中草药有机质谱分析英文论文讲解

Characterisation of Soy Isoflavones and Screening for Novel Malonyl Glycosides using High-performance Liquid Chromatography–Electrospray Ionisation–Mass SpectrometryLiwei Gu and Wenying Gu*School of Food Science and Technology,Wuxi University of Light Industry Huihe Road170,Wuxi,Jiangsu,214036,People’s Republic of ChinaHPLC combined with electrospray ionisation(ESI)-MS and photodiode array detection has been employed to study the isoflavone components of soy.All of the known soy isoflavones separated by HPLC were identified and characterised,and three novel isoflavones were detected and screened out.These minor isoflavones were deduced to be isomers of6@-O-malonyl isoflavone glycosides,based on the ESI-MS and UV data,in which the malonyl group is attached at a position other than the6@position of the glycosyl moiety of the molecule.These novel malonyl glycosides are as thermally labile as the6@-O-malonyl glycosides,being converted into known isoflavone glycosides after heating in aqueous ethanol.The advantages of HPLC-ESI-MS in detection of novel isoflavones from plant extracts are reviewed.Copyright#2001John Wiley& Sons,Ltd.Keywords:High performance liquid chromatography–electrospray ionisation–mass spectrometry;isoflavones;soy;Glycine max L.INTRODUCTIONSoy isoflavones are currently significant targets in food and nutritional research owing to their numerous health-promoting properties such as the lowering of cholesterol levels,the prevention of cancers,and the alleviation of osteoporosis and menopause syndrome(Adlercreutz and Mazur,1997).It is generally recognised that there are in total12types of isoflavones in soy,these being divided into three groups based on their aglycone moieties, namely daidzein,genistein and glycitein(Fig.1).Within each group there are four types of isoflavone,the isoflavone aglycone,the glycoside,the6@-O-acetyl glycoside and the6@-O-malonyl glycoside.Approxi-mately68%of the isoflavones in soy are in the form of 6@-O-malonyl glycosides which are highly thermally labile and decompose into glycosides when heated above 60°C in aqueous solvents(Kudou et al.,1991).In natural soy,6@-O-acetyl glycosides are absent,being derived in processed soy from6@-O-malonyl glycosides after dry heating.Less than5%of the isoflavones in natural soy are aglycones,but they might be the main forms present following fermentation(Barnes et al.,1994).Some novel polyhydroxy-isoflavones have been identified in fermen-ted soy foods,e.g.in tempe,miso and natto,but these structures are not found in the natural soy and are formed from the known isoflavones by the action of micro-organisms(Esaki et al.,1999).Recently,Hosny and Rosazza(1999)isolated three novel isoflavones from soy molasses,these being glycitein7-O-b-D-(2@,4@,6@-O-triacetyl)glucopyranoside, 8-(g-hydroxy-g,g-dimethylpropyl)genistein7-O-b-D-glu-copyranoside,and5-hydroxy-8-(g-hydroxy-g,g-di-methylpropyl)-3',4'-dimethoxyisoflavone-7-O-b-D-gluco-pyranoside.Since molasses is the syrup left after condensation of aqueous ethanol extracts of defatted soyflakes,these isoflavones might be contaminants introduced,or artefacts formed,during processing and storage,and their presence in native soybean needs to be confirmed.The coupling of MS and photodiode array detection (PAD)with liquid chromatography is a very efficient technique for the on-line identification of polarflavonoid aglycones and glycosides in plants.SeveralreportsFigure1.Structures of genistein,glycitein and the structural conversion of6@-O-malonyldaidzin.PHYTOCHEMICAL ANALYSISPhytochem.Anal.12,377–382(2001)DOI:10.1002/pca.603*Correspondence to:W.Gu,School of Food Science and Technology,Wuxi University of Light Industry,Huihe Road170,Wuxi,Jiangsu,214036, People’s Republic of China.Email:wygu@concerning HPLC-ESI-MS analysis of isoflavones have been published.Very low concentrations of genistein, daidzein and their conjugates in rat blood were quantified and identified simultaneously using HPLC-ESI-MS by Holder et al.(1999).Barnes et al.(1994,1998)compared the application of atmospheric pressure chemical ionisa-tion(APCI)and ESI for the identification of isoflavones in soy following HPLC separation;this work,however, focused mainly on identification and characterisation of known compounds in the plant(Stobiecki et al.,1999). The capacity of HPLC-ESI-MS to screen for novel compounds seems not to have been exploited fully, although Lin et al.(2000)recently detected six novel6@-O-malonyl isoflavone glycosides and their isomers in red clover using this technology.In the present study we tried to confirm the existence of the novel isoflavones isolated from soy molasses,as mentioned above,in natural soy,and to screen for other novel isoflavones.All isoflavones screened were then quantitatively determined by HPLC-ESI-MS.EXPERIMENTALStandards.Daidzin and glycitin were purified from soy germ in our laboratory,and their structures were confirmed by UV,IR,MS and NMR data.Genistin, genistein and daidzein were obtained from Sigma(St. Louis,MO,USA).All the standard isoflavones were dissolved in methanol,and their purities were confirmed to be better than97%by HPLC analysis[molar extinction coefficients:daidzein and daidzin"=104.45( max= 254nm),glycitin"=104.42( max=260nm),genistein and genistin"=104.51( max=260nm)].Thefinal concentration of the individual standard employed was adjusted based on the measured purity of that standard. For isoflavones for which no standard was available,the calibration curves were adjusted on the basis of molecu-lar weight difference between them and their correspond-ing isoflavone glycosides(Song et al.,1999).In order to study the effect of solvent matrix on electrospray ionisation of a isoflavone glycoside,a small amount of daidzin was dissolved in pure methanol,and an aliquot(2mL)of this methanol solution was diluted by adding equal volumes of pure methanol,water or water containing0.4%(v/v)modifier such as formic acid, acetic acid or trifluoroacetic acid.Processing of plant material.Germs,cotyledons and hulls were separated by a dry de-hulling procedure from soy seeds(Kennong16)harvested in the fall of1997in northeast China.All samples were stored atÀ32°C in a freezer before analysis.The solid samples were milled to pass through a30-mesh sieve prior to extraction.Soy germs(250mg)were extracted with80%aqueous ethanol(25mL)in an ultrasonic ice-water bath for2h in a screw-topflask.The solvent-to-sample weight ratio employed provides efficient extraction of isoflavones according to Song et al.(1999).In order to decompose the malonyl glycoside,thefiltered extract was heated in a sealed vial at80°C for20h,a procedure which converts virtually all of the malonyl glycosides into corresponding glycosides with minimum production of acetyl glyco-sides(Lin et al.,2000).Soy cotyledons(1g)were extracted with25mL aqueous ethanol as described for Soy germs:soy molasses derived from unknown soy varieties(dry matter content59.5Æ4.1%;obtained from the Shanghai Liantang Food Factory,Shanghai,China) were dissolved directly in aqueous ethanol.For HPLC analysis,solutions werefiltered through a0.45m mfilter unit and thefiltrate(20m L)injected without further purification.HPLC-ESI-MS assay.Analyses were performed on a Micromass electrospray mass spectrometer equipped with a Micromass electrospray interface(Micromass ZMD,Manchester,UK).An in-source collision-induced dissociation(CID)region was built in to facilitate the formation of fragment ions.Ions entering the MS were analysed over the m/z range200–1500amu.The instrument was coupled with a Waters(Bedford,MA, USA)model2690separation module.Separation was carried out using a Supelcosil LC-18-DB column (250Â4.6mm i.d.;5m m,Supelco,Bellefonte,PA, USA.)eluted with a linear gradient of methanol:water: acetic acid from10:90:0.5to60:40:0.5in35min at a flow-rate of1mL/min.At the outlet,the effluent was split 9:1so that in1min,100m L effluent was delivered to the electrospray ionisation source and900m L passed through a Waters model996PAD which scanned over the range of200–400nm.Nitrogen gas was used both as drying and auxiliary gas.For the direct injection of standard compounds and for the optimisation of the electrospray parameters,a Harvard(model II,South Natick,MA, USA)syringe pump was used at aflow rate of10m L/min. MS were recorded in the positive mode by applying the following potentials to the ESI source:capillary voltage 4000V;cone voltage80V;extractor voltage5V.The source block temperature was120°C,and the drying gas temperature200°C.The conditions employed in the negative mode were the same as those for the positive mode except that the capillary voltage was3800V and the cone potential was100V.The chromatographic and spectral data were recorded using Masslynx data system (version3.1;Micromass).RESULTS AND DISCUSSIONisoflavone glycosideDirectinjection electrospray ionisationMS of the standard isoflavone glycoside,daidzin,was measured in order to evaluate the influence of solvent composition and ion source parameters on the MS of the isoflavones. In the positive ionisation mode,the MS of daidzin did not change significantly from low(30V)to high(115V) cone potential,but was significantly affected by the composition of the solvent used to dissolve the sample.In the MS recorded after injection of a sample dissolved in methanol,the[M was the almost observed ion,with[M H] of low abundance.methanol was used,the most ion was the [M H] species.of the solvent caused a further decrease in the of the[M Na] and an increase in the[M H] species.Addingacid to the solvent rendered these ions378L.GU AND W.GUmuch weaker at high cone potential(115V)and almost unobservable at low cone potential(30V).For comparison with the ions obtained in the positive mode,and for proper assignment of molecular weight, the intense[MÀH]Àions were preferred in the negative mode.However,ionisation of an isoflavone in the negative mode at low cone potential(20V)generated intense[M HCOO]Àor[M CH3COO]Àin acidified solvent,sometimes with an intense[M Cl]Àion if the solvents were contaminated with ClÀ.Under these conditions the[MÀH]Àions were of low abundance. Such interfering ions made the MS very complicated and interfered significantly with the assignment of molecular weights of unknown compounds.However,these ions could be virtually eliminated by increasing the cone potential to100V,and the abundance of[MÀH]Àion was greatly increased at the same time.Acetic acid and trifluoroacetic acid are commonly used as modifiers in an HPLC mobile phase in order to improve resolution.The use of trifluoroacetic acid, however,does not lead to a significant improvement in resolution of the isoflavone conjugates compared to acetic acid according to Kudou et al.(1991)and, furthermore,trifluoroacetic acid(0.2%)in aqueous methanol dramatically reduced the intensity of ions in the positive mode and virtually prevented the ionisationin the negative mode.For these reasons acetic acid was more appropriate as an acidifier than trifluoroacetic acid in HPLC-ESI-MS analysis.The optimal cone potential to give both distinct quasimolecular ions and aglycone fragment ions was found to be80V in the positive mode and100V in the negative mode.The appropriate capillary potential and drying gas temperature in the ion source were3800V and 200°C,respectively.Under these conditions,the standard isoflavone glycoside,daidzin,gave rise to [aglycone H] and[aglyconeÀH]Àas the most abundant ions in the acidified solvent whilst[M H] and[MÀH]Àwere the exclusive quasimolecular ions produced with good relative abundance(about30–70%) in the positive and negative modes,respectively. Generally,ionisation in the positive mode produced more intense ions than their negative counterparts and the spectra were not so significantly influenced by change in solvent composition,such that the sensitivity of the positive mode MS of HPLC-MS analysis would be higher than that of the negative mode.In fact,only positive ESI-MS data were reported by Lin et al.(2000)and Stobiecki et al.(1999).However,the measurement of both negative and positive MS of the isoflavones separated by HPLC should greatly facilitate the identification of unknown compounds.Characterisation of soy isoflavones and screening for novel malonyl isoflavone glycosidesA linear gradient of methanol in water with acetic acid as modifier was optimised in order to give complete separation of isoflavones in soy extracts.Typical HPLC chromatograms of an extract of soy germs and cotyledons [detected by UV(260nm)and by total ion current]are shown in Figs2and3,respectively.In each case,HPLC-UV gave rise to sharper peaks than did HPLC-ESI-MS. The isoflavone composition of soy germs is distinctively different from that of soy cotyledons according to Kudou et al.(1991)in that soy germs contained all known soy isoflavones in high concentration,while soy cotyledons contained only daidzein and genistein group isoflavones, the glycitein group isoflavones being completely absent. This difference was also observed in the chromatograms obtained in the present work as can be seen from Figs2 and3in which some components present in the germ extract are absent from the cotyledon extract.Since the sequence of elution of isoflavones depends on the chromatographic conditions,and since it is not possible to obtain all of the standards required,identification of peaks for each of the isoflavones present is difficult if only a UV detector is employed.However,with the aid of ESI-MS and PAD,all isoflavone peaks could belocated Figure2.Simultaneous HPLC-UV(260nm)(A)and HPLC-ESI-MS total ion current(B)chromatography of an extract of soy germs(Kennong16).The peak assignments are listed in Table1.(For chromatographic protocol see the Experimentalsection.)Figure3.Simultaneous HPLC-UV(260nm)(A)and HPLC-ESI-MS total ion current(B)chromatography of an extract of soy cotyledons(Kennong16).The peak assignments are listed in Table1.(For chromatographic protocol see the Experimental section.)ISOFLAVONES IN SOY379even without standards.Since the ionisation parameters were optimised to give the [aglycone H] and [aglycone ÀH]Àions as the most abundant ions in the positive and the negative mode,respectively,these ions were used as diagnostic ions to monitor the peaks of isoflavones in a specific group.The [daidzein H] ion at m/z 255was set as the selective ion in the positive mode in order to screen out all components belonging to the daidzein group in soy germ extract,and five distinct peaks (1,3,7,9,12in Fig.4)were observed.Amongst these,only peak 1was singled out by selective positive ion monitoring at m/z 417([daidzin H] ),and the MS of this component showed [aglycone H] at m/z 255as the base peak with [M H] at m/z 417as the second abundant ion in the positive mode,with the respective counterparts being [aglycone ÀH]Àion at m/z 253and [M ÀH]Àat m/z 415in the negative mode.No ions other than these were observed up to m/z 1500,and hence peak 1undoubtedly corresponded to daidzin.Similarly,peaks 9and 12were identified to correspond to 6@-O -acetyldaidzin and daidzein,respectively.However,by selective ion monitoring at m/z 503for 6@-O -malonyl-daidzin,peaks 3and 7(Fig.5)were revealed.Peak 7was the main component in the extract of soy germs and cotyledons and was identified as 6@-O -malonyldaidzin by reference to its UV spectrum and other published information.The compound corresponding to minorpeak 3was suspected to be some kind of impurity initially,but its UV and MS were found to be identical to those of 6@-O -malonyldaidzin (Figs 6and 7)suggesting that the peak might be an unknown isoflavone related to the former.Through selective ion chromatography,all isoflavone peaks in the extracts of soy germs and cotyledons were identified and their UV and MS data are listed in Table 1.Within the glycitein group isoflavones,peak 5was found to possess similar UV and MS characteristics to those of 6@-O -malonylglycitin,whilst peak 6in the genistein group isoflavones showed the same UV and MS as 6@-O -malonylgenistin.These findings suggest that the peaks 3,5and 6,belonging to the three distinct isoflavone groups,might be novel isomers of 6@-O -malonyl isoflavoneglycosides.Figure 4.Selective ion chromatography at m/z 255[daidzein H] of an extract of soy germs (Kennong 16).The peak assignments are listed in Table 1.(For chromato-graphic protocol see the Experimentalsection.)Figure 5.Selective ion chromatography at m/z 503(proto-nated ion of 6@-O -malonyldiadzin)of an extract of soy germs (Kennong 16).Peak 3is an unknown malonyldiadzin isomer and peak 7is 6@-O -malonyldiadzin as assigned in Table 1.(For chromatographic protocol see Experimentalsection.)Figure 6.UV-PAD spectrum of the unknown malonyldiadzin isomer (A)compared with that of 6@-O -malonyldiadzin (B).The spectra have not been processed to subtract the back-ground.Figure 7.ESI-MS of the unknown malonyldiadzin isomer (A)compared with that of 6@-O -malonyldiadzin (B).The spectra have not been processed to subtract the background.380L.GU AND W.GUBoth the unknown isomers and their related glycosides showed identical UV absorption profiles,suggesting that the malonyl group is not attached to any of the phenolic functions in the aglycone moiety.It is thus concluded that the malonyl group is attached at a position other than the 6@position of the glycosyl moiety of the molecule,namely,position 2@,3@or 4@.The existence of such minor isomers of 6@-O -malonyl isoflavone glycosides in red clover,a plant of the same family (Leguminosae )as soy,has recently been reported by Lin et al.(2000),who postulated that the malonyl isoflavone glycosides might exist in two isomers.The co-existence of two isomers of malonyl isoflavone glycosides was also implied by Edwards et al.(1997).However,there are no reports describing the details of the structures of such minor malonyl isoflavone glycosides to date,and their presence in soy is revealed here for the first time.An HPLC-ESI-MS study was carried out using heated soy extracts to confirm the identification of malonyl isoflavone glycosides.Isoflavones of all forms in the extracts of germs and cotyledons were quantitatively determined before and after prolonged (20h)heating.The results shown in Table 2indicate that the unknown malonyl isoflavone glycosides were as thermally liable as their respective 6@-O -malonyl isoflavone glycosides and were not detectable after 20h heating in aqueous ethanol.Following heating,the amount of the respective iso-flavone glycoside had increased,but the total amount ofTable 1.Assignments of HPLC peaks detected in the extracts obtained from soy germs and cotyledons (see Figs 2and 3)Peak a R t (min)UV *max(nm)ESI MS-positive modeESI MS-negative mode Compound identi®cation [Aglycone H][M H] [Aglycone ÀH]À[M ÀH]À110.72249,310sh 255(100)417(30)253(100)415(48)Daidzin 2b 12.87259,321sh 285(100)447(29)283(100)445(45)Glycitin314.82249,305sh 255(100)503(23)253(100)501(10)Unknown malonyldaidzin 416.10259,335sh 271(100)433(27)269(100)431(28)Genistin5b 16.74259,325sh 285(100)533(19)283(100)531(11)Unknown malonyglycitin 619.59259,335sh 271(100)519(31)269(100)517(6)Unknown malonylgenistin 720.80249,305sh 255(100)503(25)253(100)501(10)6@-O -malonyldaidzin 8b 22.55259,321sh 285(100)533(29)283(100)531(8)6@-O -malonylglycitin 924.19254,305sh 255(100)459(21)253(100)457(87)6@-O -acetyldaidzin 1025.14259,335sh 271(100)519(34)269(100)517(9)6@-O -malonylgenistin 11b 25.80259,327sh 285(100)489(28)283(100)487(81)6@-O -acetylglycitin 1226.67254,310sh 255(20)c 211(100)c,d 253(100)253(100)Daidzein 13b 28.52259,320sh 285(15)211(100)d Ðe ÐeGlycitein1429.05261,330sh271(100)475(17)269(100)473(41)6@-O -acetylgenistin 1531.44261Ðc,ec,e269(50)255(100)dGenisteina Peak numbering corresponds to that shown in Figs 2and 3.bThese compounds are absent from cotyledons and exist only in germs.cUnable to measure in extract of cotyledons.dUnknown fragment ions.eUnable to measure in extract of germs.Table 2.Content of isoflavone compounds in soy seed (Kennong 16)and in molasses as determined by HPLC-ESI-MSCompoundsSoy molasses (mg/100g)aSoy germs (mg/100g)aSoy cotyledons (mg/100g)a ExtractHeated extract (20h)ExtractHeated Extract (20h)Unknown malonyldaidzin Ðb 152.2Ð20.5ÐUnknown malonylglycitin Ð43.6ÐÐÐUnknown malonylgenistin Ð39.9Ð21.8Ð6@-O -malonyldaidzein Ð1437.2Ð105.7Ð6@-O -malonylglycitin Ð514.3ÐÐÐ6@-O -malonylgenistin 0.6406.0Ð213.4ÐDaidzin 370.1306.71582.827.3139.1Glycitin 157.8220.6670.5ÐÐGenistin979.477.7439.251.5255.26@-O -acetyldaidzein 32.789.5143.2 4.47.56@-O -acetylglycitin 24.337.159.3ÐÐ6@-O -acetylgenistin 97.522.035.28.514.4Daidzein 5.9 5.5 5.80.50.6Glycitein 5.5 2.3 2.4ÐÐGenistein33.6 2.5 2.5 1.0 1.0Total iso¯avones 1707.43357.02944.9454.7420.4Total aglycones1071.71804.8A1812.6A 244.1B 258.4Ba Mean values (n =3);values in list with the same superscript are not signi®cantly different at p <0.05.bNot detected.ISOFLAVONES IN SOY 381isoflavone aglycone remained unchanged indicating that the malonyl isoflavone glycosides were being converted into the corresponding glycosides.The malonyl isofla-vone glycosides were virtually absent in soy molasses, and it is possible that they might be completely decomposed to glycosides after protracted thermal processing.It is probable that these novel malonyl isoflavone glycosides have not isolated and identified until now because of their low concentrations and unstable characters.Although thermally labile,the malonyl glycosides are not decomposed in the ES ion source because of the cooling effects caused by evaporation of solvent(as stated by Barnes et al.,1998).This suggestion is supported by the fact that the relative abundance of protonated quasimolecular ions of malonyl isoflavone glycosides was similar to that of isoflavone glycosides and6@-O-acetyl isoflavone glycosides in the positive mode,whilst in the negative mode the relative abundance of[MÀH]Àions of malonyl isoflavone glycosides (<12%)was much lower than that of glycosides and of 6@-O-acetyl glycosides(>40%).From the chemical viewpoint,the methylene between the two carbonyls in the malonyl group is partly positively charged and extremely reactive,and would be attacked by large amounts of negative charged molecules in the ion source in the negative mode and promptly decomposed.Hence abundant[MÀH]Àions from malonyl glycosides may be theoretically impossible.The novel isoflavone reportedly isolated from soy molasses by Hosny and Rosazza(1999)was genistin substituted with a g-hydroxy-g,g-dimethylpropyl group at position8of the aglycone moiety,which had the same molecular weight,and hence the same quasimolecular ion([M H] ;m/z519)as6@-O-malonylgenistin. However,this compound would give different aglycone fragment ions in the MS:its[aglycone H] and [aglyconeÀH]Àion would be present at m/z341and 339.None of the detected isoflavones formed these ions even when the ionisation parameters were changed,and no distinct peaks were observed by selective ion monitoring for the aglycone or the molecular ion of the other two novel isoflavones isolated by Hosny and Rosazza(1999)in extracts of soy germs and cotyledons. They were also absent in soy molasses.It may be that the novel isoflavones isolated by Hosny and Rosazza(1999) were artefacts of specific processing and/or storage conditions,or,possibly,the varieties of soy used may have been significantly different from those employed in the present studies.In conclusion,HPLC-ESI-MS has been shown to be a rapid,sensitive and precise method for the analysis of isoflavones in ing this method,the presence of some novel malonyl isoflavone glycosides in soy has been demonstrated for thefirst time.These novel compounds are present in such low amounts that they have not before been detected by HPLC-UV analysis.In fact the detection limit of ESI-MS(typically20ng/g)is reported to be50-fold lower than that of UV(1–5m g/g) (Barnes et al.,1998;Stobiecki et al.,1999)such that weak signals buried within the noise in a UV chromato-gram could be identified by ESI-MS.This will greatly facilitate the discovery of minor novel compounds in nature,since,although ESI-MS cannot normally be used to establish the structures of unknown compounds unambiguously,it does provide useful hints about their structures and could surely guide further isolation and identification.AcknowledgementThe authors wish to thank Professor Patricia A.Murphy(Iowa State University,Ames,IA,USA)for valuable advice during the course of this research.REFERENCESAdlercreutz H and Mazur W.1997.Phyto-estrogens and western diseases.Ann Med29:95±120.Barnes S,Kirk M and Coward L.1994.Iso¯avones and their conjugates in soy foods:extraction conditions and analysis by HPLC-ES-MS spectrometry.J Agric Food Chem42:2466±2474.Barnes S,Coward L,Kirk M and Sfakianos J.1998.HPLC-ES-MS spectrometry analysis of iso¯avones.Proc Soc Exp Biol Med127:254±262.Edwards R,Tiller SA and Parry AD.1997.The effect of plant age and nodulation on the iso¯avonoid content of red clover(Trifolium pratense).J Plant Physiol150:603±610. 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Stobiecki M,Malosse C,Kerhoas L,Wojlaszek P and Einhorn J.1999.Detection of iso¯avonoids and their glycosides by liquid chromatography-electrospray ionisation mass spectrometry in root extracts of lupin(Lupinus albus).Phytochem Anal10:198±207.382L.GU AND W.GU。