Characterisation of flavonols in broccoli ( Brassica oleracea L. var. italica) by liquid chromatography–UV diode-array detection–electrospray ionisation mass spectrometry

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Characterisation of flavonols in broccoli ( Brassica oleracea L. var. italica) by liquid chromatography–UV diode-array detection–electrospray ionisation mass spectrometry

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  Journal of Chromatography A, 1054 (2004) 181–193 Characterisation of flavonols in broccoli(  Brassica oleracea  L. var.  italica ) by liquidchromatography–UV diode-arraydetection–electrospray ionisationmass spectrometry F. Vallejo, F.A. Tomás-Barberán, F. Ferreres ∗  Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology,CEBAS-CSIC, P.O. BOX 164, E-30100 Espinardo, Murcia, Spain Available online 28 July 2004 Abstract The flavonoid composition of broccoli inflorescences has been studied by LC/UV-DAD/ESI-MS n  . A large number of hydroxycinnamicacid esters of kaempferol and quercetin glucosides has been characterised. The structures of the flavonoid glycosides were analysed afteralkalinehydrolysis,andwereidentifiedas3-sophoroside/sophorotrioside-7-glucoside/sophorosideofkaempferol,quercetinandisorhamnetin(this last found in trace amount). These complex quercetin and isorhamnetin glucosides have not been previously characterised in nature. Inaddition, several less complex glucosides based on the same aglycones have been identified. The effect of sugar substitution and acylation onchromatographic mobility and ESI ionisation and fragmentation are discussed.© 2004 Elsevier B.V. All rights reserved. Keywords:  Food analysis; Broccoli; Flavonoids; Acylated glycosides 1. Introduction Recent studies have shown the relevant biological activ-ities of flavonoids against various types of cancer and car-diovascular diseases [1–3]. Broccoli inflorescence is a goodsource of health promoting compounds since it contains glu-cosinolates, flavonoids and hydroxycinnamic acids, vitaminC and other minor compounds.Scarce information is available regarding broccolipolyphenols identification. Several studies have previouslyreported on broccoli flavonoids but none has describedmore than five flavonol glycosides and also without iden-tifying the combinations with hydroxycinnamic acids andwith glycosylations up to two glucoses [5,6]. A recent study on cauliflower flavonoids showed that Brassicaceae speciescontain very complex flavonoids with up to five sugarresidues on the flavonol nucleus [7]. Although broccoli has ∗ Corresponding author. Tel.: + 34 9683 963 24; fax: + 34 9683 962 13.  E-mail address:  federico@cebas.csic.es (F. Ferreres). been reported to be one of the main flavonol sources in thediet, very little is known about the structures, as they arecombination of hydroxycinnamic acids with highly glycosy-lated flavonoids, that make their identification very difficultby classical methods [4]. HPLC coupled to MS–MS–DAD has shown to be a very useful method in the identificationof these complex flavonoids, as have been recently reportedin cauliflower leaves [7].The purpose of this work was to identify the polyphenolsfrom broccoli inflorescences by high-performance liquidchromatogram coupled with on-line mass spectrometry withelectrospray ionisation source (LC/UV-DAD/ESI-MS n ). 2. Experimental 2.1. Plant material The broccoli (  Brassica oleracea  L. var.  italica ) (Marathoncv.) florets used for the assays were obtained from the In-stituto Murciano de Investigacion y Desarrollo Agraroali-mentario (IMIDA, Murcia, Spain). At optimum commercial 0021-9673/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.chroma.2004.05.045  182  F. Vallejo et al./J. Chromatogr. A 1054 (2004) 181–193 maturity, uniform size plants, free from insect and/or me-chanical damage, were selected at random and immediatelytransported to the laboratory where the edible portions werecut. Samples of 20g from each plant per replicate were com-bined, weighed, frozen at − 70 ◦ C and freeze-dried. This tis-sue was ground into a fine powder and stored at − 20 ◦ C forfurther analysis. 2.2. Phenolic extraction It was carried out according to methodology previouslydescribed for cauliflower [7]. Thus, freeze-dried samples(70g) were extracted by boiling with 3L of distilled waterfor 60min. This aqueous extract was then mixed with Am-berlite XAD-2 particles (Supelco, Hellefonte, PA) in suffi-cient amount to fill a column of 55cm × 4cm and stirred for4h at room temperature to retain the phenolic compoundson the surface of the non-ionic Amberlite particles [8]. TheAmberlite particles were packed into a chromatography col-umn, washed with distilled water (5L), and the absorbedphenolics eluted with methanol (1L). The methanol extractwas then taken to dryness and redissolved in methanol/water(1:1, v/v) and the phenolic fraction purified in a SephadexLH-20 column (40cm × 3cm). The phenolic fraction wasfollowed under UV light (254 and 360nm). This phenolicfraction was freeze-dried and used for the HPLC and hy-drolysis analyses. 2.3. Alkaline and acid hydrolysis This was achieved according to previously reportedmethodology described for cauliflower with slight changes[7]. This was performed by adding 1mL 4N NaOH tothe hydroalcoholic phenolic extract purified as describedabove (1mL) and keeping the mixture for 16h at roomtemperature in a stoppered test tube under N 2  atmosphere.After this step, the alkaline hydrolysis products were acid-ified with concentrated HCl (up to pH 1–2) and directlyanalysed by LC/UV-DAD/ESI-MS n . Total acid hydrolysiswas carried out by adding 1mL 4N HCl to 1mL of thehydroalcoholic phenolic extract and this solution was keptin a stoppered test tube, incubated for 30min at 85 ◦ C anddirectly analysed by LC/UV-DAD/ESI-MS n . 2.4. LC/UV-DAD/ESI-MS  n analyses Chromatographic analyses were carried out on a LiChro-CART column (250mm  ×  4mm, RP-18, 5  m particlesize, LiChrospher ® 100 stationary phase, Merck, Darmstadt,Germany) protected with a LiChroCART guard column(4mm  × 4mm, RP-18, 5  m particle size, Merck, Darm-stadt, Germany). The mobile phase consisted of two sol-vents: water–formic acid (0.1%) (A) and methanol (B). Forstudying both free flavonol glycosides and the correspond-ing acylated derivatives a linear gradient starting with 20%B was installed to reach 50% B at 35min and 80% B at37min. On the other hand, for the analysis of the acidsand the aglycones obtained after acid hydrolysis, a lineargradient starting with 15% B and reaching 65% B at 50minwas used to reach 80% B at 52min. The flow rate was1mLmin − 1 , and the injection volume ranged 10–50  L de-pending on the compound and extract assayed. Spectral datafrom all peaks were accumulated in the range 240–400nm,and chromatograms were recorded at 340nm for glycosidesand acylated derivatives, and at 330 and 360nm for hy-droxycinnamic acids and flavonoid aglycones, respectively.The LC/UV-DAD/ESI-MS n analyses were carried out inan Agilent HPLC 1100 series equipped with a diode arraydetector and mass detector in series (Agilent Technologies,Waldbronn, Germany). The HPLC consisted of a binarypump (model G1312A), an autosampler (model G1313A),a degasser (model G1322A), and a photodiode array de-tector (model G1315B). The HPLC system was controlledby a ChemStation software (Agilent, v. 08.03). The massdetector was an ion trap spectrometer (model G2445A)equipped with an electrospray ionisation interface and wascontrolled by LCMSD software (Agilent, v. 4.1). The ion-isation conditions were adjusted at 350 ◦ C and 4kV forcapillary temperature and voltage, respectively. The nebu-liser pressure and flow rate of nitrogen were 65.0psi and11Lmin − 1 , respectively. The full scan mass covered therange from  m  /   z  200 up to 2000 for free glycosides and acy-lated derivatives and from  m  /   z  90 up to 400 for acids andaglycones. Collision-induced fragmentation experimentswere performed in the ion trap using helium as the collisiongas, with voltage ramping cycles from 0.3 up to 2V. Massspectrometry data were acquired in the negative ionisationmode. MS n is carried out in the automatic mode on themore abundant fragment ion in MS n  − 1 .Tables 1–4 show the most frequent ions which charac-terise the fragmentation of these flavonoid  O -glycosides.Other ions were found but they have not been included dueto their low significance on the MS behaviour ions. Theclassical nomenclature [9] for glycoconjugates was adoptedto designate the fragment ions. Ions  k,l X j  , Y nj  , Z nj   repre-sent those fragments still containing the flavonoid aglycone,where  j  is the number of the interglycosidic bond broken,counted from the aglycone,  n  represents the position of thephenolic hydroxyl where the oligosaccharide is attached,and the  k   and  l  denote the cleavage within the carbohydraterings.The ions obtained as a consequence of a second oligosac-charide fragmentation have been labelled according toprevious reports [10,11]. Thus, ions obtained from the ion Y 70 − ( − MS 3 [ (M  − H ) → Y 70 ] − ) have been labelled startingwith the ion Y 70 − and followed by the resultant MS 3 ion,e.g. the ion Y 70 Y 32 − (MS 3 of compound  3 ) (Table 1, Fig. 3, Scheme 1) denotes the loss of terminal sugar of the trigly-coside in the position 3 (Y 32 − ) from the fragmentation of ion Y 70 − (total loss of a glycosylation in the position 7). Thelosses indicated in the MS 3 scan show that the fragment  F  . V  a l    l    e  j    o e t   a l    . /    J   . C h  r  o m a t   o  gr  .A 1   0   5  4    (   2   0   0  4    )   1   8  1  –1   9   3   1   8   3   Table 1  R t , UV, –MS: [  M  − H] − , –MS 2 [  M  − H] − and  − MS 3 [ (M  − H ) → Y 70 ] ( − 162/  − 324)] − data of flavonoid-3- O -Soph/Sophtr-7- O -Glc/SophCompounds  R t  (min) UV (nm) [  M  − H] − ( m  /   z ) –MS 2 [  M  − H] − ( m  /   z ) (%)  − MS 3 [ (M  − H ) → Y 70 ] − ( m/z ) (%)Y 70 − Y 700 , 2 X − Y 70 Y 23 − Y 70 Y 31 − Y 70 Z 31 − Y 70 Y 30 − Flavonoid-3- O -sophorotrioside-7- O -glucoside/sophoroside( − 162/  − 324) ( − 120) ( − 162) ( − 324) a ( − 342) ( − 486) 1  Querc-3-Sophtr-7-Glc 8.1 256, 266sh, 354 949.8 787 (100) 667 (100) 463 (50) a 445 (89) 301 (30) 3  Querc-3-Sophtr-7-Soph 9.0 256, 266sh, 352 1111.8 787 (100) 667 (100) 625 (22) 463 (16) a 445 (83) 301 (68) 4  Kaempf-3-Sophtr-7-Glc 9.6 266, 316sh, 348 933.8 771 (100) 609 (33) 429 (38) 285 (100) 5  Kaempf-3-Sophtr-7-Soph 10.4 266, 316sh, 349 1095.9 771 (100) 609 (18) 447 (32) a 429 (59) 285 (100) 7  Isorhmnt-3-Sophtr-7-Glc b 10.9 963.5 801 (100) 681 (37) 459 (66) 314 (100) c 9  Isorhmnt-3-Sophtr-7-Soph b 11.9 1125.5 801 (100) 681 (90) 639 (60) 459 (80) 315 (100)Flavonoid-3- O -sophoroside-7- O -glucoside/sophoroside( − 162/  − 324) ( − 120) ( − 162) ( − 180) ( − 324) 2  Querc-3-Soph-7-Glc 8.8 coelution with  3  787.7 625 (100) 463 (52) 445 (60) 300 (100) c 6  Kaempf-3-Soph-7-Glc 10.5 coelution with  5  771.6 609 (100) 489 (28) 429 (25) 284 (100) c 8  Kaempf-3-Soph-7-Soph 11.2 266, 318sh, 352 933.8 609 (100) 489 (29) 429 (18) 285 (100) a The ion [Y 70 Y 31 ] −• (Y 70 -324) has not been observed in the attached chromatograms, however it has been observed as an abundant peak in other chromatograms. b Compounds in trace amounts and hidden by other. Their UV spectra have not been observed property. c Fragments from homolytic cleavage of the glycosidic bond ([Y 70 Y 30 − H] −• ) [16].  1   8  4   F  . V  a l    l    e  j    o e t   a l    . /    J   . C h  r  o m a t   o  gr  .A 1   0   5  4    (   2   0   0  4    )   1   8  1  –1   9   3   Table 2  R t , UV, –MS: [  M  − H] − , –MS 2 [  M  − H] − and  − MS 3 [ (M  − H ) → Y 30 ] − data of flavonoid-3- O -Glc-7- O -Soph, -3,7-di- O -Glc, -3- O -Soph and -3- O -GlcCompounds  R t  (min) UV (nm) [  M  − H] − ( m  /   z ) –MS 2 [  M  − H] − ( m  /   z ) (%)  − MS 3 [ (M  − H ) → Y 30 ] − ( m/z ) (%)Flavonoid-3- O -glucoside-7- O -sophorosideY 30 − ( − 162)Y 70 − ( − 324)[Aglic − H] − ( − 486)Y 30 Y 71 − ( − 162)Y 30 Y 70 − ( − 324) 11  Querc-3-Glc-7-Soph 14.6 257, 266sh, 354 787.9 625 (26) 463 (100) 301 (14) 463 (12) 301 (100) 13  Kaempf-3-Glc-7-Soph 17.7 266, 320sh, 350 771.5 609 (26) 447 (100) 285 (30) 15  Isorhmn-3-Glc-7-Soph 18.7 255, 266sh, 350 801.9 639 (39) 477 (100) 315 (13)Flavonoid-3,7-di- O -glucoside 0 , 2 X − ( − 120)Y 70 − / Y 30 − ( − 162)[Aglic − H] − ( − 324) 10  Querc-3,7-diGlc 13.8 255, 266sh, 294sh, 354 625.7 505 (9) 463 (100) 301 (34) 12  Kaempf-3,7-diGlc 17.1 266, 318sh, 349 609.9 489 (24) 447 (100) 285 (33) 14  Isorhmn-3,7-diGlc 18.2 255, 268sh, 354 639.7 519 (11) 477 (100) 315 (17)Flavonoid-3- O -sophoroside 0 , 2 X − ( − 120)Y 31 − ( − 162)Z 31 − ( − 180)Y 30 − ( − 324) 16  Querc-3-Soph a 21.6 257, 267sh, 293, 355 625.6 505 (4) 463 (17) 445 (54) 300 (100) b 17  Kaempf-3-Soph 25.4 266, 294sh, 350 609.7 489 (2) 447 (21) 429 (55) 285 (100)Flavonoid-3- O -glucosideY 30 − ( − 162) 18  Querc-3-Glc 28.7 255, 266sh, 355 463.9 301 (100) 19  Kaempf-3-Glc 33.2 266, 294sh, 349 447.9 285 (100) a Compounds in trace amounts and hidden by other. UV spectra from deacylated extract chromatogram. b Fragments from homolytic cleavage of the glycosidic bond ([Y 70 Y 30 − H] −• ) [16].  F. Vallejo et al./J. Chromatogr. A 1054 (2004) 181–193  185 came from the trapped and fragmented ion (Y 70 − ) and notfrom the deprotonated molecular ion. 3. Results and discussion ThebroccoliphenolicfractionwasanalysedbyHPLC/UV-DAD and the characteristic sinapoyl/feruloyl gentiobio-sides were detected [4] (Fig. 1). In addition a large number of flavonoid compounds was detected in the extract andtheir UV spectra suggested that they were mainly acylatedderivatives in which sinapic, ferulic, caffeic and  p -coumaricacids were linked to the flavonoid-glycoside molecules.Their characteristic UV spectra showed a flavonol spectrumoverlapped with a hydroxycinnamic acid spectrum with abroad maximum around 330nm, and the structural informa-tion obtained by these spectra was much smaller than thatobtained from the UV spectra of non-acylated flavonoids orhydroxycinnamic acid derivatives. After alkaline hydrolysisthe flavonoid pattern was much simpler (Fig. 2) indicatingthat the naturally occurring flavonoids were different acy-lated derivatives of the same flavonol glycosides. By thisreason it was decided to start the structural study with theanalysis of the aglycones and the deacylated glycosides. 3.1. Flavonoid aglycones and hydroxycinnamic acids After acid hydrolysis of the broccoli phenolic extractthe LC/UV-DAD/ESI-MS n study, showed that the mainacids present were sinapic,  p -coumaric, caffeic and fer-ulic, and that the flavonoid aglycones were kaempferolas the main compound (3,5,7,4  -tetrahydroxyflavone),quercetin (3,5,7,3  ,4  -pentahydroxyflavone) and isorham-netin (3,5,7,4  -tetrahydroxy-3  -methoxyflavone), this last intrace amounts. They were identified by their UV spectrum,MS analysis and co-chromatography with standards. 3.2. Deacylated glycosides After saponification (Fig. 2), a large number of flavonoidhexosides (most probably glucosides due to filoge-netic similitude with the cauliflower compounds) havebeen characterised. Glycosides of quercetin, isorham-netin and kaempferol, with the following glycosylationpatterns: 3-glucoside; 3,7-diglucoside; 3-sophoroside;3-sophoroside-7-glucoside; 3-glucoside-7-sophoroside;3-sophoroside-7-sophoroside;3-sophorotrioside-7-glucosideand 3-sophorotrioside-7-sophoroside.The tentative assignation of the glycosylation positionsover the flavonol nucleus has been achieved taking into ac-count that the hydroxyl at 3 position was always blocked(UV spectra, band I  ∼=  348–355nm) (Tables 1–2) and re- leased after acid hydrolysis (aglycones UV spectra, band I ∼=  367–371nm); and that glycosylation on the hydroxyl at7 position once the hydroxyl at 3 position is substituted, is  T   a    b    l   e    3     R     t  ,   a   n    d  –    M    S   :    [     M   −     H    ]   −  ,  –    M    S     2     [     M   −     H    ]   −    a   n    d  –    M    S     3     [    (     M   −     H    )    →     (     M   −     H  -    1    6    2    )    ]   −     d   a    t   a   o    f   a   c   y    l   a    t   e    d    d   e   r    i   v   a    t    i   v   e   s    f   r   o   m    fl   a   v   o   n   o    i    d  -    3  -     O   -   s   o   p    h   o   r   o   s    i    d   e    /   s   o   p    h   o   r   o    t   r    i   o   s    i    d   e  -    7  -     O   -   g    l   u   c   o   s    i    d   e     C   o   m   p   o   u   n    d   s   a     R     t    (   m    i   n    )    [     M   −     H    ]   −     (    m    /   z     )  –    M    S    2    [     M   −     H    ]   −     (    m     /    z     )    (    %    )  –    M    S    3    [    (     M   −     H    )    →     (     M   −     H  -    1    6    2    )    ]   −     (    m     /    z     )    (    %    )   −     1    4    6    (  -    p  .    C   o   u   m    )   −     1    6    2    (  -    G    l   c    )   −     1    7    6    (  -    F   e   r    )   −     2    0    6    (  -    S    i   n   p    )   −     3    0    8    (  -    G  -    p     C    )   −     3    2    4    (  -    G  -    C    )   −     3    3    8    (  -    G  -    F    )   −     3    5    4    (  -    G  -    M    C    )   −     3    6    8    (  -    G  -    S    )   −     4    7    0    (  -    G  -    C  -    p     C    )   −     4    8    6    (  -    G  -    C  -    C    )   −     5    3    0    (  -    G  -    C  -    S    )   −     5    4    4    (  -    G  -    F  -    S    )   −     5    7    4    (  -    G  -    S  -    S    )   −     1    4    6    (  -    p  .    C   o   u   m    )   −     1    6    2    (  -    C   a    f    )   −     1    7    6    (  -    F   e   r    )   −     1    9    2    (  -    M   e    O    C   a    f    )   −     2    0    6    (  -    S    i   n   p    )   −     3    0    8    (  -    C  -    p     C    )   −     3    2    4    (  -    C  -    C    )   −     3    6    8    (  -    C  -    S    )   −     3    8    2    (  -    F  -    S    )   −     4    1    2    (  -    S  -    S    )    A   c   y    l   a    t   e    d    d   e   r    i   v   a    t    i   v   e   s    f   r   o   m         1    :   q   e   r   c   e    t    i   n  -    3  -     O   -   s   o   p    h   o   r   o    t   r    i   o   s    i    d   e  -    7  -     O   -   g    l   u   c   o   s    i    d   e         2        0     1  -    C   a    f    9 .    2    1    1    1    1 .    7    9    4    9    (    1    0    0    )    7    8    7    (    5    9    )    7    8    7    (    1    0    0    )         2        1     1  -    d    i    C   a    f    9 .    5    1    2    7    3 .    6    1    1    1    1    (    1    0    0    )    9    4    9    (    3    0    )    7    8    7    (    9    4    )    7    8    7    (    1    0    0    )         2        2     1  -    S    i   n   p    1    2 .    0    1    1    5    5 .    6    9    9    3    (    1    0    0    )    9    4    9    (    4    9    )    7    8    7    (    6    1    )    7    8    7    (    1    0    0    )         2        3     1  -    F   e   r    1    2 .    4    1    1    2    5 .    5    9    6    3    (    1    0    0    )    9    4    9    (    3    7    )    7    8    7    (    7    1    )    7    8    7    (    1    0    0    )         2        4     1  -    p  .    C   o   u   m    1    2 .    8    1    0    9    5 .    7    9    4    9    (    5    7    )    9    3    3    (    1    0    0    )    7    8    7    (    8    4    )    7    8    7    (    1    0    0    )         2        5     1  -    C   a    f    /    S    i   n   p    2    6 .    0    1    3    1    7 .    6    1    1    5    5    (    1    0    0    )    9    9    3    (    7    3    )    9    4    9    (    4    )    7    8    7    (    2    0    )    9    9    3    (    1    0    0    )    7    8    7    (    2    4    )         2        6     1  -    d    i    S    i   n   p    2    7 .    8    1    3    6    1 .    9    1    1    9    9    (    1    0    0    )    1    1    5    5    (    3    1    )    9    9    3    (    9    0    )    7    8    7    (    3    0    )    9    9    3    (    1    0    0    )    7    8    7    (    4    1    )         2        7     1  -    F   e   r    /    S    i   n   p    2    8 .    7    1    3    3    1 .    8    1    1    6    9    (    1    0    0    )    1    1    5    5    (    2    9    )    1    1    2    5    (    7    )    9    9    3    (    6    5    )    9    6    3    (    1    5    )    7    8    7    (    9    )    9    9    3    (    1    0    0    )    9    6    3    (    3    1    )    7    8    7    (    3    2    )    A   c   y    l   a    t   e    d    d   e   r    i   v   a    t    i   v   e   s    f   r   o   m         4    :    k   a   e   m   p    f   e   r   o    l  -    3  -     O   -   s   o   p    h   o   r   o    t   r    i   o   s    i    d   e  -    7  -     O   -   g    l   u   c   o   s    i    d   e         2        8     4  -    M   e    O    C   a    f    9 .    9    1    1    2    5 .    7    9    6    3    (    1    0    0    )    7    7    1    (    2    6    )    7    7    1    (    1    0    0    )         2        9     4  -    S    i   n   p    1    3 .    3    1    1    3    9 .    9    9    7    7    (    1    0    0    )    7    7    1    (    7    )    7    7    1    (    1    0    0    )         3        0     4  -    F   e   r    1    3 .    9    1    1    0    9 .    5    9    4    7    (    1    0    0    )    7    7    1    (    7    )    7    7    1    (    1    0    0    )         3        1     4  -    C   a    f    /    S    i   n   p    2    7 .    4    1    3    0    1 .    6    1    1    3    9    (    1    0    0    )    9    7    7    (    1    2    )    9    3    3    (    2    )    7    7    1    (    3    )    9    7    7    (    1    0    0    )    9    3    3    (    1    7    )    7    7    1    (    3    0    )         3        2     4  -    d    i    S    i   n   p    2    9 .    1    1    3    4    5 .    9    1    1    8    3    (    1    0    0    )    9    7    7    (    7    )    7    7    1    (    2    )    9    7    7    (    1    0    0    )    7    7    1    (    1    6    )    A   c   y    l   a    t   e    d    d   e   r    i   v   a    t    i   v   e   s    f   r   o   m         2    :   q   u   e   r   c   e    t    i   n  -    3  -     O   -   s   o   p    h   o   r   o   s    i    d   e  -    7  -     O   -   g    l   u   c   o   s    i    d   e         3        3     2  -    C   a    f    8 .    9    9    4    9 .    6    7    8    7    (    1    0    0    )    6    2    5    (    5    2    )    6    2    5    (    1    0    0    )         3        4     2  -    F   e   r    1    2 .    0    9    6    3 .    7    8    0    1    (    1    0    0    )    7    8    7    (    2    5    )    6    2    5    (    2    3    )    6    2    5    (    1    0    0    )         3        5     2  -    p  .    C   o   u   m    1    2 .    4    9    3    3 .    6    7    8    7    (    8    )    7    7    1    (    1    0    0    )    6    2    5    (    2    7    )    6    2    5    (    1    0    0    )    A   c   y    l   a    t   e    d    d   e   r    i   v   a    t    i   v   e   s    f   r   o   m         6    :    k   a   e   m   p    f   e   r   o    l  -    3  -     O   -   s   o   p    h   o   r   o   s    i    d   e  -    7  -     O   -   g    l   u   c   o   s    i    d   e         3        6     6  -    C   a    f    1    0 .    8    9    3    3 .    9    7    7    1    (    1    0    0    )    6    0    9    (    1    5    )    6    0    9    (    1    0    0    )         3        7     6  -    C   a    f    /    p  .    C   o   u   m    1    4 .    8    1    0    7    9 .    6    9    1    7    (    1    0    0    )    7    7    1    (    3    )    7    5    5    (    8    6    )    6    0    9    (    7    )    7    7    1    (    1    0    0    )    7    5    5    (    7    )    6    0    9    (    5    )         1    :   q   u   e   r   c   e    t    i   n  -    3  -     O   -   s   o   p    h   o   r   o    t   r    i   o   s    i    d   e  -    7  -     O   -   g    l   u   c   o   s    i    d   e .         2    :   q   u   e   r   c   e    t    i   n  -    3  -     O   -   s   o   p    h   o   r   o   s    i    d   e  -    7  -     O   -   g    l   u   c   o   s    i    d   e .         4    :    k   a   e   m   p    f   e   r   o    l  -    3  -     O   -   s   o   p    h   o   r   o    t   r    i   o   s    i    d   e  -     O   -    7  -   g    l   u   c   o   s    i    d   e .         6    :    k   a   e   m   p    f   e   r   o    l  -    3  -     O   -   s   o   p    h   o   r   o   s    i    d   e  -    7  -     O   -   g    l   u   c   o   s    i    d   e .   a    G    (    G    l   c    )   :    G    l   u   c   o   s   y    l .    p     C    (    p  .    C   o   u   m    )   :    p  .    C   o   u   m   a   r   o   y    l .    F    (    F   e   r    )   :    F   e   r   u    l   o   y    l .    S    (    S    i   n   p    )   :    S    i   n   a   p   o   y    l .    M    C    (    M   e    O    C   a    f    )   :    M   e    t    h   o   x   y    C   a    f   e   o   y    l .
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