Analytica Chimica Acta 450 (2001) 8197

Identication of isoavone conjugates in red clover (Trifolium pratense) by liquid chromatographymass spectrometry after two-dimensional solid-phase extraction
Boivoj Klejdus, Dagmar Vitamv sov -St rbov , Vlastimil Kub n r a a e a a
Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry in Brno, Zem d lsk 1, CZ 61300 Brno, Czech Republic e e a Received 30 May 2001; received in revised form 22 August 2001; accepted 27 August 2001

Abstract A high-performance liquid chromatographymass spectrometry (LCMS) combined with a two-dimensional solid-phase extraction (2D-SPE) enabled the identication of fourteen isoavone glycoside malonates (daidzin-6 -O-malonate, genistin-6 -O-malonate, orobol-7-O- -d-glucoside-6 -O-malonate, 3-methylorobol-7-O- -d-glucoside-6 -O-malonate, calycosin-7-O- -d-glucoside-6 -O-malonate, pratensein-7-O- -d-glucoside-6 -O-malonate, pseudobaptigenin-7-O- -d-gluformononetin-7-O- -d-glucoside-6 -O-malonate, irilone-4 -O- -d-glucoside-6 -O-malonate, coside-6 -O-malonate, afrormosin-7-O- -d-glucoside-6 -O-malonate, biochanin A-7-O- -d-glucoside-6 -O-malonate, texasin-7-O- -d-glucoside6 -O-malonate, 5,7,2 -trihydroxy-6-methoxyisoavone-7-O- -d-glucoside-6 -O-malonate, prunetin-4 -O- -d-glucoside6 -O-malonate) and six acetyl glycosides (daidzin-6 -O-acetate, formononetin-7-O- -d-glucoside-6 -O-acetate, pseudobaptigenin-7-O- -d-glucoside-6 -O-acetate, irilone-4 -O- -d-glucoside-6 -O-acetate, biochanin A-7-O- -dglucoside-6 -O-acetate and prunetin-4 -O- -d-glucoside-6 -O-acetate) isolated from red clover (Trifolium pratense). Eight isoavone glycoside malonates and six acetyl glycosides were rstly identied in red clover. The analyses of crude extracts with those using 2D-SPE extracts were compared. UV spectra, mass spectra of protonated molecules and their fragmentation and subsequent conversion to known avonoid glycosides were the basis for the identication of the substances. 2001 Elsevier Science B.V. All rights reserved.
Keywords: High-performance liquid chromatographymass spectrometry (LCMS); Solid-phase extraction (SPE); Trifolium pratense; Red clover; Isoavone glycoside 6 -O-malonates; Isoavone glycoside 6 -O-acetates

1. Introduction Isoavonoids belong to a large group of natural substances present in plants. More than 3000 avones and more than 700 known isoavones exist in plants [1]. Their structure is based on a 3-phenylbenzpyrone
Corresponding author. Tel.: +42-5-4513-3285; fax: +42-5-4521-2044. E-mail address: kuban@mendelu.cz (V. Kub n). a

(3-phenylchromone) group. The structure differs in the degree of methylation, hydroxylation and glycosylation. The synthesis of isoavones in plants is based on a carbon skeleton of an isoavonoid and on different oxidations of three central atoms [2]. The isoavones are mostly present in the -O- glucosidic form, the malonate or the methylmalonate hemi-esters forming the glucosidic part (Fig. 1). The isoavone C-glycosides are distinguished from the isoavone O-glycosides, as the name implies, by possessing a sugar which is

0003-2670/01/$ see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 1 ) 0 1 3 7 0 - 8

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Fig. 1. Structures of isoavones.

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carboncarbon linked via its anomeric (C-1) carbon to the C-6 and/or C-8 of the isoavone nucleus. Trifolium pratense L., leguminosae, well known as red, meadow, creeping or craw clover, is one of the most important forage plants. It has been used by the Oriental and the European cultures, and more recently also by the Americans, as a medicinal herb for the treatment of eczema and psoriasis. The isoavone constituents have estrogenic properties. They may play an important role in cancer prevention, moderation of menopausal symptoms, and other health effects [3,4]. A rather complex matrix complicates the identication of secondary metabolites, thus, a clean-up procedure is usually applied prior to the chromatographic separation. A solid-phase extraction is the most useful technique by which the analytes could be preferably isolated from complex matrices. Co-polymer sorbents on the basis of N-vinylpyrrolidone and divinylbenzene are suitable for extraction procedures in a wide range of pH values [5,6]. The unique properties could be used for so called two-dimensional solid-phase extractions (2D-SPE). A maximum selectivity of analyte(s) separation can be obtained by a precise adjustment of pH values according to the analyte type (acidic, basic or neutral character) and by an optimization of organic solvent concentration [7] in the washing and in the nal elution steps. The appropriate adjustment of experimental conditions in all steps allows also elimination of the most interferences from ballast substances. A combination of mass spectrometry (MS) or nuclear magnetic resonance (NMR) with liquid chromatography is a very useful on-line system for the determination and identication of secondary metabolites [811]. In the last period, a number of papers on the LCESI MS determination of glycoside malonates in crude plant extracts [1217] has appeared. Lin et al. [17] identied six isoavone glycoside malonates in crude extracts of owers and leaves of red clover. Barnes et al. [12] applied the atmospheric pressure chemical ionization (APCI) for their determination. The aim of this paper was to evaluate a 2D-SPE method on polymeric sorbents using an automated ASPEC XL system for the subsequent off-line identication of isoavone malonate glycoside conjugates in red clover by an LCESI MS system.

2. Experimental 2.1. Apparatus and chemicals An HP 1100 LC system (Hewlett Packard, Waldbronn, Germany) with a photo diode array detector set at 280 nm was coupled on-line to an HP MSD 1100 detector (Hewlett Packard, Palo Alto, CA, USA). UV spectra were registered in the range of 190400 nm (SBW 100 nm). A Zorbax Exlipse XDB C8 (150 mm 4.6 mm; 5 m, Zorbax, Hewlett Packard, Waldbronn, Germany) analytical column was used for the control of extraction efciencies and a MetaChem Polaris C18A (150 mm 2.0 mm; 3 m, MetaChem Technologies, Torrance, CA, USA) was used for the identication of the substances of interest due to its higher separation efciency and lower ow rate. The ESI MS spectra were acquired at a positive mode: gas temperature 300 C, drying gas ow 10.0 l/min, nebulizing gas 40 psi, capillary voltage 3.5 kV, scan 100800 m/z, fragmentor 70100 V (220240 V for CID). A semi-preparative LC system consisting of a binary pump Gilson 322, a UVVIS 156 detector and a 202 fraction collector controlled by a UniPoint software (Gilson, Villiers le Bel, France) using a MetaChem Polaris C18A semi-preparative column (250 mm 9.6 mm; 10 m, MetaChem Technologies, Torrance, CA, USA) was used for the purication and the isolation of the substances of interest. When necessary, mixed peak fractions were re-chromatographed on a Waters X-Terra MS (50 mm 7.8 mm; 5 m, Waters Co., Milford, MA, USA) column. The Waters X-Terra MS column was used for its higher separation efciency and an automatic fraction collector was used for preparative work. A mobile phase consisted of (A) 0.2% acetic acid (v/v) and (B) acetonitrile. A linear gradient elution from 1550% B (v/v) in 20 min, up to 55% B in the next 25 min and followed by a negative gradient up to 15% B in 30 min was used for the Zorbax Exlipse XDB C8 column. The ow rate was 0.8 ml/min and the temperature of the column oven was 40 C. A linear gradient from 15 up to 25% B in 36 min, up to 55% B in 90 min and followed by a negative gradient up to 15% B in 100 min was used for elution using the MetaChem Polaris C18A column. The ow rate was 0.3 ml/min (3 ml/min for semi-preparative columns) and the temperature of the column oven was 40 C. A

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linear gradient from 15 to 25% B (v/v) in 15 min, up to 55% B in the next 30 min and followed by a negative gradient up to 15% B in 40 min was used for elution using the Waters X-Terra MS column at the ow rate 3 ml/min and at the temperature of 40 C. A magnetic stirrer (IKAMAG RCT basic) was used for the stirred extraction at 30 C. A fex IKA Werke 50 extractor (a computer controlled, commercially available device related to a Soxhlet apparatus) was used for the extraction using a temperature program. A rotary vacuum evaporator (IKA RV 05-ST) with a water bath HB 4 (all from IKA -Werke GmbH and Co., Staufen, KG, Germany), a single ball cryogenic grinder Vibrom 2S (Jebav , Czech Repuby lic) and an ultrasonic bath (Cole-Parmer Instrument Company, Chicago, USA) were used for the sample preparation. An automated ASPEC XL system (Gilson, Villiers le Bel, France) was used for the SPE extractions in the sequential mode. 2.2. Plant material Trifolium pratense, Start variety, was collected in the Slechtitelsk Stanice Hladk Zivotice Ltd. in the a e second cut in the summer 1999. The plant material was dried at 60 C and immediately milled. The particles were sorted out through a ne-mesh screen (0.5 mm) just before the extraction. 2.3. Sample preparation Sonication, stirred extraction at 30 C and extraction using a temperature program and methanol and methanol/water 7:3 (v/v) as extraction solvent were used for the extraction of the substances from 0.5 g of the dried material. Sonication [17] was performed at the laboratory temperature for 60 min and used for comparison. The extract was ltered through a Teon disc lter (0.45 m pores and 13 mm diameter, Alltech Associates, Deereld, IL, USA). A magnetic stirrer was used for the stirred extraction at the temperature of 30 C for 60 min. A two-step extraction program (rst step: temperature of the cooling/heating block 130 C for 30 min, cooling of the cooling/heating block to 30 C for 5 min, second step: temperature of 120 C for 30 min, cooling to 30 C) was applied for the

extraction with a temperature program using the commercially available fex IKA Werke 50 extractor [18]. 2.4. Solid-phase extraction procedure The extractions were performed by loading 3 ml of a diluted sample solution (1 ml of a methanolic extract was diluted with 3 ml of water) onto pre-conditioned SPE cartridges (washed with 1 ml of methanol and equilibrated with 1 ml of water). Impurities were washed out with 1 ml of 5% methanol containing 2% acetic acid. Isoavone glycoside malonates and acetyl glycosides were eluted from the SPE cartridges (Oasis HLB, RP 105 and Abselut sorbents) with 1 ml of 60 and 80% methanol containing 2% ammonium hydroxide, respectively. All the operations were performed by the ASPEC XL automated system controlled by the 735 Sampler Software (Gilson, Villiers le Bel, France). All eluates were collected separately, evaporated to dryness in a rotary vacuum evaporator. The residues were dissolved in 500 l of the mobile phase and injected (10 l) directly into the LCESI MS system. 2.5. Isolation and purication of standards Standard solutions of isoavone conjugates were prepared by semi-preparative chromatography after the 2D-SPE. Usually, 10 g of homogenized material were extracted with aqueous methanol (methanol/water 7:3) using fex IKA Werke 50 extractor. The extraction agent was evaporated at the temperature of 40 C under vacuum. The residue was dissolved in 3 ml aqueous methanol (methanol/water 1:3) and puried by the 2D-SPE. Volumes of 1 ml of individual fractions collected after the 2D-SPE were injected on the semi-preparative MetaChem Polaris C18-A column. The fractions were collected automatically by the fraction collector Gilson 202 on the basis of integral parameters corresponding to two-third of peak heights. To separate any impurities, the selected fractions were again separated on the Waters X Terra MS C18 column, if any co-elution was conrmed. The purity and the identication of analytes were controlled by the LCESI MS at the experimental conditions given above. The combined fractions of the isolated analytes were evaporated to dryness under vacuum, dissolved in 500 ml of the mobile phase and injected into the LCESI MS system. The UV

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Fig. 2. LC UV and TIC chromatograms of standards of isoavones. For chromatographic conditions, see Sections 2.2, 2.3 and 3.2. Peak assignments are listed in Table 1.

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Table 1 Retention time (tR ) values, [M + H]+ , fragment ions, UV max , (MetaChem C18-A column, 150 mm 2.0 mm, 3 m) of the standard solutions of isoavones Peak no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 tR (min) 4.6 6.2 9.9 11.6 14.9 16.6 24.3 25.8 29.2 32.8 35.4 39.0 40.8 43.2 45.3 50.3 60.4 [M + H] (m/z) 417 447 433 463 445 431 461 285 447 447 473 271 301 283 269 299 285 Fragment ion (m/z) 255 285 271 301 283 269 299 285 285 269 max (nm) 250, 250, 260, 261, 249, 251, 270, 250, 260, 260, 250, 260, 262, 249, 249, 272, 261, 302 286 328 286, 338 292 300 339 290 326 324 298 328 284, 334 296 302 338 326 Compound Daidzin Calycosin-7-O- -d-glucoside Genistin Pratensein-7-O- -d-glucoside Pseudobaptigenin-7-O- -d-glucoside Ononin (formononetin-7-O- -d-glucoside) Irilone-4 -O- -d-glucoside Calycosin Trifoside (prunetin-4 -O- -d-glucoside) Sissotrin (biochanin A-7-O- -d-glucoside) Formononetin-7-O- -d-glucoside-6 -O-acetate Genistein Pratensein Pseudobaptigenin Formononetin Irilone Biochanin A

max and mass spectra of the standards (see Table 1 and Fig. 2) and literature data [2,16,17,1921] were used for the identication of the analytes.

3. Results and discussion 3.1. Extraction conditions Sonication, stirred extraction and extraction using the fex IKA Werke 50 extractor [18] were compared in terms of extraction efciencies of isoavone glycosides and glycoside malonates. The comparison of the extraction efciencies of these techniques (60 min extraction time was selected for all techniques) was based on comparison of peak areas of selected isoavone glycosides (see Table 2). It can be concluded from the given results that the fex IKA Werke 50 extraction gives the highest extraction yields for isoavone glycosides and glycoside malonates. Several authors [12,16,17] described the time-dependent conversion of glycoside malonates to isoavone conjugates under elevated temperature. Their observations are based on classical reux extraction procedures. The fex IKA Werke 50 extractor eliminates the exposure to the elevated

temperature by placing the sample outside the heating block [18] unlike the reux extraction. Approximately 0.5 g of a homogenized mixed sample of the plant material was used for the selection of an optimal solvent for the fex IKA Werke 50 extraction of the isoavone glycosides and the glycoside malonates. Methanol and methanol/water 9:1, 7:3, 5:5, 3:7 (v/v) mixtures were used for the extraction of the ground material. The extracts were injected directly onto the analytical column and the extraction efciencies were compared in the same way as before (see Table 3). The best results were obtained using methanol/water 7:3 (v/v) as the extraction agent. 3.2. 2D-SPE method Two factors are the most signicant for the analyte retention on a column in the reversed-phase chromatography (RP-HPLC) and of course in the 2D-SPE the organic solvent concentration and the pH value. The retention of an analyte decreases with increasing concentration of an organic solvent in a mobile phase as Bolliet and Poole [7] described for acetonitrile, methanol and isopropanol on poly-(styrene-divinylbenzene) sorbents.

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Table 2 Comparison of extraction (stirred, ultrasonic and fex IKA) techniques (methanol/water 7:3 v/v). Column Zorbax XDB C8 150 mm4.6 mm, 5 m Peak no. 1 2 3 4 5 7 8 9 10 11 12 13 14 15 16 17 18 19
a

tR (min)

[M + H]+ (m/z) 433 463 519 445 431 461 517 447 447 547 271 533 533 283 269 299 285 285

Fragment ion (m/z) 271 301 271 283 269 299 269 285 285 299 285 285

Compound

Extraction peak area (mAU s)a Stirred Ultrasonic 2770 3535 1690 2545 15000 6911 10627 5405 3047 4419 2497 5470 2211 1344 12827 2855 1083 4005 fex IKA 3558 4269 1877 3358 21080 9184 11730 7598 4186 4518 2830 5940 2273 1571 13500 3528 1637 3671

9.6 11.9 14.2 14.6 15.3 18.6 18.9 20.7 21.6 21.8 22.6 23.6 24.6 26.3 26.7 29.6 32.3 32.6

Genistin Pratensein-7-O- -d-glucoside Gemosteom-7-O- -d-glucoside-6 -O-malonate Pseudobaptigenin-7-O- -d-glucoside Ononin (formononetine-7-O- -d-glucoside) Irilone-4 -O- -d-glucoside Formononetin-7-O- -d-glucoside-6 -O-malonate Trifoside (prunetin-4 -O- -d-glucoside) Sissotrin (biochanin A-7-O- -d-glucoside) Irilon-4 -O- -d-glucoside-6 -O-malonate Genistein Prunetin-4 -O- -d-glucoside-6 -O-malonate Biochanin A-7-O- -d-glucoside-6 -O-malonate Pseudobaptigenin Formononetin Irilone Prunetin Biochanin A

2769 3531 1760 2598 15018 7001 10987 5371 3099 4700 2625 6277 2350 1393 13917 3034 1052 4300

The best efciencies are given in bold. RSDs intervals: (n = 3) 4.35.8.

Table 3 The inuence of methanol content on isoavone extraction using fex IKA Werke 50 extractor (Zorbax XDB C8 column, 150 mm4.6 mm, 5 m) Peak no. tR (min) [M + H]+ (m/z) Fragment ion (m/z) Identication Peak area (mAU s) methanol/water (v/v) 3:7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 5.3 6.5 9.6 11.9 14.2 14.6 15.3 18.6 18.9 20.7 21.6 21.8 22.6 23.6 24.6 26.3 26.7 29.6 32.3 32.6 417 447 433 463 519 445 431 461 517 447 447 547 271 533 533 283 269 299 285 285 255 285 271 301 271 283 269 299 269 285 285 299 285 285 Daidzin Calycosin-7-O- -d-glucoside Genistin Pratensein-7-O- -d-glucoside Genistein-7-O- -d-glucoside-6 -O-malonate Pseudobaptigenin-7-O- -d-glucoside Ononin (formononetin-7-O- -d-glucoside) Irilone-4 -O- -d-glucoside Formononetin-7-O- -d-glucoside-6 -O-malonate Trifoside (prunetin-4 -O- -d-glucoside) Sissotrin (biochanin A-7-O- -d-glucoside) Irilone-4 -O- -d-glucoside-6 -O-malonate Genistein Prunetin-4 -O- -d-glucoside-6 -O-malonate Biochanin A-7-O- -d-glucoside-6 -O-malonate Pseudobaptigenin Formononetin Irilone Prunetin Biochanin A 1141 923 3348 1156 1062 1202 6333 2138 5532 1684 2907 2536 590 1739 1238 571 3905 058 321 1000 5:5 1233 966 3740 1255 1163 1353 7569 3415 6002 2298 3293 2861 557 2662 1370 314 3015 1114 380 570 7:3 1198 914 3512 1179 1095 1402 7830 3620 6352 2460 3284 2852 773 2716 1497 758 4964 2054 620 1980 9:1 1016 827 3015 1126 921 1271 7472 2989 5898 2332 2983 2677 549 2554 1318 533 4059 1771 530 1417

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Another factor is pH [6]. Retention of an analyte depends on the acidic/basic character of a substance. At a lower pH, acidic analytes are present in a non-ionized form and they exhibit higher retention on reversed-phase columns. At a higher pH, the analytes are in an ionized form and they exhibit lower retention. The retention of basic analytes is reverse to the acidic analytes. Divinylbenzene-based polymeric sorbents exhibit excellent stability over the whole pH range from 0 to 14 unlike classical modied silica gel sorbents C18 and C8. Thus, the applicability of silica gel sorbents for the 2D-SPE is rather limited. Strategy of a 2D-SPE method [22,23] is based on the sample adjustment and mainly on the selection of experimental conditions in individual washing and elution steps. The maximum separation selectivity of analyte(s) can be obtained by proper selection of pH and organic solvent concentration. The SPE study was performed to optimize the isolation of the substances. Alcoholic extracts (1 ml typically) diluted with 3 ml of water were applied for the SPE extractions. Ten cartridges of Oasis HLB 3 cm3 /60 mg (Waters, Milford, USA), RP 105 3 cm3 /200 mg (Applied Separations, Allentown, USA) and Abselut nexus 3 cm3 /60 mg (Varian, Harbor City, CA, USA) were used. The sorbents were conditioned with 1 ml of methanol and equilibrated with 1 ml of water. In our case, both parameters were combined to optimize the separation selectivity of the analytes of interest. Usually, 3 ml of a sample were loaded onto an initialized and equilibrated column. Interferents were washed with 1 ml 5% methanol followed by 1 ml 30% methanol containing 2% acetic acid. The analytes of interest were eluted with methanol/ water mixture with the increasing methanol content and higher pH values (090% methanol with 10% increments) containing 2% ammonium hydroxide. The content of an organic solvent has to be optimized for both washing and elution steps. The elution of analytes was not observed for methanol up to 40% containing 2% acetic acid, thus, 35% methanol containing 2% acetic acid was used for washing HLB, RP105 and Abselut cartridges. A mixture of methanol/water containing 2% ammonium hydroxide was applied in each step of the elution studies. Analytes were eluted over 60% methanol containing 2% ammonium hydroxide. The mixture

of 60% methanol containing 2% ammonium hydroxide was used for the elution of isoavone glycoside malonates while 80% methanol containing 2% ammonium hydroxide was used for the elution of isoavone glycosides from HLB, RP105 and Abselut cartridges. Complete elution of all analytes was obtained for the solvents containing more than 80% methanol and 2% ammonium hydroxide. The results of the elution studies for the isoavone glycoside malonates and the isoavone glycosides are presented in Fig. 3. 3.3. LCMS study LC UV and total ion chromatograms of the crude extract (Fig. 4) and retention times (tR ), absorption spectra (wavelength of absorption maximum in UV max ) and molecular masses of the substances in individual peaks are presented in Table 4. A total of 34 avonoid substances were identied in the crude extracts. Hyperosid, isoquercitrin, tetrahydroxyavones, 3-methyl-quercetin and their glycosides were identied using the UV max , the [M + H]+ and the published characteristics [19]. The isoavones and their conjugates were identied by comparing their retention times with the retention times tR , the wavelengths of absorption maximum in UV max and the [M + H]+ parameters of the separated compounds. A serious overlapping of peaks occurred in many cases, thus, the identication of the avonoid substances was complicated or even impossible. Interpretation of UV spectra was not adequate in many cases despite the fact that the spectra of avones and isoavones differ seriously [23]. More precise identication of the co-eluting compounds was done on the basis of their mass spectra and mainly using an extract ion system and peak purity procedure for mass spectra in the overlapping peaks. Identication of glycetin, calycosin-7-O- -d-glucoside, genistin, daidzein-7-O- -d-glucoside-6 -O-malonate, 3-methylorobol-7-O- -d-glucoside, pratensein7-O- -d-glucoside, calycosin-7-O- -d-glucoside-6 O-malonate and pseudobaptigenin was complicated by the presence of the avones eluted in the front part of chromatograms of the crude extracts. The 2D-SPE was applied to simplify the sample matrix and to separate the avones from the sample. The LC UV and the total ion chromatograms (Figs. 5 and 6) conrmed the separation efciency of the 2D-SPE

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Fig. 3. Elution study of isoavone glucoside malonates and isoavone glucosides with Abselut (A) and Oasis HLB (B) SPE extraction cartridges. Curves description: (1) daidzein-7-O- -d-glucoside-6 -O-malonate; (2) genistien-7-O- -d-glucoside-6 -O-malonate; (3) pseudobaptigenin-7-O- -d-glucoside-6 -O-malonate; (4) formononetin-7-O- -d-glucoside-6 -O-malonate; (5) irilone-4 -O- -d-glucoside6 -O-malonate; (6) prunetin-4 -O- -d-glucoside-6 -O-malonate; (7) biochanin A-7-O- -d-glucoside-6 -O-malonate; (8) daidzein-7-O-d-glucoside; (9) genistien-7-O- -d-glucoside; (10) pseudobaptigenin-7-O- -d-glucoside; (11) formononetin-7-O- -d-glucoside; (12) irilone-4 -O- -d-glucoside; (13) prunetin-4 -O- -d-glucoside; (14) biochanin A-7-O- -d-glucoside.

procedures in terms of the purity of the samples. The simplication of the sample matrix allowed much better identication of isoavones and their glycosides, malonate and acetyl glycosides using the 2D-SPE. Total separation of interfering hyperoside, isoquercitrin,

tetrahydroxyavones, 3-methylquercetin and their glycosides was obtained using washing with 35% methanol containing 2% acetic acid. After the separation step the following isoavones (see Table 5) were identied on the basis of their mass and UV spectra.

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Fig. 4. LC UV and TIC chromatograms of a crude extract of T. pratense (methanol/water 7:3 v/v). For chromatographic conditions, see Sections 2.2, 2.3 and 3.2. Peak assignments are listed in Table 4.

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Fig. 5. LC UV and TIC chromatograms of isoavones after 2D-SPE extraction (60% methanol with 2% ammonium hydroxide) for the Abselut cartridges. For chromatographic conditions, see Sections 2.2, 2.3 and 3.2. Peak assignments are listed in Table 5.

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Fig. 6. LC UV and TIC chromatograms of isoavones after dual SPE extraction (80% methanol with 2% ammonium hydroxide) for Abselut cartridges. For chromatographic conditions, see Sections 2.2, 2.3 and 3.2. Peak assignments are listed in Table 5.

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Table 4 Compounds identied in a crude extract of red clover (methanol/water 7:3 v/v) and their spectral characteristics, MetaChem Polaris C18-A analytical column, 150 mm 2.0 mm, 3 m Peak no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 tR (min) 4.6 9.5 12.6 12.8 13.0 16.6 17.5 18.6 18.9 19.0 20.1 20.4 22.7 24.3 24.6 26.3 29.2 32.1 32.8 33.0 35.4 36.0 36.2 36.4 39.6 40.8 41.3 43.2 45.3 49.5 50.1 50.3 57.0 60.4 [M + H]+ (m/z) 417 465 533 551 551 431 535 519 535 565 535 549 517 461 531 517 447 547 447 547 473 533 533 549 533 301 533 283 269 285 489 299 285 285 Fragment ion (m/z) 255 303 285 303 303 269 287 271 287 317 287 301 269 299 283 269 285 299 285 299 269 285 285 301 285 285 max (nm) 250, 256, 259, 255, 255, 251, 266, 260, 265, 264, 266, 261, 250, 270, 249, 250, 260, 271, 260, 270, 250, 260, 260, 262, 260, 262, 260, 249, 249, 260, 285 272, 261, 261, 302 355 286 354 355 300 348 328 350 352 348 286, 326 298 339 293 298 326 340 324 337 298 326 326 287, 320 326 284, 334 325 296 302 330 338 327 326 Compound identied Daidzin Hyperoside Calycosin-7-O- -d-glucoside-6 -O-malonate Hyperoside 6 -O-malonate Isoquercitrin 6 -O-malonate Ononin (forrnononetin-7-O- -d-glucoside) Unknown tetrahydroxyavone malonate Genistein-7-O- -d-glucoside-6 -O-malonate Unknown tetrahydroxyavone malonate 3-Methylquercetin 7-O- -d-glucoside-6 -O-malonate Unknown tetrahydroxyavone malonate Pratensein-7-O- -d-glucoside-6 -O-malonate Formononetin-7-O- -d-glucoside-6 -O-malonate-isomer Irilone-4 -O- -d-glucoside Pseudobaptigenin-7-O- -d-glucoside-6 -O-malonate Formononetin-7-O- -d-glucoside-6 -O-malonate Trifoside (prunetin-4 -O- -d-glucoside) Irilone-4 -O- -d-glucoside-6 -O-malonate Sissotrin (biochanin A-7-O- -d-glucoside) Afrormosin-7-O- -d-glucoside-6 -O-malonate Formononetin-7-O- -d-glucoside-6 -O-acetate Texasin-7-O- -d-glucoside-6 -O-malonate Unknown malonate (isomer) Irilin B-5,7,2 -trihydroxy-6-methoxyisoavon-7-O-d-glucoside-6 -O-malonate Biochanin A-7-O- -d-glucoside-6 -O-malonate Pratensein Prunetin-4 -O- -d-glucoside-6 -O-malonate Pseudobaptigenin Formononetin Texasin Biochanin A-7-O- -d-glucoside-6 -O-acetate Irilone Prunetin Biochanin A

The avonoid glycoside malonates and acetates showed the same UV spectra as their related glycosides. That means the malonyl group is not bounded to any functional group at the aglycon part [21]. The facts also conrm the malonyl (acetyl) group is located only on glycosidic part of the molecule, especially in its C6-position as 6 -O-malonylglucoside or 6 -O-acetylglucoside. The difference of 248 amu exists among the malonate glycoside and the mass ions of the parent compounds. The difference of 204 amu exists for acetate glycosides. The value of 248 amu for the malonate glycosides is 86 amu larger than that

of the glucosidic group (162 amu) of the glycoside and value 204 amu for acetate glycosides is 42 amu larger than that of the glucosidic group (162 amu). The difference of 86 amu corresponds to the malonyl group and the value of 42 corresponds to the acetyl group. The above facts led to the conclusion that the malonyl or acetyl group should be attached only to the glycosidic part of the molecule. Related glycosides can be obtained by liberation of malonate according the Lin et al. [16,17] method. The presence of glucosidic group could be veried by specic enzymatic reaction with -glucosidase [2,13]. The observations

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Table 5 Compounds identied in 2D-SPE red clover extracts and their spectral characteristics (methanol/water 7:3 v/v), ABS, HLB and RP-105 SPE columns, MetaChem Polaris C18-A analytical column, 150 mm 2.0 mm, 3 m Peak no. tR 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 4.6 5.3 6.2 9.9 10.2 11.1 11.6 12.6 14.9 15.7 16.6 18.6 18.7 19.5 20.4 22.7 23.1 23.7 24.3 24.6 24.7 25.4 25.8 26.3 26.6 27.8 29.2 29.5 [M + H]+ Fragment ion (m/z) 417 447 447 433 503 463 463 533 445 459 431 519 535 549 549 517 255 517 461 531 285 287 285 517 461 473 447 463 255 285 285 271 255 301 301 285 283 255 269 271 287 301 301 269 269 299 283 max (nm) 250, 258, 250, 260, 250, 264, 261, 259, 249, 251, 251, 260, 260, 264, 261, 250, 250, 250, 270, 249, 258, 261, 250, 250, 302 286 286 328 300 330 286, 286 292 301 300 328 288, 332 286, 298 302 298 339 293 288 288, 290 298 Compound identied Daidzin Glycetin-7-O- -d-glucoside Calycosin-7-O- -d-glucoside Genistin Daidzien-7-O- -d-glueoside-6 -O-malonate 3-Methylorobol-7-O- -d-glucoside Pratensein-7-O- -d-glucoside Calycosin-7-O- -d-glucoside-6 -O-malonate Pseudobaptigenin-7-O- -d-glucoside Daidzein-7-O- -d-glueoside-6 -O-acetate Ononin formononetin-7-O- -d-glucoside Genistein-7-O- -d-glucoside-6 -O-malonate Orobol-7-O- -d-glucoside-6 -O-malonate 3-Methyloronol-7-O- -d-glucoside-6 -O-malonate Pratensein-7-O- -d-glucoside-6 -O-malonate Isomer (formononetin-7-O- -d-glucoside-6 -O-malonate) Daidzein Isomer (formononetin-7-O- -d-glucoside-6 -O-malonate) Irilone-4 -O- -d-glucoside Pseudobaptigenin-7-O- -d-glueoside-6 -O-malonate Glycitein Orobol Calycosin Formononetin-7-O- -d-glucoside-6 -O-malonate Afrormosin-7-O- -d-glucoside Isomer (formononetin-7-O- -d-glucoside-6 -O-acetate) Trifoside (prunetin-4 -O- -d-glucoside) Irilin B-5,7,2 -trihydroxy-6-methoxyisoavon7-O- -d-glucoside Irilone-4 -O- -d-glucoside-6 -O-malonate Sissotrin (biochanin A-7-O- -d-glucoside) Afrormosin-7-O- -d-glucoside-6 -O-malonate Pseudobaptigenin7-O- -d-glucoside-6 -O-acetate Formononetin-7-O- -d-glucoside-6 -acetate Texasin-7-O- -d-glucoside-6 -O-malonate Irilin B-5,7,2 -trihydroxy-6-methoxyisoavon7-O- -d-glucoside-6 -O-malonate 3-Methylorobol Genistein Biochanin-7-O- -d-glucoside-6 -O-malonate Pratensein Prunetin-4 -O- -d-glucoside-6 -O-maIonate Pseudobaptigenin Irilone-4 -O- -d-glucoside-6 -O-acetate Formononetin Prunetin-4 -O- -d-glucoside-6 -O-acetate Texasin Biochanin A-7-O- -d-glucoside 6 -O-acetate Irilone Prunetin Biochanin A ABS HLB RP 105 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

337

340 338

340

269 299 269 285 301

260, 326 262, 288, 320

29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

32.1 32.8 33.0 33.2 35.4 36.0 36.4 36.6 39.0 39.6 40.8 41.3 43.2 43.5 45.3 49.3 49.5 50.1 50.3 57.0 60.4

547 447 547 487 473 533 549 301 271 533 301 533 283 503 269 489 285 489 299 285 285

299 285 299 283 269 285 301

271, 260, 270, 262, 250, 260, 262, 264, 260, 260, 262, 260, 249, 249, 260, 261, 260, 272, 261, 261,

340 324 337 288, 340 298 326 287, 320 334 330 326 284, 334 325 296 302 327 325 325 338 327 326

285 285 299 285 285

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are conrmed by the fact the avonoid glycoside malonates from plants have the 6 -O-malonylglucosyl unit [2]. A higher fragmentation voltage (220 and 240 V in some cases) on a skimmer (CID) was used to increase the fragmentation of the original protonated molecules of the analytes (Table 6). The information on the fragments improved the data on the structures of some isoavones due to more precise localization of substituents on A and B rings. The identied isoavonoids can be divided into eight groups depending on the molecular mass of their aglycones (MW 254, 268, 270, 282, 284, 286, 298 and 300). The isoavones of Trifolium pratense can be classied into ve sub-groups depending on the fragments of the A ring substitution: 136, 152, 166, 180 and 182. The compounds substituted on C7 by OH group giving RDA fragment 136 belong to the rst sub-group. The fragmentation was easy at the lower fragmentation voltage of 180 V when C4 atom on the B ring was substituted by OH group for daidzein or by OCH3 group for formononetin. The fragmentation voltage of 240 V on the skimmer was applied for the fragmentation of calycosin with OH and OCH3 substituents on C3 and C4 carbon atoms of the B ring and for the fragmentation of pseudobaptigenin with methylendioxy-group on C3 , C4 atoms. The fragmentation of the A ring was about 82% for calycosin, but only 17% for pseudobaptigenin. Methylendioxy-group on C3 and C4 carbon atoms of the B ring probably reduces the fragmentation efciency on the A ring. Dihydroxy-substituted compounds on C7, C5 and C7, C6 on the A ring belong to the second sub-group. An RDA fragmentation on the A ring was 62% for biochanin A, 50% for texasin and 54% for pratensein at 180 V. The fragmentation was 83% for orobol (7,5,3 ,4 -tetrahydroxyisoavon) and 28% for 3 -methylorobol (7,5,4 -trihydroxy-3 -methoxyisoavon) when the fragmentation voltage was increased up to 240 V. The compounds substituted on C7 carbon atom by OH group and on C6 (or C5) atom by OCH3 group form the third sub-group. A C7 hydroxy-, C6 methoxy- (glycitein, afrormosin) substitution on the A ring strongly reduced the fragmentation. The fragmentation of the A ring was 24% for glycitein

and 14% for afrormosin at 240 V on the skimmer while the fragmentation of the B ring was 41% for afrormosin. Irilone (7,6-methylendioxy-5,4 -dihydroxy-isoavon) forms the fourth sub-group. The substitution of 7,6-methylendioxy-5-hydroxy on the A ring strongly reduced the fragmentation and also the formation of 180 ion. The fragmentation on the A ring was 14% for the higher fragmentation voltage of 240 V. The compound of a molecular mass 300 belongs to the fth sub-group. An RDA fragmentation producing fragments [M-H2 O]+ and [M-43]+ and the absence of fragments the A are typical for 5,7-dihydroxy-6-methoxy group on the A ring. The presence of hydroxy-group located on 2 position on the B ring is highly probable on the basis of the information. The presence of 5,7,2 -trihydroxy-6-methoxyisoavone [19] is the most probable.

4. Conclusions A lot of studies used the crude extracts for the fast identication of secondary metabolites [1217]. Lin et al. [17] identied six isoavone glycoside malonates in crude extracts. The number of identied substances in crude extracts is often reduced due to the co-elution of two or more compounds. Purication procedures are necessary for more detailed studies due to interferences of other substances. The solid-phase extraction is the most useful extraction procedure [23,24]. The advantages of the 2D-SPE for the clean-up of a sample, mainly for more precise identication of isoavones, are presented in this paper. The presence of some of glycoside malonates and acetyl glycosides for different leguminoses [12,25] has already been described, but the presence of the others in Trifolium pratense has been not published until now. Additionally, the existence of six acetyl glycoside isoavones (daidzin-6 -O-acetate, formononetin-7-O- -d-glucoside-6 -O-acetate, pseudobaptigenin-7-O- -d-glucoside-6 - O - acetate, irilone -4-O- -d-glucoside-6 -O-acetate, biochanin A7-O- -d-glucoside-6 -O-acetate and prunetin-4-O- d-glucoside-6 -O-acetate) and of eight 6 -O-malonates (daidzin-6 -O-malonate, orobol-7-O- -d-gluco-

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side-6 -O-malonate, 3-methylorobol-7-O- -d-glucoside-6 -O-malonate, calycosin-7-O- -d-glucosidepseudobaptigenin-7-O- -d-gluco6 -O-malonate, side-6 -O-malonate, afrormosin-7-O- -d-glucoside6 -O-malonate, texasin-7-O- -d-glucoside-6 -O-malonate, 5,7, 2 - trihydroxy - 6-methoxyisoavone-7-O-d-glucoside-6 -O-malonate) in Trifolium pratense was found and conrmed for the given time by the LCMS. The combination of the 2D-SPE and the LCMS techniques improves the procedures and allows more precise and exact identication of secondary metabolites in the fundamental and applied research due to the reduced interferences and co-elution of other compounds. The application of the 2D-SPE leads to the more simple chromatograms, but the sensitivity is sacriced. Acknowledgements The nancial support from the Grant Agency of the Czech Republic (GA CR, 521/99/0863), and from the Ministry of Education, Youth and Sports of the Czech Republic (MSMT CR, MSM 432100001) is gratefully acknowledged. References
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[5] E.S.P. Bouvier, D.M. Martin, P.C. Iraneta, M. Capparella, Y.F. Cheng, D.J. Phillips, LCGC 15 (1997) 152. [6] U.D. Neue, Am. Lab. 29 (1997) 334. [7] D. Bolliet, C.F. Poole, Chromatographia 46 (1997) 381. [8] K. Hostettman, J.L. Wolfender, S. Rodriguez, Planta Med. 63 (1997) 2. [9] M.P. Balogh, LCGC 15 (1997) 456. [10] J.L. Wolfender, S. Rodriguez, K. Hostettmann, W. WagnerRedeker, J. Mass Spectrom. 30 (1995) S35. [11] X.-G. He, L.-Z. Lin, L.-Z. Lian, J. Chromatogr. A 755 (1996) 127. [12] S. Barnes, M. Kirk, L. Coward, J. Agric. Food Chem. 42 (1994) 2466. [13] M. Stobiecki, C. Malosse, L. Kerhoas, P. Wojtaszek, J. Einhorn. Phytochem. Anal. 10 (1999) 198. [14] L.W. Summer, N.L. Paiva, R.A. Dixon, P.W. Geno, J. Mass Spectrom. 31 (1996) 472. [15] S. Barnes, L. Coward, M. Kirk, J. Sfakianos, Proc. Soc. Exp. Biol. Med. 217 (1998) 254. [16] L.-Z. Lin, X.-G. He, M. Lindenmaier, G. Nolan, J. Yang, M. Cleary, S.-X. Qiu, G.A. Cordell, J. Chromatogr. A 876 (2000) 87. [17] L.-Z. Lin, X.-G. He, M. Lindenmaier, J. Yang, M. Cleary, S.X. Qiu, G.A. Cordell, J. Agric. Food Chem. 48 (2000) 354. [18] J. Redeker, Patent Application, DEP 1961, 12037.3, DEG 8411 672. [19] F. Hanawa, S. Tahara, J. Mizutani, Phytochemistry 1 (1991) 157. [20] A.K. Jain, V.K. Saxena, Natl. Acad. Sci. Lett. (India) 9 (1986) 379. [21] T.J. Mabry, K.R. Markham, M.B. Thomas, The ultraviolet spectra of avones and avonols: the ultraviolet spectra of isoavones, avanones, and dihydroavones, in: A.Y. Mabry, K.R. Markham, M.B. Thomas, (Eds.), The Systematic Identication of Flavonoids, Springer-Verlag, New York, 1970 (Chapters V and VI). [22] Y.-F. Cheng, U.D. Neue, L. Bean, J. Chromatogr. A 828 (1998) 273. [23] Y.-F. Cheng, D.J. Phillips, U.D. Neue, L.L. Bean, J. Liq. Chromatogr. 20 (1997) 2461. [24] B. Klejdus, D. Vitamv sov , V. Kub , J. Chromatogr. A 839 a a a (1999) 261. [25] S. Kodou, Y. Fleury, D. Welti, D. Magnolato, T. Uchida, K. Kitamura, K. Okubo, Agric. Biol. Chem. 55 (1991) 2227.