ce in a sampling cone. The process is greatly enhanced if the analyte is itself ionic or by the presence of a volatile electrolyte. Ammonium acetate is the best general-purpose electrolyte for ionising neutral analytes although other volatile salts, acids, bases or no electrolyte at all may be preferred in certain applications. Reverse-phase eluents containing a high percentage of water are pre-
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ferred for good sensitivity at conventional LC £ow rates, conditions well suited to the analysis of polar phenolic species [ 42 ]. However, TSI is not without its dif¢culties, in particular, the ef¢ciency of ion produc-
tion varies widely with compound type and the £ow rate and temperature of the inlet tube must be optimised for each different compound class. Moreover, each class of compound requires different conditions
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Fig. 3. Chromatograms comparing mass spectrometric detection with ultraviolet detection at 280 nm for the reverse-phase chromatography 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 spectra were collected on a Quattro II quadrupole mass spectrometer (MS ) (Micromass, Altrincham, Cheshire, UK ) by electrospray ionisation ( ESI ). An injection volume of 10 Wl and a constant £ow rate of 0.200 ml / min was used for each analysis with a split ratio of approximately 10:1 (UV detector:MS ). Chromatograms are shown for total ion current (TIC ) and extracted ion monitoring 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
for optimal ionisation and this is further complicated by gradient elution. TSI continues to ¢nd applications despite its limitations [ 42,43 ] but will probably be gradually phased out by atmospheric pressure chemical ionisation ( APCI ) and electrospray ( see Fig. 1 in [ 44 ]). In the mean time, TSI LC^MS^MS has provided [ 43 ] characterisation of catechins and £avonoids from their collision induced dissociation spectra of the quasi-molecular ion. Flavonoids exhibited three types of ring cleavage in the pyran ring and differentiation among £avanone, £avone and £avonol was possible. In TSI, the fundamental problem of coupling a liquid chromatograph and a mass spectrometer, namely, that of pressure reduction, is addressed by connecting an additional pumping line directly to the ion source. The majority of the vaporised solvent and mist goes to this auxiliary vacuum pump after it traverses a skimmer aperture to the mass spectrometer. A more elegant solution which obviates the problem entirely is to eliminate the vacuum altogether. In atmospheric pressure ionisation ( API ) [ 38,39,45 ], pumping at the ion source is eliminated and atmospheric pressure operation of the ion source facilitates easier installation, ¢tting and cleaning. In APCI, a combination of a heated capillary and a corona discharge is used to promote the formation of ions from the nebulised sample. Ionisation occurs in the gas phase by ion molecule reactions and follows the sequence Sample in solution!sample vapour!sample ions In coupled mode, the eluant from the HPLC is evaporated completely and the mixture of solvent and sample vapour is then ionised by chemical ionisation involving proton transfer, adduct formation and charge exchange reactions in positive ion mode or proton abstraction, anion attachment and electron capture reactions in the negative mode. APCI is compatible with 100% aqueous or 100% organic mobile phases at £ow rates of up to 2 ml / min [ 23 ] and is
therefore ideal for normal or reverse-phase operation with conventional HPLC columns. In this fashion it has been used to determine various iso£avones. Negative ion mode provided quality mass spectra which gave not only the molecular mass of the iso£avones, but also their molecular structures. Deuterium oxide was used to induce peak shifts in the mass spectra to determine the number of exchangeable hydrogen atoms in each molecule. Aramend|èa et al. [ 11 ] reported the LC^APCI^MS of phenolics in olive mill wastewater. Analytes were separated on a C18 phase by gradient elution with methanol^water containing formic acid. Mass spectral conditions were optimised by direct infusion of standards in £ow injection mode into the APCI source. The study was restricted to negative ion mode with detection limits in total ion current mode ranging from 0.5 ng to 500 ng. These detection limits were about 20 times better when working in selected ion monitoring mode and monitoring the [ M-H ]3 ion. Mass spectra were recorded with soft ( 315 V ) and strong ( 350 V ) voltages applied at the ion source of the mass spectrometer. With the smaller voltages, deprotonated molecular species [ M-H ]3 were the major ions observed in the mass spectra with the appearance of very few fragment ions which were all of low intensity. The presence of substantial fragmentation from collisionally induced dissociation processes which became evident on increasing the voltage applied at the source ( extraction and cone ) voltages, gave structural information about the molecules. Structures were assigned to major eluent cluster ions from methanol^water^formic acid mixtures occurring at m / z 91, 113, 137, 159, 181 and 183. APCI still has a major drawback for polar thermolabile plant phenols: volatilisation of sample must occur before ionisation. The newer soft ionisation methods overcome lack of analyte volatility by direct formation or emission of ions from the surface of a condensed phase and sample ions are collected from the condensed phase inside the ion source and transferred to the mass analyser. Hence, they eliminate the
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Table 4 Application areas of various mass spectrometric methods with respect to analyte polarity and molecular mass
Sample polarity Molecular mass range HPLC compatability Ease of use Sensitivity Structural information
Thermospray
Particle beam
Electrospray
APCI
medium^high 100^2000 ( polarity )
low^medium 100^2000 ( volatility ) good medium low high ( EI and CI )
medium^high 100^ca 100 000 ( multiple charges )
low^medium 100^1500 ( thermal lability ) good medium high medium ( with CID )
medium low medium medium ( with fragmentor )
need for neutral molecule volatilisation prior to ionisation and generally minimise thermal degradation of the molecular species. The process is termed electrospray [ 46 ] and it is most suited to compounds that either exist as ions in solution, can be ionised at an appropriate pH or polar neutral molecules that can associate with small ions such as Na , ammonium or chloride. It is therefore ideally suited to the plant phenols. Electrospray is used as a generic term that also covers several variants of the basic technique that differ in the precise manner in which charged droplets of sample are produced. These techniques collectively have revolutionised the ¢eld of mass spectrometry [ 47 ]. Electrospray ion production requires two steps: dispersal of highly charged droplets at near atmospheric pressure, followed by conditions resulting in droplet evaporation. An electrospray is generally produced by injection of a solution of the analyte through a metal capillary maintained at a potential of several kV relative to the surrounding chamber walls. There are two widely debated mechanisms for the formation of gasphase ions from solute species in charged droplets. In the charged residue model [ 47^50 ], a strong electric ¢eld generated by the potential difference causes the eluate to be expelled from the capillary as a plume of charged droplets. As solvent evaporates from the small droplets, a critical size ^ the Rayleigh limit ^ is reached where Coulomb repulsion between the charged entities in the droplet ( mobile phase, electrolyte and sample ions ) becomes greater than the surface tension forces. At this point the droplet breaks into several smaller droplets and the process is repeated until the droplets become so small that they contain only one analyte molecule. This molecule retains some of the droplet charge when the last solvent molecules evaporate. The ionised species enter the mass analyser through a skimmer cone. The same sequence of evaporation and Coulomb ¢ssion steps is envisaged
good medium high medium ( with CID )
in the ion desorption ( evaporation ) model [ 47,51,52 ]. However, at some intermediate stage before the droplets are so small that they contain only one analyte molecule, the surface ¢eld is suf¢ciently intense to cause expulsion of a charged analyte ion from the droplet surface. Thus, the fundamental difference between theories is in how the analyte ion becomes separated from other species in the droplet. The process of ESI appears reminiscent of what occurs in TSI. The difference is that in ESI most or all of the nebulisation work is done by electrostatic forces while that work is performed by gas dynamic forces in TSI. Electrospray ideally operates with £ow rates at less than 10 Wl / min using microbore columns or a conventional column equipped with an ef£uent splitter. However, conventional columns and £ow rates are compatible with ESI without the need for splitting by use of pneumatic nebulisers or thermal input. The latter enhance the spray process and increase the rate of solvent evaporation from the highly charged droplets and hence counter to some degree the decreasing ionisation ef¢ciency at higher £ows. ESI spectra of glycosidic compounds typically show a pseudomolecular ion ( e.g., [ M+H ] ), aglycone ion and ions associated with the solvent although fragmentation can often be induced by raising the cone voltage. Acid ( acetic or formic ) is often added to mobile phases in positive ion electrospray as a source of protons to assist ionisation. Sensitivity is improved when the organic content in the mobile phase exceeds 20%. The type of information required in an analysis ( e.g., structural elucidation ^ molecular mass versus fragmentation data; quantitative determination ) and the nature of the analyte, and more speci¢cally its polarity, are the prime factors in the choice of the LC^MS interface (Table 4 ). Nonetheless, electrospray is undoubtedly the fastest developing approach [ 44 ]. The power of electrospray as an alternative, highly
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sensitive soft ionisation technique for investigation of polar, non-volatile and thermolabile molecules such as anthocyanins has been demonstrated [ 53 ]. Ion spray ionisation modi¢cation [ 54 ] incorporated pneumatic nebulisation with true electrospray to improve the ef¢ciency of the spray generation process. This change enabled operation with higher £ow rates of 2^200 Wl / min [ 54 ] when used with conventional ( e.g., 2U100 mm ) reverse-phase columns as illustrated by the structure determination of anthocyanins [ 53 ] and the identi¢cation [ 7 ] of the phenolic glucoside content of olive leaves. In one application, an anthocyanic extract of blueberry was applied [ 19 ] to a solid-phase cartridge and lipophilic substances ( e.g., chlorophyll and carotenoids ) were removed with hexane following which £avonoids and phenolic acids were eluted with ethyl acetate. Hydrophilic species including anthocyanins, sugars and organic acids were then desorbed with methanol and after further clean-up the anthocyanin fraction was analysed in the £avylium cationic form by ion spray MS. Strong ( quasi-)molecular glycoside ions were observed and the API ion spray interface enabled the use of the same conditions as in conventional HPLC with ultraviolet detection. On rare occasions, MS can provide data suf¢cient for full structure analysis but more generally it is used to determine molecular mass and to establish the distribution of substituents on the phenolic ring( s ). This situation will improve with new developments. Tandem mass spectrometry (MS^MS ) has been applied successfully to problems involving trace analysis of citrus £avanones and metabolite identi¢cation [ 2 ]. Positive CI MS^MS was superior to EI MS^MS for the detection of a common daughter ion for £avanones at m / z 153. Using this approach, the £avanones naringenin and hesperitin were detected in human urine after citrus ingestion. Glycosides were labile under the experimental conditions, probably during ionisation. MS^MS particularly in combination with LC and soft ionisation techniques [ 43 ] can be expected to improve signi¢cantly separations of complex samples. The information available from such methods can be expected to increase with the development of newer technologies including collisionally induced dissociation spectra [ 43 ]. For the latter, alternating low and high ori¢ce voltages are used in which no fragmentation occurs at low voltage and fragmentation is induced at the high ori¢ce voltage. This permits simultaneous measurement of molecular mass and structural characterisation.
5. Future directions An increasing application of multistage ion analysers coupled with various MS ionisation methods will facilitate the identi¢cation of an increasing range of plant phenolics. In routine applications, mass-selective detection will provide rapid trace analysis of phenolics and the selectivity of MS^MS and collisioninduced dissociation spectra will be used to advantage to facilitate dif¢cult separations. Developments still in their infancy, at least in applications to phenolic analysis, include the coupling of capillary electrophoresis with MS ( CE^MS ). In combination with the inherent sensitivity and selectivity of mass spectrometry, CE^MS [ 38,39 ] becomes a very powerful technique. The correspondence between CE and electrospray ionisation £ow rates provides the basis for an extremely attractive technique. Hence, various iso£avones were separated [ 13 ] on an uncoated fused-silica column using 25 mM ammonium acetate buffer and negative ion electrospray ionisation mass spectrometric detection. This approach permitted the determination of molecular mass of the iso£avones as well as the presence of various functional groups according to observed losses from the [ M-H ]3 ion during collision-induced dissociation effected by adjusting MS parameters.
References [ 1 ] F. Angerosa, N. d'Alessandro, P. Konstantinou, L. Di Giacinto, 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. [ 3 ] G.J. Soleas, J. Dam, M. Carey, D.M. Goldberg, J. Agric. Food Chem. 45 ( 1997 ) 3871. [ 4 ] M. Careri, A. Mangia, M. Musci, J. Chromatogr. 794 ( 1998 ) 263. [ 5 ] L. Cinquanta, M. Esti, E. Lanotte, J. Am. Oil Chem. Soc. 74 ( 1997 ) 1259. [ 6 ] R.M. Facino, M. Carini, G. Aldini, L. Deangelis, Rapid Commun. Mass Spectrom. 11 ( 1997 ) 1303. [ 7 ] A. De Nino, N. Lombardo, E. Perri, A. Procopio, A. Raffaelli, G. Sindona, J. Mass Spectrom. 32 ( 1997 ) 533. [ 8 ] Y.M. Xing, P.J. White, J. Am. Oil Chem. Soc. 74 ( 1997 ) 303. [ 9 ] C. Kraus, G. Spiteller, Phytochemistry 44 ( 1997 ) 59. [ 10 ] C. Bocchi, M. Careri, F. Groppi, A. Mangia, P. Manini, G. Mori, J. Chromatogr. 753 ( 1996 ) 157. [ 11 ] M.A. Aramend|èa, V. Boraèu, I. Garc|èa, C. Jimeènez, F. Lafont, J.M. Marinas, F.J. Urbano, Rapid Commun. Mass Spectrom. 10 ( 1996 ) 1585. [ 12 ] V. Ossipov, K. Nurmi, J. Loponen, E. Haukioja, K. Pihlaja, J. Chromatogr. 721 ( 1996 ) 59.
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[ 13 ] M.A. Aramend|èa, I. Garc|èa, F. Lafont, J.M. Marinas, J. Chromatogr. A 707 ( 1995 ) 327. [ 14 ] Y.K. Park, C.Y. Lee, Colloq. Inst. Natl. Rech. Agron. 69 (Polyphenols 94 ) ( 1995 ) 265. [ 15 ] N. Mulinacci, F.F. Vincieri, A. Baldi, A. Romani, D. Favretto, P. Traldi, Rapid Commun. Mass Spectrom. 9 ( 1995 ) 963. [ 16 ] P.A. Tarantilis, G. Tsoupras, M. Polissiou, J. Chromatogr. 699 ( 1995 ) 107. [ 17 ] B. Ducrey, J.L. Wolfender, A. Marston, K. Hostettmann, Phytochemistry 38 ( 1995 ) 129. [ 18 ] A. Baldi, R.T. Rosen, E.K. Fukuda, C.T. Ho, J. Chromatogr. 718 ( 1995 ) 89. [ 19 ] A. Baldi, A. Romani, N. Mulinacci, F.F. Vincieri, B. Casetta, J. Agric. Food Chem. 43 ( 1995 ) 2104. [ 20 ] A. Rehwald, B. Meier, O. Sticher, Pharm. Acta Helv. 69 ( 1994 ) 1531. [ 21 ] R.G. Bailey, H.E. Nursten, I. Mcdowell, J. Sci. Food Agric. 66 ( 1994 ) 203. [ 22 ] K.V. Wood, C. Bonham, J. Hipskind, R.L. Nicholson, Phytochemistry 37 ( 1994 ) 557. [ 23 ] S. Barnes, M. Kirk, L. Coward, J. Agric. Food Chem. 42 ( 1994 ) 2466. [ 24 ] T. Berahia, E.M. Gaydou, C. Cerrati, J.C. Wallet, J. Agric. Food Chem. 42 ( 1994 ) 1697. [ 25 ] P.A. Hedin, V.A. Phillips, J. Agric. Food Chem. 40 ( 1992 ) 607. [ 26 ] K.R. Markham, in J.B. Harborne ( Editor ), The Flavonoids: Advances in Research, Chapman and Hall, London, 1988, p. 427. [ 27 ] R.J. Grayer, in J.B. Harborne ( Editor ), Plant Biochemistry, Vol. 1, Plant Phenolics, Academic Press, San Diego, 1989, pp. 223, 283, 347. [ 28 ] H. Itokawa, Y. Oshida, A. Ikuta, Y. Shida, Chem. Lett. 1 ( 1982 ) 49. [ 29 ] A.G. Harrison, Chemical Ionization Mass Spectrometry, 2nd edn., CRC Press, Boca Raton, FL, 1992. [ 30 ] T. Nakanishi, A. Inada, K. Kambayashi, K. Yoneda, Phytochemistry 24 ( 1985 ) 339. [ 31 ] J. Wolfender, M. Mailard, A. Marston, K. Hostettmann, Phytochem. Anal. 3 ( 1992 ) 193. [ 32 ] B. Domon, K. Hostettmann, Phytochemistry 24 ( 1985 ) 575. [ 33 ] R. Gujer, D. Magnolato, R. Self, Phytochemistry 25 ( 1986 ) 1431. [ 34 ] M. Claeys, Q.M. Li, H. Van den Heuvel and L. Dillen, in R.P. Newton and T.J. Watton ( Editors ), Applications of Modern Mass Spectrometry in Plant Science Research, Clarendon Press, Oxford, 1996. [ 35 ] T.J. Schmidt, I. Merfort, U. Matthiesen, J. Chromatogr. 634 ( 1993 ) 350. [ 36 ] F. Angerosa, N. d'Alessandro, F. Corana, G. Mellerio, J. Chromatogr. 736 ( 1996 ) 195. [ 37 ] C.S. Creaser, M.R. Koupai-Abyazani, G.R. Stephenson, Analyst 117 ( 1992 ) 1105. [ 38 ] A.P. Bruins, Trends Anal. Chem. 13 ( 1994 ) 37. [ 39 ] A.P. Bruins, Trends Anal. Chem. 13 ( 1994 ) 81. [ 40 ] J.F. Garcia, D. Barcelo, J. High Resolut. Chromatogr. 16 ( 1993 ) 633. [ 41 ] P. Arpino, Mass Spectrom. Rev. 9 ( 1990 ) 631.
[ 42 ] P. Pietta, R.M. Facino, M. Carini, P. Mauri, J. Chromatogr. 661 ( 1994 ) 121. [ 43 ] Y.Y. Lin, K.J. Ng, S. Yang, J. Chromatogr. 629 ( 1993 ) 389. [ 44 ] J. van der Greef, W.M.A. Niessen, Int. J. Mass Spectrom. Ion Process. 118 / 119 ( 1992 ) 857. [ 45 ] A.P. Bruins, Mass Spectrom. Rev. 10 ( 1991 ) 53. [ 46 ] R.D. Smith, J.A. Loo, C.G. Edmonds, C.J. Barinaga, H.R. Udseth, Anal. Chem. 62 ( 1990 ) 882. [ 47 ] A.P. Snyder ( Editor ), Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry, American Chemical Society, Washington, DC, 1996. [ 48 ] R.B. Cole, Editor, Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation, and Applications, Wiley, New York, 1997. [ 49 ] M. Dole, L.L. Mack, R.L. Hines, R.C. Mobley, L.D. Ferguson, M.B. Alice, J. Chem. Phys. 49 ( 1968 ) 2240. [ 50 ] P. Kebarle, L. Tang, Anal. Chem. 65 ( 1993 ) 972A. [ 51 ] J.V. Iribarne, B.A. Thomson, J. Chem. Phys. 64 ( 1976 ) 2287. [ 52 ] J.B. Fenn, J. Am. Soc. Mass Spectrom. 4 ( 1993 ) 524. [ 53 ] W.E. Glassgen, H.U. Seitz, J.M. Metzger, Biol. Mass Spectrom. 21 ( 1992 ) 271. [ 54 ] A.P. Bruins, T.R. Covey, J.D. Henion, Anal. Chem. 59 ( 1987 ) 2642. Danielle Ryan is currently in the ¢rst year of her Ph.D. at Charles Sturt University Riverina. Her Ph.D. project concerns phenolic compounds in olive fruits. She obtained her undergraduate degree in analytical chemistry and her Honours project concerned the role of phenolic compounds in olive fruits as a function of physiological development. Kevin Robards is Associate Professor of Chemistry at Charles Sturt University Riverina. He obtained his Ph.D. in analytical chemistry from the University of New South Wales in 1979. His research interests are focused on the application of analytical chemistry to food science and in particular the identi¢cation and role of naturally occurring phenolic compounds in fruits. Paul Prenzler is a lecturer in Chemistry at Charles Sturt University Riverina. He received his Ph.D. in 1992 from the University of Queensland and has held postdoctoral appointments with Hitachi Ltd in Japan and the Research School of Chemistry, Australian National University. Prior to joining CSU he was a Research Fellow with Prof. Tony Wedd at the University of Melbourne. His current research interests include applications of electrospray mass spectrometry to metal complexes, particularly those with phenolic compounds. Michael Antolovich is a lecturer at Charles Sturt University Riverina. He completed his Ph.D. in 1989 at the University of New South Wales, followed by a postdoctoral position at Princeton University working on metalloporphyrins. He returned to Australia on a Research Fellowship. His current interests are in computational chemistry and its application to food and environmental sciences.
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Applications of mass spectrometry to plant phenols Danielle Ryan, Kevin Robards*, Paul Prenzler, Michael Antolovich
School of Science and Technology, P.O. Box 588, Wagga Wagga 2678, Australia Plant phenols embrace a considerable range of compounds and are de¢ned as those substances derived from the shikimate acid pathway and phenylpropanoid metabolism. The present article examines the application of mass spectrometry to the analysis of these compounds and traces the chronological development of analyte ionisation methods. z1999 Elsevier Science B.V. All rights reserved. Keywords: Soft ionization; Phenols, Plants
1. Introduction Terminology relating to the plant phenols is often confusing and a brief overview is presented before considering aspects of their mass spectrometry. The plant phenols (Table 1 ) are a diverse group of secondary metabolites possessing an aromatic ring bearing one or more hydroxy substituents. However, this de¢nition is not entirely satisfactory since it inevitably includes compounds such as oestrone, the female sex hormone which is principally terpenoid in origin. For this reason, a de¢nition based on metabolic origin is preferable, the plant phenols being regarded as those substances derived from the shikimate pathway and phenylpropanoid metabolism ( Fig. 1 ). Some members are characterised as `polyphenols', an unfortunate term since not all are polyhydroxy derivatives. In particular, a number of compounds, for example, cinnamic acid, elenolic acid, shikimic acid and quinic acid, are treated in the present discussion as phenolics because of metabolic considerations although they lack a phenolic group or even an aromatic ring. Plant phenols (Table 2 ) have been classi¢ed into major groupings distinguished by the number of constitutive carbon atoms in conjunction with the structure of the basic phenolic skeleton. The most widespread and *Corresponding author. E-mail: [email protected]
diverse of the phenolics are the £avonoids which are built upon a C6 -C3 -C6 £avone skeleton in which the three-carbon bridge between the phenyl groups is commonly cyclised with oxygen. Several classes of £avonoid ( Fig. 2 ) are differentiated on the degree of unsaturation and degree of oxidation of the three-carbon segment. Within the various classes, further differentiation is possible based on the number and nature of substituent groups attached to the rings. The range of known phenolics is thus vast and also includes polymeric lignins and condensed tannins although these species are not considered in the present review. Additional structural complexity is introduced by the common occurrence of certain phenolics as the O-glycosides ( or, less commonly, as C-glycosides ) in which one or more of the phenolic hydroxyl groups is bound to a sugar or sugars by an acid-labile hemiacetal bond. Glucose is the most commonly encountered sugar with rhamnose and the disaccharide, rutinose ( 6-O-K-L-rhamnosyl-D-glucose ), is also encountered. Anthocyanins are intensely coloured plant pigments in which an anthocyanidin aglycone is glycosidically linked to a sugar( s ). Acylation of the glycosides in which one or more of the sugar hydroxyls is derivatised with an acid such as acetic or ferulic acid is occasionally observed. Although the number of identi¢ed phenols is increasing exponentially, the phenolic content of most plants constitutes a complex mixture, the complete chemical nature of which has not, as yet, been elucidated for any species. For example, there are many phenolics present in low concentrations which remain unidenti¢ed but whose signi¢cance may far outweigh their concentration level. Isolation and structure elucidation of these compounds are the initial steps to understanding their signi¢cance and action. Information on their biosynthesis is essential to understanding the interaction between plants and the environment. It will also facilitate genetic manipulation ( both qualitative and quantitative ) of the phenolic content of plants for dietary modulation of disease and to provide an environmentally sustainable feedstock for the petrochemical industry.
0165-9936/99/$ ^ see front matter PII: S 0 1 6 5 - 9 9 3 6 ( 9 8 ) 0 0 1 1 8 - 6
ß 1999 Elsevier Science B.V. All rights reserved.
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Table 1 De¢nition of terms used Plant phenols
Flavonoids
Glycoside
Aglycone API APCI Electrospray ionisation
Aromatic substances bearing one or more hydroxy groups on an aromatic ring and derived from the shikimate pathway or phenylpropanoid metabolism A diverse class of plant phenols based on a C6 ^C3 ^C6 £avone skeleton in which the three-carbon bridge between the phenyl groups is commonly cyclised with oxygen In the current context, a molecule formed by linking a phenol and a sugar( s ) by an acidlabile hemiacetal bond between the phenolic hydroxyl group( s ) and the sugar( s ) The phenolic portion of a glycoside Atmospheric pressure ionisation ( generic term ) Atmospheric pressure ionisation by chemical ionisation Ion formation from samples in solution by dispersion of liquids into an electrically charged aerosol due to the action of an electric ¢eld
Methods of characterisation and identi¢cation of plant phenols follow those in general use for natural substances. Hence, preparation of an extract, biological screening, bioguided fractionation, isolation and structure elucidation is the usual approach. For the latter, physical methods based on spectral characteristics feature prominently although older chemical and biochemical approaches should be considered particularly as adjuncts to spectral analysis. Of these techniques, none has been more useful in providing structural information than mass spectrometry (MS ). However, its use has not been restricted to this role for mass spectrometry is increasingly viewed as a mass-speci¢c detector [ 1 ] for both qualitative and
quantitative applications in high resolution chromatography. Tandem mass spectrometry (MS^MS ) is particularly attractive for trace analysis where high selectivity is essential [ 2 ]. In another role, MS of phenolic compounds meets the multivariate criteria for ¢ngerprinting food samples in adulteration testing [ 3 ]. Representative applications of MS in the analysis of plant phenols are presented in Table 3.
2. Classical ionisation techniques The traditional mode of MS involves electron impact ionisation ( EI ) in which the neutral sample molecule is impacted in the gas phase with an electron beam of energy 70^100 eV. EI mass spectrometry ( EI^MS ) is used extensively in structural studies of a number of plant phenols including various £avonoids. EI produces a positive radical ion Mc which if detected provides information on the molecular mass of the analyte. Comprehensive information about the mass spectra of £avonoids has been published [ 26,27 ]. In general, the EI mass spectra of £avonoid aglycones are characterised [ 24,25 ] by intense molecular ion peaks plus signi¢cant fragments from both A and B rings. The fragmentations often provide suf¢cient information to determine molecular mass, elemental formula, substitution patterns in the A and B rings, and the class of £avonoid. For example, the EI mass spectrum of substituted £avones shows the typical fragmentation pattern to yield two fragments characteristic of the A and B rings. In the case of a disubstituted £avone there are three possibilities, either both substituents are located on the A or B ring or one is located on each ring and the mass spectrum will provide unambiguous information on the substitution pattern. In less favourable situations
Table 2 Classi¢cation of plant phenols Class
Base structure
Examples
Comments
Simple phenols Phenolic acids
C6 C6 C1
Hydroxycinnamic acids Lignans Coumarins
C6 C3
catechol, resorcinol p-hydroxybenzoic acid, salicylic acid, gallic acid caffeic acid, ferulic acid
among the most common of all phenolic compounds
( C6 C3 ) 2 C6 C3
nordihydroguaiaretic acid umbelliferone
Chromones Flavonoids
C6 C3 C6 C3 C 6
eugenin quercitin, cyanidin
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Table 3 Mass spectrometric techniques used in the analysis of phenolic compounds in plants and related samples Sample
Phenolic compounds
Technique
Ref.
Wines Food Olive oil Krameria triandra roots
phenolic acids, £avonoids review
GC^MS LC^MS GC^MS LC^MS (PB, EI ); FAB^MS ( negative ion mode ) and FAB^MS^MS
[3] [4] [5] [6]
ESI-LC^MS GC^MS GC^MS (TMS derivatives ) PB-LC^MS ( EI ) LC^APCI-MS ( negative ion )
FAB^MS ( negative ion ) LC^MS Thermospray LC^MS Thermospray LC^MS
[7] [8] [9] [ 10 ] [ 11 ] [ 12 ] [ 13 ] [ 14 ] [ 15 ] [ 16 ] [ 17 ] [ 18 ]
API ion spray LC^MS
[ 19 ]
Plasmaspray^LC^MS Plasma desorption MS APCI-LC^MS ( positive ion ) EI-GC^MS EI and CI-MS
[ 20 ] [ 21 ] [ 22 ] [ 23 ] [ 24 ] [ 25 ]
low to intermediate molecular weight polyphenol constituents with radical scavenging activity Olive leaf oleuropein, ligstroside Oat groats and hulls phenolic acids Spruce shoots and galls 47 phenolics and phenolic glucosides Not applied 15 benzoic and cinnamic acid derivatives Olive mill wastewater 15 phenolic compounds White and silver birch gallic and chlorogenic acids iso£avones Onions £avonoids Juice of Sedum telephium leaves 6 £avonol glycosides Plant extracts £avonoid precursors ( cis trans isomers ) Epilobium species 19 £avonol glycosides Lemon peel coumarins, phenyl propanoid glycosides, £avone-C-glucosides, £avonols, £avone-Oglycosides and £avonones Vitis vinifera L. anthocyanins: 3-glucosides, the 3-acetylglucosides, and the 3-p-coumaroylglucosides of delphinidin, cyanidin, petunidin, peonidin, and malvidin; also 3,5-diglucosides Passionfruit swertisin Black tea £avanols, £avonol glycosides, chlorogenic acid Sorghum bicolor anthocyanins and anthocyanidins Soy foods iso£avone glycosides (Orange oil ) 39 polymethoxylated £avones Not applied 43 £avones and £avonols, 7 iso£avones, 18 £avanones and dihydro£avonols, and 11 chalcones and dihydrochalcones
where there are many substituents, the mass spectrum will assist in structural elucidation although other techniques such as nuclear magnetic resonance will be required for a de¢nitive assignment of the substitution. EI more commonly produces an internally excited parent ion, Mc , which fragments so extensively that the parent radical cation is not observed. This is the situation with glycosidically linked phenols where the EI ( and chemical ionisation, CI ) mass spectra [ 28 ] are dominated by the same ions as for the corresponding aglycones [ 2,28 ], the protonated aglycone invariably being the base peak in the mass spectrum. A distinction with the mass spectra of the aglycones is the relatively weak fragments from ¢ssion of the A and B rings. However, EI is generally unsuitable for glycosidically bound phenols because these compounds are polar and thermolabile. Similarly, CI [ 29 ] has limited application for the broader range of plant phenols
CE^ESI-MS ( negative ion )
because vaporisation of the analyte prior to ionisation is once again a prerequisite. Chemical derivatisation overcomes the limitations of restricted volatility and thermal stability of polar phenols but presents further dif¢culties by increasing the molecular mass of the analyte possibly beyond the range of the mass analyser. This is a major consideration for glycosidic species with numerous hydroxyl groups although permethylation or perdeuteromethylation may be suitable. However, these methods often produce mixtures of partially derivatised compounds and, in some cases, permethylation can produce artefacts, e.g., when ester groups are present. Usually only weak molecular ion signals are observed when permethylated compounds are studied under EI conditions. The application of chemical derivatisation is extended in the discussion of coupled techniques. The development of desorption ionisation techniques in which ionisation of thermolabile molecules
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occurs directly from the condensed phase provides a solution to the problem of limited analyte volatility and stability. Field desorption ( FD ) was the ¢rst technique employed for direct analysis of polar and thermolabile phenolic species. FD has been applied to the analysis of £avonoid glycosides [ 30 ] and provides molecular mass data but usually little structural information and is ``notorious for the transient nature of the spectra'' [ 31 ]. Desorption chemical ionisation ( DCI ) uses a probe consisting of an electrically heated tungsten wire introduced into the CI source. DCI provides rapid heating of the analyte and overcomes the problem of thermal decomposition inherent in conventional CI but it was the development of particle induced desorption or sputtering techniques that truly extended the range of analyte polarities and sizes. Fast atom bombardment ( FAB ) [ 32 ] is the most successful and in this technique the analyte is solubilised in a non-volatile polar matrix ( e.g., glycerol, thioglycerol ) and deposited on a copper target which is bombarded with fast neutral energised particles such as xenon or argon thereby inducing the desorption and ionisation. The value of FAB-MS is demonstrated by the molecular mass and structural information provided for large polar glycosides in both 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. Furthermore, background matrix signals ( from glycerol or thioglycerol ) can complicate interpretation of the spectra. Nevertheless, when combined with collisionally induced dissociation of positive ions and tandem mass spectrometric techniques, FAB-MS can provide information on the aglycone moiety, the carbohydrate sequence and the glycosylation position of glycosides [ 34 ].
3. Coupled techniques The on-line coupling of methods is of enormous potential because the selectivity can then be tuned in an optimal way, which in turn can be translated to either a faster analysis or an improved determination limit. Coupled gas chromatography-mass spectrometry ( GC^MS ) is now well established as a routine technique carried out with either EI or CI sources, since these are appropriate for the introduction of volatile compounds. Thus, Berahia et al. [ 24 ] analysed 39 polymethoxylated £avones by GC^MS. In addition to common behaviour of £avones under electron impact, such as a retro-Diels^Alder reaction which gives a characteristic fragment from the phenyl group 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 characteristic of various substitution patterns were also identi¢ed. Improvements in GC column technology have increased the range of £avonoids amenable to GC^MS as the underivatised compounds. For instance, Schmidt et al. [ 35 ] analysed 49 £avones, £avonols, £avanones and chalcones without derivatisation by GC^MS in EI mode. Compared with direct inlet mass spectra, the GC^MS data exhibited the same typical fragmentation patterns but with slight differences in intensities. However, because of limited volatility, analysis of phenolic compounds and their glycosides, in particular, by GC^MS has not generally found favour. Two approaches have been used, namely, hydrolysis of the glycosides to their corresponding aglycones and / or chemical derivatisation. Angerosa et al. [ 36 ] have shown GC^MS with CI to be an effective tool for phenol identi¢cation after extraction from olive oil with methanol and derivatisation with bis( trimethylsilyl )tri£uoracetamide. Peaks in the mass spectra at m / z 192 or at m / z 280 were useful for assigning the phenolic nature to minor components. The advantages of CI with ammonia for providing molecular masses of the aglycones were also demonstrated. One of the problems associated with derivatisation, namely, the formation of several derivatives from a single analyte, was highlighted by this work. Derivatisation has also been applied to the analysis of the phenolic components of wine which were extracted and separated [ 3 ] as trimethylsilyl derivatives on a DB-5HT capillary column using MS detection with one target and two qualifying ions for each compound in a total run time of 26 min. Excellent resolution of 15 phenolic compounds was achieved. Flavonoid glycosides in fruit juices have been characterised [ 37 ] by GC^MS as the corresponding aglycones following extraction, hydrolysis and derivatisation to TMS ethers. In the case of anthocyanins, derivatisation is an essential step for GC^MS [ 19 ]. The on-line coupling of LC and MS [ 4,38^40 ] is of enormous potential as demonstrated in Fig. 3. The fundamental limitations relate to the problem of interfacing a high pressure LC system with a mass spectrometer ion source located inside a high-vacuum envelope and of producing gas-phase ions, particularly intact molecular ion species, without the application of heat. First-generation LC^MS instruments such as the moving wire or belt interface [ 31 ] overcame incompatibility between high vacuum and the introduction of solvent by removing the liquid. The moving belt system enabled EI, CI and FAB ionisation
but here also the classical mass spectrometric gasphase ionisation techniques were of limited application to more polar compounds such as the plant phenols and these systems never achieved widespread acceptance. A recent report [ 10 ] however compared particle beam (PB ) EI-MS with ultraviolet and electrochemical detection of phenolic acids. In the second generation, for example, continuous £ow FAB ( or dynamic FAB ), soft ionisation techniques were coupled with liquid introduction. When introduced, continuous £ow FAB-MS rapidly superseded all other ionisation methods for £avonoids and, in particular, anthocyanin studies as it provided an ideal technique for the analysis of highly polar compounds, without the need for derivatisation. It had the advantage of producing a molecular ion plus various fragmentation ions which provided structural information. Nowadays, interfacing and ionisation have merged in third-generation instruments such as the thermospray and electrospray mass spectrometers.
4. Development of newer soft ionisation techniques Thermospray ionisation (TSI ) [ 41 ] was the ¢rst method to combine true LC^MS compatibility with the ability to determine non-volatile thermally labile compounds. In this approach, the chromatographic eluate passes through a resistively heated stainless steel capillary tube located in the thermospray probe. A supersonic jet of vapour is created by adjusting the temperature of the capillary to a level where the solvent is partially vaporised and expansion of the resulting vapour provides the gas dynamic forces needed for atomisation of the remaining liquid. The vapour jet contains an entrained `mist' of small, statistically generated electrically charged droplets that ¢nally as a result of mechanisms detailed later give rise to the analyte ions. There is no conventional external means of ionisation which is accomplished during the solvent vaporisation and desolvation of the small droplets. The analyte ions leave the thermospray source through an ori¢ce in a sampling cone. The process is greatly enhanced if the analyte is itself ionic or by the presence of a volatile electrolyte. Ammonium acetate is the best general-purpose electrolyte for ionising neutral analytes although other volatile salts, acids, bases or no electrolyte at all may be preferred in certain applications. Reverse-phase eluents containing a high percentage of water are pre-
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ferred for good sensitivity at conventional LC £ow rates, conditions well suited to the analysis of polar phenolic species [ 42 ]. However, TSI is not without its dif¢culties, in particular, the ef¢ciency of ion produc-
tion varies widely with compound type and the £ow rate and temperature of the inlet tube must be optimised for each different compound class. Moreover, each class of compound requires different conditions
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Fig. 3. Chromatograms comparing mass spectrometric detection with ultraviolet detection at 280 nm for the reverse-phase chromatography 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 spectra were collected on a Quattro II quadrupole mass spectrometer (MS ) (Micromass, Altrincham, Cheshire, UK ) by electrospray ionisation ( ESI ). An injection volume of 10 Wl and a constant £ow rate of 0.200 ml / min was used for each analysis with a split ratio of approximately 10:1 (UV detector:MS ). Chromatograms are shown for total ion current (TIC ) and extracted ion monitoring 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
for optimal ionisation and this is further complicated by gradient elution. TSI continues to ¢nd applications despite its limitations [ 42,43 ] but will probably be gradually phased out by atmospheric pressure chemical ionisation ( APCI ) and electrospray ( see Fig. 1 in [ 44 ]). In the mean time, TSI LC^MS^MS has provided [ 43 ] characterisation of catechins and £avonoids from their collision induced dissociation spectra of the quasi-molecular ion. Flavonoids exhibited three types of ring cleavage in the pyran ring and differentiation among £avanone, £avone and £avonol was possible. In TSI, the fundamental problem of coupling a liquid chromatograph and a mass spectrometer, namely, that of pressure reduction, is addressed by connecting an additional pumping line directly to the ion source. The majority of the vaporised solvent and mist goes to this auxiliary vacuum pump after it traverses a skimmer aperture to the mass spectrometer. A more elegant solution which obviates the problem entirely is to eliminate the vacuum altogether. In atmospheric pressure ionisation ( API ) [ 38,39,45 ], pumping at the ion source is eliminated and atmospheric pressure operation of the ion source facilitates easier installation, ¢tting and cleaning. In APCI, a combination of a heated capillary and a corona discharge is used to promote the formation of ions from the nebulised sample. Ionisation occurs in the gas phase by ion molecule reactions and follows the sequence Sample in solution!sample vapour!sample ions In coupled mode, the eluant from the HPLC is evaporated completely and the mixture of solvent and sample vapour is then ionised by chemical ionisation involving proton transfer, adduct formation and charge exchange reactions in positive ion mode or proton abstraction, anion attachment and electron capture reactions in the negative mode. APCI is compatible with 100% aqueous or 100% organic mobile phases at £ow rates of up to 2 ml / min [ 23 ] and is
therefore ideal for normal or reverse-phase operation with conventional HPLC columns. In this fashion it has been used to determine various iso£avones. Negative ion mode provided quality mass spectra which gave not only the molecular mass of the iso£avones, but also their molecular structures. Deuterium oxide was used to induce peak shifts in the mass spectra to determine the number of exchangeable hydrogen atoms in each molecule. Aramend|èa et al. [ 11 ] reported the LC^APCI^MS of phenolics in olive mill wastewater. Analytes were separated on a C18 phase by gradient elution with methanol^water containing formic acid. Mass spectral conditions were optimised by direct infusion of standards in £ow injection mode into the APCI source. The study was restricted to negative ion mode with detection limits in total ion current mode ranging from 0.5 ng to 500 ng. These detection limits were about 20 times better when working in selected ion monitoring mode and monitoring the [ M-H ]3 ion. Mass spectra were recorded with soft ( 315 V ) and strong ( 350 V ) voltages applied at the ion source of the mass spectrometer. With the smaller voltages, deprotonated molecular species [ M-H ]3 were the major ions observed in the mass spectra with the appearance of very few fragment ions which were all of low intensity. The presence of substantial fragmentation from collisionally induced dissociation processes which became evident on increasing the voltage applied at the source ( extraction and cone ) voltages, gave structural information about the molecules. Structures were assigned to major eluent cluster ions from methanol^water^formic acid mixtures occurring at m / z 91, 113, 137, 159, 181 and 183. APCI still has a major drawback for polar thermolabile plant phenols: volatilisation of sample must occur before ionisation. The newer soft ionisation methods overcome lack of analyte volatility by direct formation or emission of ions from the surface of a condensed phase and sample ions are collected from the condensed phase inside the ion source and transferred to the mass analyser. Hence, they eliminate the
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Table 4 Application areas of various mass spectrometric methods with respect to analyte polarity and molecular mass
Sample polarity Molecular mass range HPLC compatability Ease of use Sensitivity Structural information
Thermospray
Particle beam
Electrospray
APCI
medium^high 100^2000 ( polarity )
low^medium 100^2000 ( volatility ) good medium low high ( EI and CI )
medium^high 100^ca 100 000 ( multiple charges )
low^medium 100^1500 ( thermal lability ) good medium high medium ( with CID )
medium low medium medium ( with fragmentor )
need for neutral molecule volatilisation prior to ionisation and generally minimise thermal degradation of the molecular species. The process is termed electrospray [ 46 ] and it is most suited to compounds that either exist as ions in solution, can be ionised at an appropriate pH or polar neutral molecules that can associate with small ions such as Na , ammonium or chloride. It is therefore ideally suited to the plant phenols. Electrospray is used as a generic term that also covers several variants of the basic technique that differ in the precise manner in which charged droplets of sample are produced. These techniques collectively have revolutionised the ¢eld of mass spectrometry [ 47 ]. Electrospray ion production requires two steps: dispersal of highly charged droplets at near atmospheric pressure, followed by conditions resulting in droplet evaporation. An electrospray is generally produced by injection of a solution of the analyte through a metal capillary maintained at a potential of several kV relative to the surrounding chamber walls. There are two widely debated mechanisms for the formation of gasphase ions from solute species in charged droplets. In the charged residue model [ 47^50 ], a strong electric ¢eld generated by the potential difference causes the eluate to be expelled from the capillary as a plume of charged droplets. As solvent evaporates from the small droplets, a critical size ^ the Rayleigh limit ^ is reached where Coulomb repulsion between the charged entities in the droplet ( mobile phase, electrolyte and sample ions ) becomes greater than the surface tension forces. At this point the droplet breaks into several smaller droplets and the process is repeated until the droplets become so small that they contain only one analyte molecule. This molecule retains some of the droplet charge when the last solvent molecules evaporate. The ionised species enter the mass analyser through a skimmer cone. The same sequence of evaporation and Coulomb ¢ssion steps is envisaged
good medium high medium ( with CID )
in the ion desorption ( evaporation ) model [ 47,51,52 ]. However, at some intermediate stage before the droplets are so small that they contain only one analyte molecule, the surface ¢eld is suf¢ciently intense to cause expulsion of a charged analyte ion from the droplet surface. Thus, the fundamental difference between theories is in how the analyte ion becomes separated from other species in the droplet. The process of ESI appears reminiscent of what occurs in TSI. The difference is that in ESI most or all of the nebulisation work is done by electrostatic forces while that work is performed by gas dynamic forces in TSI. Electrospray ideally operates with £ow rates at less than 10 Wl / min using microbore columns or a conventional column equipped with an ef£uent splitter. However, conventional columns and £ow rates are compatible with ESI without the need for splitting by use of pneumatic nebulisers or thermal input. The latter enhance the spray process and increase the rate of solvent evaporation from the highly charged droplets and hence counter to some degree the decreasing ionisation ef¢ciency at higher £ows. ESI spectra of glycosidic compounds typically show a pseudomolecular ion ( e.g., [ M+H ] ), aglycone ion and ions associated with the solvent although fragmentation can often be induced by raising the cone voltage. Acid ( acetic or formic ) is often added to mobile phases in positive ion electrospray as a source of protons to assist ionisation. Sensitivity is improved when the organic content in the mobile phase exceeds 20%. The type of information required in an analysis ( e.g., structural elucidation ^ molecular mass versus fragmentation data; quantitative determination ) and the nature of the analyte, and more speci¢cally its polarity, are the prime factors in the choice of the LC^MS interface (Table 4 ). Nonetheless, electrospray is undoubtedly the fastest developing approach [ 44 ]. The power of electrospray as an alternative, highly
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sensitive soft ionisation technique for investigation of polar, non-volatile and thermolabile molecules such as anthocyanins has been demonstrated [ 53 ]. Ion spray ionisation modi¢cation [ 54 ] incorporated pneumatic nebulisation with true electrospray to improve the ef¢ciency of the spray generation process. This change enabled operation with higher £ow rates of 2^200 Wl / min [ 54 ] when used with conventional ( e.g., 2U100 mm ) reverse-phase columns as illustrated by the structure determination of anthocyanins [ 53 ] and the identi¢cation [ 7 ] of the phenolic glucoside content of olive leaves. In one application, an anthocyanic extract of blueberry was applied [ 19 ] to a solid-phase cartridge and lipophilic substances ( e.g., chlorophyll and carotenoids ) were removed with hexane following which £avonoids and phenolic acids were eluted with ethyl acetate. Hydrophilic species including anthocyanins, sugars and organic acids were then desorbed with methanol and after further clean-up the anthocyanin fraction was analysed in the £avylium cationic form by ion spray MS. Strong ( quasi-)molecular glycoside ions were observed and the API ion spray interface enabled the use of the same conditions as in conventional HPLC with ultraviolet detection. On rare occasions, MS can provide data suf¢cient for full structure analysis but more generally it is used to determine molecular mass and to establish the distribution of substituents on the phenolic ring( s ). This situation will improve with new developments. Tandem mass spectrometry (MS^MS ) has been applied successfully to problems involving trace analysis of citrus £avanones and metabolite identi¢cation [ 2 ]. Positive CI MS^MS was superior to EI MS^MS for the detection of a common daughter ion for £avanones at m / z 153. Using this approach, the £avanones naringenin and hesperitin were detected in human urine after citrus ingestion. Glycosides were labile under the experimental conditions, probably during ionisation. MS^MS particularly in combination with LC and soft ionisation techniques [ 43 ] can be expected to improve signi¢cantly separations of complex samples. The information available from such methods can be expected to increase with the development of newer technologies including collisionally induced dissociation spectra [ 43 ]. For the latter, alternating low and high ori¢ce voltages are used in which no fragmentation occurs at low voltage and fragmentation is induced at the high ori¢ce voltage. This permits simultaneous measurement of molecular mass and structural characterisation.
5. Future directions An increasing application of multistage ion analysers coupled with various MS ionisation methods will facilitate the identi¢cation of an increasing range of plant phenolics. In routine applications, mass-selective detection will provide rapid trace analysis of phenolics and the selectivity of MS^MS and collisioninduced dissociation spectra will be used to advantage to facilitate dif¢cult separations. Developments still in their infancy, at least in applications to phenolic analysis, include the coupling of capillary electrophoresis with MS ( CE^MS ). In combination with the inherent sensitivity and selectivity of mass spectrometry, CE^MS [ 38,39 ] becomes a very powerful technique. The correspondence between CE and electrospray ionisation £ow rates provides the basis for an extremely attractive technique. Hence, various iso£avones were separated [ 13 ] on an uncoated fused-silica column using 25 mM ammonium acetate buffer and negative ion electrospray ionisation mass spectrometric detection. This approach permitted the determination of molecular mass of the iso£avones as well as the presence of various functional groups according to observed losses from the [ M-H ]3 ion during collision-induced dissociation effected by adjusting MS parameters.
References [ 1 ] F. Angerosa, N. d'Alessandro, P. Konstantinou, L. Di Giacinto, 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. [ 3 ] G.J. Soleas, J. Dam, M. Carey, D.M. Goldberg, J. Agric. Food Chem. 45 ( 1997 ) 3871. [ 4 ] M. Careri, A. Mangia, M. Musci, J. Chromatogr. 794 ( 1998 ) 263. [ 5 ] L. Cinquanta, M. Esti, E. Lanotte, J. Am. Oil Chem. Soc. 74 ( 1997 ) 1259. [ 6 ] R.M. Facino, M. Carini, G. Aldini, L. Deangelis, Rapid Commun. Mass Spectrom. 11 ( 1997 ) 1303. [ 7 ] A. De Nino, N. Lombardo, E. Perri, A. Procopio, A. Raffaelli, G. Sindona, J. Mass Spectrom. 32 ( 1997 ) 533. [ 8 ] Y.M. Xing, P.J. White, J. Am. Oil Chem. Soc. 74 ( 1997 ) 303. [ 9 ] C. Kraus, G. Spiteller, Phytochemistry 44 ( 1997 ) 59. [ 10 ] C. Bocchi, M. Careri, F. Groppi, A. Mangia, P. Manini, G. Mori, J. Chromatogr. 753 ( 1996 ) 157. [ 11 ] M.A. Aramend|èa, V. Boraèu, I. Garc|èa, C. Jimeènez, F. Lafont, J.M. Marinas, F.J. Urbano, Rapid Commun. Mass Spectrom. 10 ( 1996 ) 1585. [ 12 ] V. Ossipov, K. Nurmi, J. Loponen, E. Haukioja, K. Pihlaja, J. Chromatogr. 721 ( 1996 ) 59.
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[ 13 ] M.A. Aramend|èa, I. Garc|èa, F. Lafont, J.M. Marinas, J. Chromatogr. A 707 ( 1995 ) 327. [ 14 ] Y.K. Park, C.Y. Lee, Colloq. Inst. Natl. Rech. Agron. 69 (Polyphenols 94 ) ( 1995 ) 265. [ 15 ] N. Mulinacci, F.F. Vincieri, A. Baldi, A. Romani, D. Favretto, P. Traldi, Rapid Commun. Mass Spectrom. 9 ( 1995 ) 963. [ 16 ] P.A. Tarantilis, G. Tsoupras, M. Polissiou, J. Chromatogr. 699 ( 1995 ) 107. [ 17 ] B. Ducrey, J.L. Wolfender, A. Marston, K. Hostettmann, Phytochemistry 38 ( 1995 ) 129. [ 18 ] A. Baldi, R.T. Rosen, E.K. Fukuda, C.T. Ho, J. Chromatogr. 718 ( 1995 ) 89. [ 19 ] A. Baldi, A. Romani, N. Mulinacci, F.F. Vincieri, B. Casetta, J. Agric. Food Chem. 43 ( 1995 ) 2104. [ 20 ] A. Rehwald, B. Meier, O. Sticher, Pharm. Acta Helv. 69 ( 1994 ) 1531. [ 21 ] R.G. Bailey, H.E. Nursten, I. Mcdowell, J. Sci. Food Agric. 66 ( 1994 ) 203. [ 22 ] K.V. Wood, C. Bonham, J. Hipskind, R.L. Nicholson, Phytochemistry 37 ( 1994 ) 557. [ 23 ] S. Barnes, M. Kirk, L. Coward, J. Agric. Food Chem. 42 ( 1994 ) 2466. [ 24 ] T. Berahia, E.M. Gaydou, C. Cerrati, J.C. Wallet, J. Agric. Food Chem. 42 ( 1994 ) 1697. [ 25 ] P.A. Hedin, V.A. Phillips, J. Agric. Food Chem. 40 ( 1992 ) 607. [ 26 ] K.R. Markham, in J.B. Harborne ( Editor ), The Flavonoids: Advances in Research, Chapman and Hall, London, 1988, p. 427. [ 27 ] R.J. Grayer, in J.B. Harborne ( Editor ), Plant Biochemistry, Vol. 1, Plant Phenolics, Academic Press, San Diego, 1989, pp. 223, 283, 347. [ 28 ] H. Itokawa, Y. Oshida, A. Ikuta, Y. Shida, Chem. Lett. 1 ( 1982 ) 49. [ 29 ] A.G. Harrison, Chemical Ionization Mass Spectrometry, 2nd edn., CRC Press, Boca Raton, FL, 1992. [ 30 ] T. Nakanishi, A. Inada, K. Kambayashi, K. Yoneda, Phytochemistry 24 ( 1985 ) 339. [ 31 ] J. Wolfender, M. Mailard, A. Marston, K. Hostettmann, Phytochem. Anal. 3 ( 1992 ) 193. [ 32 ] B. Domon, K. Hostettmann, Phytochemistry 24 ( 1985 ) 575. [ 33 ] R. Gujer, D. Magnolato, R. Self, Phytochemistry 25 ( 1986 ) 1431. [ 34 ] M. Claeys, Q.M. Li, H. Van den Heuvel and L. Dillen, in R.P. Newton and T.J. Watton ( Editors ), Applications of Modern Mass Spectrometry in Plant Science Research, Clarendon Press, Oxford, 1996. [ 35 ] T.J. Schmidt, I. Merfort, U. Matthiesen, J. Chromatogr. 634 ( 1993 ) 350. [ 36 ] F. Angerosa, N. d'Alessandro, F. Corana, G. Mellerio, J. Chromatogr. 736 ( 1996 ) 195. [ 37 ] C.S. Creaser, M.R. Koupai-Abyazani, G.R. Stephenson, Analyst 117 ( 1992 ) 1105. [ 38 ] A.P. Bruins, Trends Anal. Chem. 13 ( 1994 ) 37. [ 39 ] A.P. Bruins, Trends Anal. Chem. 13 ( 1994 ) 81. [ 40 ] J.F. Garcia, D. Barcelo, J. High Resolut. Chromatogr. 16 ( 1993 ) 633. [ 41 ] P. Arpino, Mass Spectrom. Rev. 9 ( 1990 ) 631.
[ 42 ] P. Pietta, R.M. Facino, M. Carini, P. Mauri, J. Chromatogr. 661 ( 1994 ) 121. [ 43 ] Y.Y. Lin, K.J. Ng, S. Yang, J. Chromatogr. 629 ( 1993 ) 389. [ 44 ] J. van der Greef, W.M.A. Niessen, Int. J. Mass Spectrom. Ion Process. 118 / 119 ( 1992 ) 857. [ 45 ] A.P. Bruins, Mass Spectrom. Rev. 10 ( 1991 ) 53. [ 46 ] R.D. Smith, J.A. Loo, C.G. Edmonds, C.J. Barinaga, H.R. Udseth, Anal. Chem. 62 ( 1990 ) 882. [ 47 ] A.P. Snyder ( Editor ), Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry, American Chemical Society, Washington, DC, 1996. [ 48 ] R.B. Cole, Editor, Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation, and Applications, Wiley, New York, 1997. [ 49 ] M. Dole, L.L. Mack, R.L. Hines, R.C. Mobley, L.D. Ferguson, M.B. Alice, J. Chem. Phys. 49 ( 1968 ) 2240. [ 50 ] P. Kebarle, L. Tang, Anal. Chem. 65 ( 1993 ) 972A. [ 51 ] J.V. Iribarne, B.A. Thomson, J. Chem. Phys. 64 ( 1976 ) 2287. [ 52 ] J.B. Fenn, J. Am. Soc. Mass Spectrom. 4 ( 1993 ) 524. [ 53 ] W.E. Glassgen, H.U. Seitz, J.M. Metzger, Biol. Mass Spectrom. 21 ( 1992 ) 271. [ 54 ] A.P. Bruins, T.R. Covey, J.D. Henion, Anal. Chem. 59 ( 1987 ) 2642. Danielle Ryan is currently in the ¢rst year of her Ph.D. at Charles Sturt University Riverina. Her Ph.D. project concerns phenolic compounds in olive fruits. She obtained her undergraduate degree in analytical chemistry and her Honours project concerned the role of phenolic compounds in olive fruits as a function of physiological development. Kevin Robards is Associate Professor of Chemistry at Charles Sturt University Riverina. He obtained his Ph.D. in analytical chemistry from the University of New South Wales in 1979. His research interests are focused on the application of analytical chemistry to food science and in particular the identi¢cation and role of naturally occurring phenolic compounds in fruits. Paul Prenzler is a lecturer in Chemistry at Charles Sturt University Riverina. He received his Ph.D. in 1992 from the University of Queensland and has held postdoctoral appointments with Hitachi Ltd in Japan and the Research School of Chemistry, Australian National University. Prior to joining CSU he was a Research Fellow with Prof. Tony Wedd at the University of Melbourne. His current research interests include applications of electrospray mass spectrometry to metal complexes, particularly those with phenolic compounds. Michael Antolovich is a lecturer at Charles Sturt University Riverina. He completed his Ph.D. in 1989 at the University of New South Wales, followed by a postdoctoral position at Princeton University working on metalloporphyrins. He returned to Australia on a Research Fellowship. His current interests are in computational chemistry and its application to food and environmental sciences.
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