High-performance Liquid Chromatography/Continuous-flow Liquid Secondary Ion Mass Spectrometry of Flavonoid Glycosides in Leguminous Plant Extracts

JOURNAL O F MASS SPECTROMETRY, VOL. 31, 472-485 (1996)

High-performance Liquid Chromatography/Continuous=flow Liquid Secondary Ion Mass Spectrometry of Flavonoid Glycosides in Leguminous Plant Extracts

Lloyd W. Surnner,'*t Nancy L. Paiva,' Richard A. Dixon' and Paul W. Geno' I Department of Chemistry, Oklahoma State University, Stillwater, OK 74078 * Plant Biology Division, Samuel Roberts Noble Foundation, P.O. Box 2180, Ardmore, OK 73402

Department of Environmental Chemistry, Southwest Research Institute, San Antonio, TX 78228

A method for the identification of flavonoid glycosides utilizing continuous-flow liquid secondary ion mass spectrometry (CF-LSIMS) is presented. Minimum detectable quantities (MDQs) were determined for three model flavonoid glycosides (rutin, naringin and esculin) by both positive ion direct insertion probe (DIP)-LSIMS (1.6 nmol, 1.7 nmol and 730 pmol, respectively) and positive ion CF-LSIMS (330 pmol, 340 pmol and 290 pmol, respectively). Optimization of CF-LSIMS instrumental parameters was performed using the model compound rutin. Parameters optimized included mobile phase composition, glycerol concentration, mobile phase flow rate, ion source temperature, acceleration lens potential (amplitude and polarity) and Cs' primary ion energy. Final instrumental optimization yielded an MDQ of 1.0 ng (1.6 pmol) for rutin by flow-injection CF-LSIMS. The optimization parameters were utilized in the identification of flavonoid glucosides in alfalfa (Medicugo sativa L) and chickpea (Cicer urietinum) extracts by high-performance liquid chromatography/CF-LSIMS. The results support the controversial identification of a major extract component as formononetin-7-O-glucoside-6"-malonate as opposed to afrormosin-7-O-glucoside-6"-malona te.

KEYWORDS : continuous-flow liquid secondary ion mass spectrometry; flavonoid glycosides; optimization; alfalfa (Medicago sativa L.); chickpea (Cicer arietinum)

INTRODUCTION

Flavonoids are recognized as one of the most abundant groups of natural products found in plants. To date, over 4000 flavonoid compounds have been identified and documented.' The flavonoid family consists of many subclasses which include, but are not limited to, flavones, flavanones, isoflavones, aurones, anthocyanins and pterocarpans. The functions of flavonoids vary as much as their structure and include such roles as flower pigments, UV protectants, growth regulators, nodu- lation inducers and phyt~alexins .~-~ Further studies suggest that some flavonoids may even have medicinal properties such as anticancer a~ t iv i ly .~

Alfalfa and other leguminous plants accumulate a variety of flavonoids in response to the presence of pathogens. Those flavonoids of particular interest in alfalfa are the isoflavonoids and the pterocarpans. The pterocarpan medicarpin has been identified as a major phytoalexin present in alfalfa6v7 and its biosynthetic pathway has been determined.* Phytoalexins are see-

* Present address: Texas A&M University, Department of Chem- istry, Laboratory for Biological Mass Spectrometry, College Station, TX 77843, USA.

t Author to whom correspondence should be addressed.

ondary metabolites synthesized in response to microbial infection and accumulate to levels sufficient to limit microbial growth within plant tissue^.^^^ Although the aglycone or nonconjugated form of isoflavonoids and pterocarpans have been identified as possessing the antimicrobial activity, it has been reported that some of these compounds are stored in the cell vacuole as glycosides or malonated glycosides.'O~" Acidic flavonoid glycosides are of biological interest owing to the ability of plants to utilize this form of conjugation to store less soluble flavonoid aglycones. Upon infection of the cell, the isoflavonoid aglycones may be synthesized from precursors, but are more likely generated by the hydrolysis of the stored glycosides into their phytoalexic aglycone form.'0*'' Thus, further study and identification of these conjugates will help provide a better understand- ing of the molecular biology associated with phytoalexins.

Mass spectral identification of flavonoids by electron impact (EI) ionization mass spectrometry (MS) has been extensively Although EI/MS and chemical ionization MS have been successful in the determination of flavonoid aglycones (non-conjugated form), these techniques have found little utility in the mass spectral determination of flavonoid glycosides without extensive derivatization.".' The development of particle desorption techniques such as fast atom

CCC 1076-5174/96/050472-14 0 1996 by John Wiley & Sons, Ltd.

Received 15 November 1995 Accepted 5 January 1996

HPLC-CF-LSIMS OF FLAVONOID GLYCOSIDES 473

bombardment (FAB)14 and liquid secondary ion mass spectrometry (LSIMS)l has provided the means for lower energy mass spectral analyses of many non- volatile, thermally labile, polar and ionic species without derivatization.16.' To date, direct insertion probe FAB (DIP-FAB) has been used almost exclu- sively for the identification of flavonoid g l y c ~ s i d e s . ' ~ - ~ ~ These reports establish that molecular mass and valu- able structural information can be obtained from DIP and tandem FAB mass spectra.

Although FAB was a revolutionary addition to mass spectrometry, the introduction of continuous-flow FAB (CF-FAB)Z6.z7 provided many advantages over the con- ventional DIP technique. These advantages have been summarized by Caprioli.28 The adaptation of the continuous-flow technique to LSIMS (CF-LSIMS) soon followed, and differs only in the nature of the primary particle. 29

Initial attempts to utilize DIP-LSIMS for the identification of suspect isoflavonoid glucosides in alfalfa had limited success owing to sample decomposition of the more sensitive suspect glucosides. Therefore, to minimize possible sources of sample decomposition (extraction processes, fraction collection, concentration, elapsed time and sample handling), the feasibility of an on-line separation and analysis method was considered. The literature offers a few reports of such a method using high-performance liquid chromatography (HPLC) interfaced with thermospray ionization mass spectrometry for the analysis of flavonoid glycoside mixtures, but not malonated isoflavonoid glycosides (our target analyte~) .~ '*~l Since thermospray utilizes high temperatures, typically 100-200 "C, we reasoned that the high temperatures would further aggravate the sample decomposition problem encountered with the more labile intermediates. A study of decomposition of bio- molecules as a function of thermospray vaporizer temperature has been presented by Heeremans et aL3' Barnes et al.33 also report the detrimental effects of heat on the decomposition of isoflavonoid malonylglucosides in heated nebulizer atmospheric pressure chemical ionization (APCI), but also show that strong [M + H]+ ion peaks may be obtained using electrospray ionization (ESI).

Capitalizing on the benefits of CF-LSIMS and its ability to interface with HPLC, we investigated the use of HPLC/CF-LSIMS for the determination of flavonoid glycosides. Such a method has been suggested by Wolf- ender et a1." but, to the best of the authors' knowledge, none have yet been reported. We further utilize this method to shed new light on the controversial identification of formononetin-7-O-glucoside-6-rnalonate versus afrormosin-7-O-glucoside-6"-malonate as a major extractable component of alfalfa.

EXPERIMENTAL

Chemicals and reagents

Glycerol, trifluoroacetic acid (TFA), acetic acid (HOAc), rutin and naringin were obtained from Sigma Chemical

(St Louis, MO, USA). Esculin was acquired from Fluka Chemical (Ronkonkoma, NY, USA). Daidzin and genistin were obtained from Apin Chemicals (Abingdon, UK). All flavonoid conjugates were used as received. Burdick & Jackson GC/MS-grade methanol and acetonitrile were obtained from Baxter Health (McGraw Park, IL, USA). Water was distilled and deionized using an 8 in x 44 in mixed-bed ion-exchange column (Continental Waters Systems, San Antonio, TX, USA). Solvents were vacuum filtered through a 0.2 pm nylon 66 membrane filter (Alltech Associates, Deerfield, IL, USA) to remove any particulate matter and to degas the solvents. HPLC mobile phases were further sparged for -15 min at 25 ml min-l with chromatographic- grade helium prior to use to ensure complete degassing.

Mass spectrometry

Mass spectral analyses were performed on a VG ZAB2-SE double-focusing mass spectrometer (VG Ana- lytical, Manchester, UK) equipped with a VG continuous-flow probe with stainless-steel hemispherical probe tip. The CF-LSIMS probe was slightly modified for convenience and consisted of mounting an internal loop injector (Model CWW, Valco, Houston, TX, USA) or a splitting device previously described by Kok- konen et al.34directly on to the CF probe. Sample introduction for CF-LSIMS optimization experiments was performed in the flow-injection mode, whereas an HPLC/CF-LSIMS mode was utilized on plant extracts.28 The flow-injection mode utilized an 1x0 pLC-500 Micro Flow syringe pump in conjunction with the Valco internal loop injector to deliver an isocratic mobile phase and sample to the probe tip through a 1 m x 75 Fm id. fused-silica transfer capillary. HPLC/ CF-LSIMS experiments with plant extracts utilized a Waters 600MS high-performance liquid chromato- graph. Reversed-phase HPLC/CF-LSIMS was performed with a 4.6 mm x 250 mm Bakerbond (J. T. Baker, Phillipsburg, NJ, USA) 5 pm, 120 A pore size octadecylsilane (ODS) column. The column eluent was first passed through an ISCO pLC-10 variable- wavelength detector (A 287 nm, which corresponds to the A,,, of medicarpin or 254 nm) that had been placed in series with the mass spectrometer. The eluate from the UV detector was then delivered to the splitting device through a 25 cm x 0.178 mm i.d. (0.007 in id.) stainless-steel capillary. The eluent was split 160: 134 such that 5 p1 min-' was delivered to the continuous- flow probe tip through a 50 pm i.d. fused-silica capillary (Polymicro Technologies, Phoenix, AZ, USA). The stainless-steel capillary entering the splitting tee was counter-bored -500 pm id . by 5 mm in depth. The 50 pm i.d. transfer fused-silica capillary was then recessed into this counter bore to minimize band spreading in the dead volume of the tee.

Reversed-phase separations of plant extracts were achieved with a linear gradient of 80 : 20 (solvent A : B) to 40:60 over 45 min at 0.8 ml min-I. Solvent A consisted of 97.9% water, 2.0% glycerol and 0.1% TFA. Solvent B consisted of 97.9% acetonitrile, 2.0% glycerol and 0.1 % TFA. Experiments were also performed with 1.0% acetic acid modifier in both solvents A and B in

474 L. W. SUMNER ET AL.

place of the TFA while still maintaining 2.0% glycerol in each. The CF-LSIMS source was operated using a 30-35 keV Cs+ primary ion beam. Positive or negative secondary ions for the LSIMS experiments were extracted and accelerated to 6 keV unless stated other- wise. All data were acquired using a mass spectral resolving power in excess of 2000 and the data were recorded using a VG 11-2505 data system. Two modes of data acquisition were utilized: selected-ion monitoring (SIM) and magnetic scanning. The SIM data were acquired using a dwell time of 80 ms and a delay time of 20 ms. Magnetic scanning was performed by scanning the magnetic field exponentially downward at a rate of 10 or 15 s per decade from m/z 800 to 200. Mass cali- bration was performed using known values for glycerol clusters originating from the mobile phase. The Cs' primary ion beam and the CF-LSIMS probe were aligned as described elsewhere.'*

Determination of minimum detectable quantities and CF-LSIMS characterization

Minimum detectable quantities (MDQs) were determined for DIP-LSIMS and CF-LSIMS using a series of methanol solutions ranging in concentration from 0.10 to 50 pg pl-' of the model flavonoid glycosides. Samples for DIP-LSIMS were prepared by the separate addition of 1 pl aliquots of the flavonoid glycoside solutions to 1 pl of a 1.0% TFA in thioglycerol matrix and analyzed using a 35 keV Cs' primary ion energy. Cf- LSIMS analyses were performed by injection of 1.0 pl samples into a mobile phase consisting of 49% CH,OH, 49% H'O, 2.0% glycerol and 0.10% TFA operating at a flow rate of 5.0 pl min-' while the source/probe temperature was maintained at 38 "C. MDQs were recorded for the minimum amount of analyte sufficient to generate an [M + H]+ ion peak having a signal-to-background (S/B) ratio of three. The S/B ratio was determined as described by Watson35 for FAB MDQ calculations. Negative ion CF-LSIMS characterization of the model phenolic conjugates was performed using the above-described mobile phase/source conditions, acceleration lens potential of -6 kV and a primary ion energy of 30 keV.

CF-LSIMS reproducibility experiments using selected-ion monitoring

Ten repetitive 1 pl injections were made with a methanol solution containing 2.5 pg p1-' of rutin. The mobile phase and other conditions used were as described above. Injections were made every 2 min, alternating between rutin and a blank consisting of the mobile phase. The injector was further rinsed with methanol 30 s prior to all rutin and blank injections. Chromatograms were acquired in the SIM mode while monitoring the [M + H]+ ion of rutin at m/z 611. The area of each SIM peak was integrated using a computer function available on the VG 11-2505 data system.

MGM reference material isolation and hydrolysis

Medicarpin-3-O-glucoside-6"-malonate (MGM) was isolated from alfalfa cell suspension cultures (Medicago sativa L., cultivar Calwest) that had been elicited with the cell walls of the phytopathogenic fungus, Cotletotri- chum lindemuthianum, by preparative HPLC.36*37 After isolation, MGM was hydrolyzed by autoclaving the isolated fraction at 121 "C for 3 h in a solution of 10% acetic acid in 60 : 40 water-acetonitrile. This resulted in a conversion of -60% of the MGM to MG and free medicarpin as determined by HPLC. This crude reference mixture was then analyzed by negative ion HPLC/ CF-LSIMS in which simultaneous UV and mass spectral detection were performed.

Extraction of isoflavonoid conjugates from alfalfa

New callus cultures (non-differentiated plant cells) were initiated for alfalfa (cultivars Calwest and Apollo) and were used to generate cell suspension Approximately 3-week-old (late growth phase) cell cultures were harvested, immediately frozen in liquid nitrogen and stored at a maximum of -10°C until extracted. Suspect isoflavonoid conjugates were isolated from alfalfa cell suspension cultures by a modification of the method reported by Kessmann et This method extracted smaller amounts of plant material and was approximately three times faster than the column method reported.37 The smaller sample isolation method was preferred since the large amount of extract material generated from the previously reported method could not be used in a timely manner to ensure that isoflavonoid conjugate decomposition was kept to a minimum. The modified method consisted of freezing -30 g of plant material in liquid nitrogen followed by immediate grinding with a liquid nitrogen-cooled mortar and pestle, The sample was then extracted twice with 50-200 ml portions of ice-cold acetone for - 4 h each depending on the quantity of original plant tissue. The extraction was performed while the mixture was magnetically stirred and contained in an insulated ice- bath. The extracts were pooled and sequentially filtered through Whatman No. 4 and No. 40 filter-papers. The pooled extract was then concentrated on a rotary evaporator and loaded on to a fresh polyamide (Polyamide 6 or Polyclar AT) column.

Polyamide particles were prepared as reported prior to column preparati01-1.~~ A 25 ml bed volume of polyamide was placed in to a Pyrex coarse (40-60 pm pores) fritted disc glass funnel (Fisher Scientific, Pittsburgh, PA, USA). The column was then washed with successive 75 ml portions of water, methanol and a 1:500 (v/v) solution of concentrated (30%) ammonia in methanol. A slight vacuum generated by water aspiration was used to increase the flow rate through the column, although care was needed to keep the column bedding from cavitating. The column was then re-equilibrated with water and the extract loaded.

The extract was eluted with a step gradient (batches) of 75 ml each water, methanol and the basic methanol solution described above. The basic fraction was collected and taken to dryness using a rotary evaporator.


The remaining oily residue was then resuspended in a minimal amount of methanol for transfer to a vial. The resulting solution was concentrated under a stream of Ar or N, at 40 "C. The basic fraction was then analyzed by HPLC/CF-LSIMS. Typical of the above-described extraction process, a 35 g sample of extracted Calwest alfalfa cells generated 360 mg of total extract that after fractionation on the polyamide column yielded 9.7 mg of the basic extract. This was then diluted in methanol to generate a 45 pg pl- basic extract solution of which 25 pl were injected on to the HPLC column.

Chickpea (Cicer arietinum) plant growth and extraction

Chickpea seeds were disinfected for 2-5 min in ethanol followed by -20 min in a 20% bleach solution. The seeds were then washed three times in sterile water and placed on the surface of a water-agarose (0.5%) medium contained in plastic boxes (Magenta, Chicago, IL, USA). The seeds were allowed to germinate in a lighted culture room at 25 "C. Contaminated seedlings were removed and discarded. A 1.948 g amount of 9-day-old chickpea hypocotyls and cotyledons were collected, frozen in liquid nitrogen and immediately finely ground with a mortar and pestle. The plant material was extracted with 25 ml of ice-cold acetone on an orbital shaker. The acetone phase was decanted from the residual plant tissue and taken to dryness under a stream of nitrogen in a heated block maintained at 40°C. The residue was then dissolved in 1 ml of methanol and centrifuged to remove any remaining plant tissue. Half of this extract was set aside for LC/MS analysis while the remaining portion of the extract (0.5 ml) was subjected to fl-glucosidase (from almonds, Sigma Chemical) hydrolysis. fl-Glucosidase hydrolysis was performed by taking the sample to dryness under a stream of nitrogen in a heated block maintained at 40 "C. A 1.5 ml aliquot of a citric acid-phosphate buffer (pH 5.2) containing 3 mg ml-' of fl-glucosidase was then added to the dry extract material and this solution was incubated for -24 h. The aqueous solution was extracted twice with 2 ml portions of ethyl acetate, pooled and taken to dryness under a stream of nitrogen

in a heated block maintained at 40°C. The hydrolyzed extract was then dissolved in methanol and analyzed by LC/MS.

RESULTS AND DISCUSSION

Positive ion direct insertion probe LSIMS of model flavonoid conjugates

Positive ion DIP-LSIMS was performed to help charac- terize the LSIMS mass spectra of model flavonoid glycosides and for comparison with CF-LSIMS mass spectra. DIP-LSIMS mass spectra were obtained for 20-30 pg of the commercially available model compounds rutin (3,3',4,5,7-pentahydroxyflavone- 3-rutinoside), naringin (4',5,7-trihydroxyflavanone 7-rhamnoglucoside) and esculin (6,7-dihydro- xycoumarin-6-glucoside). Additional isoflavonoid conjugates, i.e. genistin (4',5,7-trihydroxyisoflavone 7-glucoside) and daidzin (4',7-dihydroxyisoflavone 7- glucoside), supplemented our model compound list. Molecular structures of these compounds are provided in Fig. 1 and their mass spectra summarized in Table 1 while a representative DIP-LSIMS mass spectrum of rutin is illustrated in Fig. 2(a). From a survey of common LSIMS matrices (i.e. glycerol, glycerol with 1.0% TFA, thioglycerol, thioglycerol with 1.0% TFA, nitrobenzyl alcohol, a 50: 50 mixture of glycerol and thioglycerol and a 50:50 mixture of glycerol and thioglycerol containing 1.0% TFA), it was experimen- tally determined that thioglycerol containing 1.0% TFA yielded the best matrix for positive ion DIP-LSIMS and is in agreement with that reported by Beechi and Fraisse' for C-glycosidic flavonoids.

We have adopted a nomenclature for the identification of positive and negative ion flavonoid fragments similar to that described by Crow et a1." The positive ion notation is illustrated in the inset of Fig. 2(a) with the proposed fragmentation for rutin where the [M + HI+ and [M + Na]' ions correspond to proton and sodium adducts with the molecular species. The [FIH,]+ ion corresponds to the doubly protonated

Table 1. Summary of the positive ion DIP-LSI and CF-LSI mass spectra lm/z with relative intensity ("A) in parentheses1

Method Compound [M + H I + [M +Na]* [F,H,]+ CAHJ' Others

DIP-LSIMS Rutin 6111 (34) 633 (14) 465 (11) 303 (100) 593 (3)$413 (5). 287 (14); 317 (5), 273 (5), 239 (5) Naringin 581 (12) 603 (2) 435 (5) 273 (100) 551 (7). 523 (5). 435 (4), 257 (9)' Esculin 341 (100) 363 (2) 179 (67) 323 (3),' 163 (7). 149 (6) Daidzin 417 (29) 439 (3) 255 (100) 281 (lo), 267 (7), 239 (7); 221 (8) Genistin 433 (14) 455 (6) 271 (100) 255 (22); 237 (22)

CF-LSIMS Rutin 611 (45) 633 (8) 465 (18) 303 (100) 595 (4): 441 (5). 425 (18). 287 (20)' Naringin 581 (10) 603 (3) 435 (2) 273 (100) 419 (5), 403 (17). 257 (15),'287 (18) Esculin 433 (14) - a.b 681 (6): 433 (8)'

a

a

a

a

a [F,H,]+ =[AH,]+ for monosaccharides. Lower m/z limit abovemlz value for [AH,]+. [M + H -18]+ =[M + H -H,O]+.

d[M + H - 161' =[M -OH +2H]+. [2M + HI+.

'[AH, - 161'. [M + Gly + HI+.


FIAVONES

OH OH Rdh (nvu = 610) 4,?-dty&xylauna (mw = 254)

FLAVANONES CO UMARINS

- M.W. B B B s( ISOFLAVONES Daidzein 254 OH H H OH Daidzin 416 Glu H H OH

432 Glu H OH OH Genistin Formononetin 268 OH H H OCH, Fonnononetin-7-0-glucoside (FG) 430 Glu H H OCH, Formononetin-7-O-glucoside-6”-malonate (FGM) 516 GluMal H H OCH, Biochanin A-7-0-glucoside (BG) 446 Glu H OH OCH, Biochanin A-7-O-glucoside-6”-malonate (BGM) 532 GluMal H OH OCH, Afrormosin-7-O-glucoside-6”-malonak (AGM) 546 GluMal OCH, H OCH,

R4

PTEROCARPANS hzw. B B

Medicarpin 270 OH OCH, Medicarpin-3 -0-glucoside 432 Glu OCH, Medicarpin-3-0-glucoside-6” -acetate 474 GluOAc OCH, Medicarpin-3 -O-glucoside-6”-malonate 518 GluMal OCH,

RL

Figure 1. Molecular structures of model and extractable flavonoid glycosidss.

fragment ion resulting from cleavage between the first and second sugars whereas the [AHz] + ion corresponds to the doubly protonated aglycone fragment. The adduction of the first proton regains charge neutrality for the fragmented species while the second imparts a positive charge. It is proposed that the additional protons originate from the acidified matrix.

Our results were similar to those reported in the literature for positive ion direct probe FAB utilizing 8 keV xenon atoms and a glycerol or acidic glycerol matrix.” Fragment ion peaks corresponding to the successive

cleavage of the sugars from the molecular species were observed in all LSI mass spectra with the aglycone, [AH,]’, ion yielding the base peak in all but the case of esculin. For esculin, a coumarin monoglycoside, the mass spectrum yielded the [M + HI+ ion peak as the base peak. These findings differ slightly from the reported FAB mass spectra of naringin and apiin, which did not include aglycone peaks.20 Sodium ion adducts with the molecular species, [M + Na]’, were observed at relative abundances ranging from 2% to 14%. Positive ion DIP-LSI mass spectra of genistin and daidzin,

HPLC-CF-LSIMS OF FLAVONOID GLYCOSIDES

100

90

80

70

477

90

80

70

5 60

9 50 Y 5 40 ' 30

20

10

0 200

[M+H]+ 61 I

4 0 0

ni I z ni I 7.

(M-H). 609

500 600 700

200 300 400 500 600 700

M i z

Figure 2. (a) Positive ion DIP-LSI mass spectrum of 40 pg of rutin with the fragmentation illustrated in the inset. Note that the origin of the ion peak at m/z 595 ([M + H - 161' =[M -OH +2H]+) could result from the cleavage of any one of the hydroxyl groups and not necessarily the exact one illustrated. (b) Positive ion CF-LSI mass spectrum of 2.5 pg of rutin. (c) Negative ion CF-LSI mass spectrum of 2.0 pg of rutin.

two isoflavonoid conjugates, are also reported in Table 1. LSI mass spectra of these compounds have not been reported previously. These isoflavonoid glycosides yielded mass spectra similar to those of the other model flavonoid conjugates (i.e. a base peak corresponding to the [AH2]+, [M + HI+ and [M + Na]+ ion peaks).

MDQs of the three model conjugates were determined to evaluate the sensitivity of positive ion DIP- LSIMS for flavonoid glycosides. MDQs for rutin, naringin and esculin were determined to be 1.0 pg (1.6 nmol), 1.0 pg (1.7 nmol) and 0.25 pg (0.73 nmol), respectively, for positive ion DIP-LSIMS.

The linearity of the LSIMS response for the flavonoid glycosides was also evaluated. It was found that the intensity ratio of the [M + H]+ ion peak of esculin at m/z 341 relative to the background matrix ion peak intensity at m/z 391 (i.e. Im/z=341/Im,z=391) was linear with respect to the esculin concentration. A relative intensity method was chosen over an absolute intensity method owing to the many instrumental variations that can alter the absolute intensities of the analyte ion peak. A response curve of I+= 341/Im,r= 391 vs. concentration was then constructed. For the range of 730 pmol-58.6 nmol(0.25 pg-20 pg) esculin, the response curve showed good linearity with a correlation coefficient of 0.998.

Instrumental optimization of CF-LSLMS

During initial CF-LSIMS experiments, it was observed that variations in several instrumental parameters had significant effects on ion source ~ t a b i l i t y , ~ ~ , ~ ~ repro- duciblility of CF-LSIMS mass spectra and abundance of secondary ions. Because of these observations, an in- depth study of the instrumental parameters was initiated to determine optimum conditions for the CF-LSIMS determination of phenolic conjugates. Instrumental optimizations were performed using the model compound rutin and evaluated by determining the MDQ of rutin as a function of the instrumental variables.

Optimization began with the matrix and mobile phase. Although thioglycerol yielded higher positive secondary ion currents than glycerol, its use in the CF- LSIMS interface made source stability significantly more difficult to attain owing to thioglycerol's higher volatility. Therefore, glycerol was chosen as the CF- LSIMS matrix additive and the glycerol concentration in the mobile phase was optimized. Experiments investigated the use of 0-10% glycerol in acidic solutions of varying concentrations of methanol and water. It was


determined that a mobile phase consisting of 49% CH,OH, 49% H,O, 2.0% glycerol and 0.1% trifluoroacetic acid (TFA) produced stable source conditions at a flow rate of 5.0 p1 min-' and a source/probe temperature of 38 "C. The low glycerol concentration allowed for a low source/probe temperature which minimized the possibility of thermal degradation of the phenolic conjugates. An acid was incorporated in the mobile phase since the final objective was to perform gradient HPLC/CF-LSIMS of flavonoid extracts which requires the acid modifier to achieve adequate chromatographic r e ~ o l u t i o n . ~ ~ . ~ ~

Positive and negative secondary ion CF-LSIMS experiments were conducted using secondary ion acceleration lens potentials of + 8 and - 8 kV, respectively. The MDQ of rutin was determined to be 200 ng (330 pmol) in the positive ion mode and 50 ng (82 pmol) in the negative ion mode. It should be noted, however, that the absolute ion abundances were greater in the positive ion mode but an increased S/N ratio for the [M - HI- ion peak yielded lower MDQs. Although the preference of negative ion LSIMS mode is only illustrated for rutin, Beechi and Fraisse" reported that negative ion DIP-FAB provided lower detection limits for 23 C-glycosidic flavonoids. As a result, further optimizations were performed in the negative ion mode.

Operation of the CF-LSIMS interface at a mobile phase flow rate of 5 pl min- resulted in a source pressure of 2.5 x mbar, which is higher than normally encountered during EI or DIP-LSIMS operation. Owing to the increased pressure, the CF-LSIMS ionization source voltage had a tendency to discharge when the instrument was operated at acceleration lens potentials of + 8 kV or higher. It was determined that the instrument could be operated at an acceleration lens potential of - 6 kV with greater stability and no significant decrease in sensitivity as reflected in the equal MDQ values of 50 ng (82 pmol) rutin, which were obtained at acceleration lens potentials of both - 6 and

During instrument tuning, it was observed that variations in the Cs' primary ion energy produced changes in the abundance of negative secondary ions of both the glycerol matrix clusters and the sample analyte. It was also noted that a broad primary ion beam produced more stable source conditions than a narrowly focused primary ion beam. Observations further indicated that increases in the absolute glycerol ion yield were directly related to increases in the absolute secondary ion yield of the analyte. Thus, experiments were performed to determine the effect of Cs+ ion energy on the secondary ion yield. The Cs' primary ion energy was varied from 15-40 keV while measuring the absolute ion peak intensities of m/z 91 and 367 which correspond to the glycerol ions [Gly - HI- and [4Gly - H]-, respectively. Absolute ion abundances were obtained by measure- ment of the peak intensity directly from the oscilloscope of the VG ZABZSE and then multiplying by the appro- priate amplification setting. A broad maximum was observed in the range of 28-31 keV for the glycerol ion peak at m/z 91 while the higher mass glycerol cluster ion (m/z 367) showed similar results with more variance. Therefore, an average Cs' primary ion energy of 30 keV was selected and an MDQ of 5.0 ng was deter-

- 8 kV.

mined for the [M - HI- ion of rutin that had been extracted and accelerated to 6 keV. These results are similar to those reported by Aberth and Burlingame4' for DIP-LSIMS experiments with bovine insulin and Barber and Green42 for small proteins.

Reversed-phase HPLC separation methods for flavonoids typically utilize an acidified mobile phase to improve chromatographic r e ~ o l u t i o n . ~ ~ - ~ ~ Therefore, an acid modifier consisting of 0.1% TFA was added to the mobile phase in all the above experiments. However, the addition of the acid provides a source of excess protons and, therefore, could hinder the formation of negative ions by proton abstraction based on the acid- base mechanism of ion formation.16 It was postulated that a mobile phase without TFA would yield a lower MDQ for rutin in the negative ion mode than a mobiie phase containing TFA. This postulate was investigated and mass spectra were acquired for rutin without TFA in the mobile phase and a MDQ of 1.0 ng (1.6 pmol) was determined for rutin. Thus, if chromatographic separation were not a consideration, ultimate detection limits could be obtained by omitting the acid modifier from the mobile phase. Final instrumentation optimization yielded an MDQ of 1.0 ng or 1.6 pmol for rutin by flow-injection CF-LSIMS.

Positive ion CF-LSIMS of model phenolic conjugates

Positive ion CF-LSIMS mass spectra were acquired for the model compounds. The positive ion CF-LSIMS mass spectrum of 2.5 pg of rutin is shown in Fig. 2(b) and the mass spectra of rutin, naringin and esculin are given in Table 2. The lower limit of the acquisition mass range (m/z 200) was chosen to avoid the abundant protonated glycerol dimer ion, [2Gly + HI', at m/z 185. Thus, the acquisition mass range was above the m/z value of 179 for the esculin aglycone, [AH,]', which also equals [FIHz] + for this monosaccharide. The positive ion CF-LSIMS spectra obtained for rutin, naringin and esculin contained similar information to those obtained by positive ion DIP-LSIMS. Character- istic ion peaks observed for positive ion CF-LSIMS of the model compounds correspond to [M + HI', [M + Na]', [M + H - 16]+, [F1H2]', [AH,]' and [AH, - 161'. It is believed that the ion peak identified as the [M + H - 161' results from the cleavage of one of the several hydroxyl groups. Thus, the identification would be [M - OH + 2H]+. The exact hydroxyl group cleaved, however, is unknown. A reviewer suggested that these ions could also result from impurities containing one less hydroxyl group. An example of this contamination would be kaempferol, a tetrahydroxy species, present with quercetin, a pentahydroxide. Although the contamination theory is plausible in the model compounds, similar [M - OH + 2H]+ fragment ions were observed in the mass spectra of isoflavonoid glucosides obtained by positive ion HPLC/CF-LSIMS analyses of plant extracts (data not shown). This sup- ports the identification of these ion peaks as fragment ions as opposed to contaminants, since the HPLC/CF- LSIMS system separates/purifies the sample prior to MS analysis. It is suggested that ion peak identified as [AH, - 161' results from the cleavage of the glycosidic


~

Table 2. Summary of negative-ion CF-LSI mass spectra [m/z with relative intensity (%) in parentheses]

Compound

Rutin Naringin Esculin

Compound

Daidzein Medicarpin Medicarpin-3-0-glucoside (MG)

Medicarpin-3-glucoside-6"-rnalonate (MGM) Medicarpin-3-glucoside-6"-acetate (MGOAc)

Medicarpin-3-glucoside-6"-acetate (MGOAc) + 1 % HOAc acid modifier

+ 0.1 % TFA acid modifier

Model flavonoid glycosides [ M -HI- CFJ CAI- Others

609 (92) 463 (11) 301 (100) 610 (58); 593 (5); 285 (22)d 579 (100) 433 (6) 271 (78) 580 (50); 563 (6): 255 (19)d 339 (100) n/a a.b 299 (24), 265 (24)

MGM reference material [M - HI- [A]-

253 (1 00) nla 269 (100) n/a 431 (6) 269 (1 00)

517 (5) 473 (3)

269 (1 00) 269 (1 00)

473 (3) 269 (1 00)

Others

507 (5),* 345 (7),' 243 (18). 151 (23) 539 (2): 254 (29),w 239 (5), 121 (6), 73 (6) 701 (3)) 523 (3),i 491 (5),h 432 (7),

254 (27),' 121 (9), 91 (21). 73 (8) 473 (2); 254 (IS)? 121 (8), 103 (29) 743 (2)) 565 (5),i 533 (8),h, 474 (1 1 ), a 254 (30)" 587 (12),k 565 (2),' 474 (4); 301 (13), 187 (9), 91 (53), 73 (19)

Chickpea extract Compound [M - HI- CAI - Others

Peak 3 FGM 515 (19) 267 (100) 607 516 (2): 471 (11): 309 (8)

Peak 4 BG 445 (33) 283 (100) 537 (lo), 446 (23); 268 (45)? 252 (5)

Peak 6 BGM

Formononetin-7-glucoside-6"-malonate

Biochanin A-7-glucoside

Biochanin A7-glucoside-6"-malonate 252 (5) 531 (2) 283 (100) 532 (4),' 487 (3); 31 1 (28). 268 (1 3),

* Significant [MI-' observed. Low m/z limit abovernlz value of [F,]- =[A]-.

E[M-H-16]-=[M-OH]-..

' [2M - H]-. ' [M - CO,H]-. '[A -15]-.

I [M + Gly - HI-. ' [M +A]-. Ir [M + TFA - H]-. n/a = not applicable.

[A - 161- =[A - 01-.

[M + HOAc - H]-.

oxygen on the aglycone ring side instead of the sugar side. The above identification of the fragment ion peaks correlates with the extensive positive and negative ion DIP-FAB tandem mass spectra presented by Crow et dzo The mass spectrum of esculin also includes an ion peak corresponding to a protonated adduct of glycerol with the analyte, [M + Gly -t HI', at m/z 433. Further- more, a dimer ion, [2(esculIn) + HI', at m/z 681 was observed. Dimer ions were observed in the CF-LSI mass spectra of many other aglycones such as formononetin (data not shown) and daidzein (see Table 2). The presence of these additional ion peaks can help elu- cidate the molecular masses of unknown conjugates.

A study of CF-LSIMS reproducibility was performed using selected-ion monitoring (SIM). The SIM chromatogram obtained is presented in Fig. 3. The area of each SIM peak was integrated using a computer function available on the VG 11-25OJ data system. The relative standard deviation (RSD) of the peak areas was calculated to be 6.90%. The RSD is within the 10% suggested by the and indicates a high degree of reproducibility.

To evaluate the sensitivity of the CF-LSIMS technique, minimum detectable quantities (MDQ) were determined for rutin, naringin and esculin by positive ion CF-LSIMS and were found to be 200 ng (330 pmol),

200 ng (340 pmol) and 100 ng (290 pmol) for rutin, naringin and esculin, respectively. Upon comparison, the data show a fivefold decrease in the MDQs of rutin and naringin for the CF-LSIMS experiments over the DIP- LSIMS experiments, whereas the MDQ enhancement for esculin was a factor of 2.5. Although there is a significant decrease in the MDQ's of the CF-LSIMS technique, it should be noted that the DIP-LSIMS utilized an acidified thioglycerol matrix. This is in contrast to the less efficient acidified glycerol matrix used in the CF-LSIMS experiments.

Negative ion CF-LSIMS of model phenolic conjugates

Negative ion CF-LSIMS mass spectra of the model flavonoid glycosides were acquired using the optimized conditions described above and a representative mass spectrum of 2.0 pg of rutin is provided in Fig. 2(c). A summary of the negative ions observed for rutin, naringin and esculin is provided in Table 2. A series of characteristic negative ions similar to those observed in the positive ion mode were observed, consisting of [M - H]-, [M - H - 16]-, [FJ, [A]- and [A - 16]-. The [M - H - 161- is believed to be a negative radical ion resulting from the cleavage of a

480

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90

80

70

k .% 60

2 50. u > .- * j 40.

30.

20.

10. 1

L. W. SUMNER ET AL.

method is based on that presented by Edwards and K e ~ s m a n n . ~ ~ It should be noted that octadecylsilane (ODS) stationary phases with smaller pore sizes are necessary to yield adequate chromatographic resolution (i.e. 120 A Bakerbond or a 100 A Zorbax C, stationary phases as compared to a 300 8, pore size CI8 column). Gradient elutions were performed with an acid modifier consisting of either 1.0% acetic acid (pH 2.5) or 0.1% TFA (pH 2.4). These were substituted for the reported modifier, phosphoric acid, owing to their volatile nature. It was found that these two weak acids could be used alternatively without significantly altering the separation efficiency. The use of other inorganic strong acids such as HC1 was found to be detrimental to the sample even at a similar pH value owing to substantial hydrolysis of the conjugates.

0 1O:OO 2O:OO 30:OO 4O:OO

Time (minutes) -. - _ . . . . . I -. - -. . Figure 3. Selected-ion monitoring (SIM) chromatogram ot r n p 61 1, [M +HI+, generated by ten repetitive injections of 2.5 pg of rutin in the positive ion CF-LSlMS mode with a relative standard deviation of 6.9%.

hydroxyl group, [M - OH]-', similar to those ion peaks observed in positive ion LSI mass spectra. The [A - 16)- ion is believed to arise from the cleavage of the glycosidic oxygen on the aglycone ring side instead of the sugar side. Thus this ion would be labeled [A - 01-. Similar to the discussion presented earlier, the plausibility of an impurity must be considered, but the probability is unlikely based on similar fragmentation characteristics observed in mass spectra of phenolic conjugates purified by negative ion HPLC/CF-LSIMS.

A comparison of the mass spectra of rutin obtained by positive ion DIP-LSIMS [Fig. 2(a)] and negative ion CF-LSIMS [Fig. 2(c)] shows a dramatic difference. Although the amount of rutin used for the negative ion CF-LSIMS mass spectra was approximately ten times less than that used for the positive ion DIP-LSIMS mass spectrum, a significantly more intense relative [M - HI- ion peak (97%) was observed compared with the relative DIP-LSIMS [M +HI ' ion peak intensity (34%). The positive ion CF-LSIMS mass spectrum yielded a relative [M + HI' ion peak intensity of 45%. In addition, both naringin and esculin yielded [M - H] - ion peaks as the base peaks in negative ion CF-LSIMS. The increased relative molecular ion abundance resulted in the lower minimum detectable quantities observed in the negative ion CF-LSIMS experiments.

HPLC/CF-LSIMS

Chromatography

The literature reports several HPLC methods for the . . " " - % . a . . , - i c I 3 Q A n . . ~ - .... analysis of navonoias in piant extracts--'--' ' - anu uur

HPLC/CF-LSIMS of alfalfa

Initial developmental HPLC/CF-LSIMS experiments were performed using a hydrolyzed MGM reference mixture since purified malonated conjugates are not commercially available. Separate negative ion HPLC/ CF-LSIMS analyses were performed on the hydrolyzed crude MGM reference material using 0.1% TFA and 1.0% acetic acid modifiers. The UV chromatogram and the combined reconstructed ion chromatogram (RIC) of m/z 253 and 269 obtained for the crude MGM reference material using a 1.0% acetic acid modifier are presented in Fig. 4. The results were almost identical except that a different series of acid adducts were observed with both the analytes and the glycerol matrix. The mass spectra of the five major components are summarized in Table 2. The components have been identified as daidzein (4',7-dihydroxyflavone), MGM, medicarpin-3-0- glucoside (MG), medicarpin-3-O-glucoside-6-O-acetate (MGOAc) and medicarpin. Structures of these compounds are presented in Fig. 1.

The first component has been identified as daidzein (4,7 dihydroxyisoflavone) or possibly its isomer, 4,7 dihydroxyflavone, by both its retention time and its mass spectrum. Both daidzein and 4,7 dihydroxyisoflavone are present in alfalfa extracts. This component was present as an impurity in the initial MGM fraction isolated by preparative HPLC which was used to generate the crude sample. The mass spectrum of daidzein was characterized by a base peak at m/z 253 rationalized as the deprotonated aglycone, [M - H]-, a glycerol adduct with the molecular species ion peak, [M + Gly - H]-, and a daidzein dimer ion peak, [2M - H]-, at m/z 507.

The second component has been identified as medicarpin-3-0-glucoside (MG). The mass spectrum of MG was characterized by a large proportion of MG present as a radical molecular anion, [MG]-'. The presence of MG as a radical molecular anion is unique since the above optimization experiments did not yield radical molecular ions for the model flavonoid glycosides (rutin, naringin or esculin) more abundant than the protonated or deprotonated molecular species, although the negative ion mass spectrum of rutin [Fig. 2(c)] does contain a radical molecular ion peak (m/z 610) with an abundance greater than that predicted by

HPLC-CF-LSIMS OF FLAVONOID GLYCOSIDES 48 1

Time (miu:sec)

Niiniher 000 I 2 30 24 5 1 37 12 4933 7 1 S4 Time (minrec)

Figure 4. (a) On-line UV and (b) reconstructed ion (m/z 253 and 269) chromatograms of MGM reference material obtained by negative ion HPLC/CF- LSIMS.

isotopic ratios. In addition, radical molecular ions seem to be prevalent in flavonoid glycosides that lack acidic or phenolic protons. This trend is observed in both positive and negative ion modes for MG. The formation of radical anions of flavonoid glycosides in FAB mass spectra has also been discussed by Crow et al.” The identity of MG is further supported by the presence of ion peaks corresponding to adducts between the molecular species and both acetic acid (HOAc) and glycerol (Gly) which originated from the mobile phase, i.e. [M + HOAc - HI- and [M + Gly - H]-. Another feature of the MG mass spectrum is the presence of an adduct ion of MG with the deprotonated aglycone giving rise to an [MG + A]- ion peak. Since each peak is HPLC purified and the aglycone elutes at a much longer retention time (see Fig. 4), it is proposed that prompt fragmentation occurring during the particle desorption event generates a high abundance of the [A]- species, as verified by a base peak corresponding to [A]-. A portion of the aglycone anions then recom- bine with the molecular species in the solution and/or gas phase to generate the [MG + A]- anion similar to the [M + Gly - H] - and [M + HOAc - H] - ions. Also observed are ion peaks rationalized as the loss of methyl, [A - 151- = [A - CHJ at m/z 254 and methoxy groups, [A - 301 - = [A - OCH,] - at m/z 239, remembering that the A fragment is already proton deficient. This fragmentation is slightly different than

that observed for the hydroxy-substituted model compounds and it is interesting that the methyl and methoxy substituents are preferentially cleaved over the OH or 0 species (i.e. [M -OH]- and [A - 01-) encountered when only hydroxyl substituents are present.

The third major component has been identified as medicarpin-3-O-glucoside-6”-O-malonate (MGM). The deprotonated molecular ion, [M - H]-, was observed at m/z 517 whereas the aglycone fragment, [A]-, was observed as the base peak at m/z 269. The observed fragment ions at m/z 473 and 431 correspond to [M - CO,H]- and [M - CO,HCH,CO]- ions respec-

tively. Similarly, the fourth component of the mixture was identified as medicarpin-3-O-glucoside-6-acetate (MGOAc). The acetate species was mostly likely generated by a substitution or cleavage reaction involving the malonate group of MGM that occurred during the hydrolysis procedure using 10% acetic acid. The identification of MGOAc was based on the presence of a radical molecular anion peak at m/z 474 and multiple anion peaks identified as adducts formed from MGOAc with acetic acid (m/z 533), glycerol (m/z 565) and the medicarpin aglycone (m/z 743). These species are similar to those adducts observed in the mass spectrum of MG. One might suggest that the MGOAc radical molecular anion could arise from MG and an adduct with acetic acid or an acetic acid fragment ion originating from the


MFM

1 20

t I 1 1 I I 1 8 8 8 8

D 0 0 0 0 8 8 X

0 x n

TIME

P N

9 !2

I

10. m I

0 50 I on I50 200 250 300 Scan Number ..

0 46 1922 2858 18 34 48 10 5 7 % Tme (mm mc)

Figure 5. (a) On-line UV and (b) reconstructed ion (m/z 267 +269) chromatograms acquired for an alfalfa (cultivar Apollo) extract by negative ion HPLC/CF-LSIMS. Mass spectra of two major extractable components isolated from alfalfa: (c) scan No. 107 identified as FGM =formononetin-7-0-glucoside-6”-malonate; (d) scan No. 127 identified as M G M = medicarpin-3-O-glucoside-6-malonate.

acid modifier in the mobile phase, but this ambiguity can be eliminated by repeating the experiment with an alternative acid modifier such as 0.1% TFA. This was performed and the mass spectrum of MGOAc obtained with a 0.1% TFA acid modifier is summarized in Table 2. Again, a radical molecular anion of MGOAc was observed at m/z 474 and a similar series of adduct anions between MGOAc and glycerol and TFA were observed. The [MGOAc + Gly - HI- ion peak were observed at m/z 565 and while the [MGOAc + TFA - HI- ion peak was observed at m/z 587. This negates the postulate of an adduct ion formed between MG and acetic acid and verifies the identification of MGOAc. The identification of the other major components were verified in a similar manner. This

example illustrates the complementary mass spectral information that can be acquired simply by changing the acid modifier in the mobile phase. The fifth component of the crude MGM reference material has been identified as the medicarpin aglycone. The mass spectrum of medicarpin was characterized by an [M - H] - ion peak at m/z 269, a dimer ion peak, [2M - H]-, at m/z 539 and ion peaks rationalized as the loss of methane and methoxy groups, [M - CHJ at m/z 254 and [M - OCHJ at m/z 239.

The information gained from the HPLC/CF-LSIMS analysis of the crude MGM reference material provided more characteristic mass spectra of the flavonoid glucosides found in actual leguminous extracts than the model flavonoids. The knowledge gained from the

HPLC-CF-LSIMS OF FLAVONOID GLYCOSIDES

100.

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80.

70.

.$ 60.

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a. .c . - 2 40

3 0

483

[ A 26

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0 I00 200 300 400 500 700

WZ

Figure 5. (continued)

developmental studies were then applied to more complex extracts.

Negative ion HPLC/CF-LSIMS analysis was performed on a basic fraction of an alfalfa cell suspension extract (Medicago sativa L., cultivar Apollo). Our atten- tion focused on the peaks eluting at approximately 17-25 min since enzyme hydrolysis experiments (data not shown) with almond /?-glucosidase indicated this was the elution region of /?-glucosides. The recorded UV chromatogram and the reconstructed ion chromatogram generated by the summation of the ions at m/z 267 and 269 are provided in Fig. 5(a) and (b). Two major peaks were observed in both the UV and RIC chromatograms. In reverse order, the background- subtracted mass spectra of the second peak (scan No. 127) is presented in Fig. 5(d) and has been identified as MGM. This mass spectrum matches that of the MGM described above in the crude MGM reference material.

The first peak (scan No. 107) has been identified as formononetin-7-O-glucoside-6"-O-malonate (FGM) and its mass spectrum is presented in Fig. 5(c). The negative ion HPLC/CF-LSI mass spectrum of FGM yielded an [M - HI- ion peak at m/z 515 and an [A]- ion peak at mlz 267 as the base peak. An ion peak at m/z 471 corresponding to [M - CO,H] - was also observed, which we have shown to be characteristic of nialonated glycosides. Further, [A - 151 - and [A - 301 - anion peaks were observed, which we have shown to be characteristic fragment ions of methoxy-substituted aglycones.

The literature offers conflicting identification of the FGM component. Previously, this major component of alfalfa extracts (cultivar Calwest) had been identified as afrormosin-7-O-glucoside-6"-O-rnalonate3' (AGM M, = 546, see Fig. 1 for structure) while Maxwell and Phillips3 identified it as FGM using another cultivar of

484 L. W. SUMNER ET ,415.

alfalfa, Moapa 69. In addition to the above mass spectral data, the fact that formononetin is found in the metabolic pathway of medicarpin and MGM whereas afrormosin is not* further support the correct identification of the suspect component as FGM. Also, lesser components of the mixture have been identified as formononetin-7-0-glucoside (FG) and formononetin (data not shown), providing evidence of the presence of formononetin conjugates. The HPLC/CF-LSIMS identification of FGM has been confirmed by additional negative and positive ion HPLC-CF-LSIMS analyses of many different alfalfa cultivar extracts including the cultivar used to identify AGM (i.e. extracts of cell suspen- sions and field grown roots of alfalfa cultivars such as Calwest, Apollo, RegenSY and Cimarron).

The identification of AGM was based in part on the positive ion DIP-LSI mass spectrum obtained with a 1% TFA acidified thioglycerol matrix36 in which an [M + HI+ ion peak was reported at m/z 547. The authors also reported an ion peak at m/z 517. Our expe- rience has shown that MG has an elution time close to FGM (19-20 min). Thus, these two components could have been isolated as a mixture. We suggest that the ion peak at m/z 547 correlates with [MG + TFA + HI+ = 432 + 114 + 1 = 517 and that the ion at m/z 517 correlates with [FGM + HI'. These identifications are supported by our data which show a strong tendency for the generation of adduct ions between the analyte and acid modifier.

HPLC/CF-LSIMS of Chickpea (Cicer arietimm).

To further support our identification of FGM, negative ion HPLC/CF-LSIMS analyses were performed on chickpea extracts since previous reports identified FGM as a major extract component based on HPLC, UV, NMR and field desorption MS.39 HPLC/CF-LSIMS analyses were performed using a crude unhydrolyzed and a b-glucosidase-hydrolyzed (data not shown) chickpea extract utilizing on-line UV detection. The UV chromatogram and RIC of m/z 255, 267, 281, 283 and 297 are provided for the unhydrolyzed extract in Fig. 6.

Two intense peaks (peaks 3 and 6) appear in the UV/RIC chromatograms of the crude extract that undergo a shift in the hydrolyzed UV/RIC chromatogram. This indicates that these peaks correspond to /3- glucosides. The recorded background-subtracted HPLC/CF-LSIMS mass spectra are summarized in Table 2. Peak 3 produced a mass spectrum consistent with formononetin-7-O-glucoside-6"-malonate (FGM) and peak 6 was identified as biochanin A-7-0- glu~oside-6~~-rnalonate (BGM, see Fig. 2), the two major isoflavonoid conjugates previously identified in chickpea.39 The mass spectrum of FGM obtained for chickpea extracts is identical with that obtained for alfalfa,

Time (minutes)

6 I

Time (minutes)

Figure 6. (a) On-line UV and (b) reconstructed ion chromatograms (RIC of m/z 255, 267, 281, 283 and 297) acquired for a basic chickpea extract by negative ion HPLC/CF-LSIMS.

therefore further supporting the identification of FGM in alfalfa. FGM yielded an [M - HI- ion peak at m/z 515 whereas BGM yielded a radical molecular anion at m/z 532. Both components yielded a base peak corresponding to a proton-deficient aglycone, [A] -. The mass spectra of both FGM and BGM yielded a fragment ion corresponding to the loss of a carboxylate group, [M - CO,H]-. Also, both mass spectra produced ion peaks consistent with an adduct formed between glycerol and the molecular moiety, [M + Gly - H]-. A third component (peak 4) of this mixture has also been identified as biochanin A-7-0-glucoside (BG). The mass spectrum of BG, summarized in Table 2, yielded [BG + Gly - H]-, [BG - HI- and [Biol- ion peaks that formed the basis for its identification. The identification of BG and BGM in chickpea further exemplifies the utility of the HPLC/CF-LSIMS technique.

Acknowledgements

The Oklahoma State University Mass Spectrometry Facility was funded in part by NSF Instrumentation Grant BBS-8704089. Lloyd Sumner was supported in part through The Samuel Roberts Noble Foundation Plant Biology Collaborative Research Program.

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Documents

High-performance Liquid Chromatography/Continuous-flow Liquid Secondary Ion Mass Spectrometry of Flavonoid Glycosides in Leguminous Plant Extracts