The largest and most ubiquitous flavonoid subgroup in the plant kingdom is the flavonols. Common flavonol aglycones are listed in Table 1. They have a 3-hydroxy group (Figure 1) and are usually found in their glycoside form in fruits, vegetables, spices and herbs [2,4,6,34-43,51,67]. Flavonol glycosides are usually present at relatively low concentrations, i.e. at 15-30 mg /kg (fresh weight) [1,2, 40]. The concentration of flavonols (and flavones, flavanones, flavan-3-ols, and anthocyanidins), as aglycones, in 59 US fruits, nut and vegetables has been reported [17]. These data have been incorporated, as a supplemental database, in the USDA National Nutrient Database for Standard Reference (16). The flavonoid supplemental database contains values collected from published literature after critical evaluation. These data are updated routinely and are available online.
More than 80 sugars, including 10 mono-saccharides, 50 di-saccharides, 30 tri-saccharides and one tetra-saccharde, have been reported [2,4,6,34-43] with linkages at one, two, or more positions on the flavonols. Moreover, some glycosylated flavonols have one or more different acyl groups (such as acetyl, malonyl and the acyls of phenolic acids) attached to the saccharides. Consequently, more than 200 glycosides have been reported for both quercetin and kaempferol [35].
The most common glycoside is glucose, followed, in decreasing frequency, by rhamnose and rutinose (6-O-α-L-rhamnosyl-D-glucose). The glycosides formed from D-saccharides (D-glucose, D-g lactose, D-glucuronic acid, D-xylose, and D-apiose) are known as β-D-glycosides while those formed from L-saccharides (L-rhamnose and L-arabinose) are known as α-L-glycosides. Most of the glycosides, however, are β-D-glycosides. The glycosylated flavonols most commonly have saccharides at the C3 position, a small number at the C7 position, fewer still at the C3’, or C4’ position, and very rarely at the C5 position.
Diglycosides (at the 3,3’-, 3,4’-, and 7,4’- positions) and triglycosides (3,7,4’-) have been reported.
The glycosides of quercetin and kaempferol, including quercetin 3-O-glucoside, quercetin 3-O-rutinosides (rutin), kaempferol 3-O-glucoside, and kaempferol 3-O-rutinoside, are the most abundant flavonoids in the plant kingdom. Rutin is one of the most common flavonoids found in foods, such as asparagus sprout (Asparagus officinalis L.) (Liliaceae) [126, 127], buckwheat (Fagopyrum esculentum Moenth) (Poligonaceae) [128, 129], grape
Long-Ze Lin and James M. Harnly 24
tomato and several other tomatos (Lycopersicon spp.) [130-132], and avocado leaf (Persea americana Mill.). Two other aglycones, isorhamnetin and myricetin, are the second most common flavonoids in vegetables. The glycosides of some methylated flavonols, such as 3,5,7,3’,4’-pentahydroxy-6-methoxyflavone (patuletin), 3,5,2', 3'-tetrahydroxy-4'-methyoxy-6,7-methylenedioxyflavone, and their isomers, have been reported as the main flavonol glycosides of baby spinach (Spinacia oleracea L.) (Chenodiaceae), a common vegetable throughout the world [133-135].
Most fruit and many vegetables contain 5-10 different flavonol or flavone glycosides as their major flavonoid constituents. In general, the flavonols accumulate in the outer and aerial tissues (peel and leaves) and the content changes significantly with maturation [2,57,67].
Flavonoid content is always highest in vegetables grown in the summer months and in the green outer leaves, as compared to the light-colored inner leaves, of leafy vegetables such as lettuce and cabbage.
Flavones are the second largest group of flavonoids found in fruit and vegetables. Some common non-glycosylated flavones are listed in Table 1. Glycosylated flavones are usually found with saccharides at the C7 position. The O-glycosides of luteolin and apigenin are among the chief members of this group. Some vegetables, spices, and herbs, such as celery (Apium graveolens L) (Umbelliferae or Apiaceae) [122], parsley (Petroselinum crispum Mill) (Umbelliferae) [122,136], cumin (Cuminum cyminum L.) (Umbelliferae) [137], chrysanthemum flowers (Chrysanthemum morifolium Ramat) (Compositae) [138], and red bell peppers (Capsicum anumuum L.) [122, 139-141] contain glycosylated flavones as their main flavonoids. The most important edible sources of flavones, however, come from a relatively small group of foods, such as celery, mung bean [142], and parsley. Some foods, such as lemon grass (Cymbopogon citrates (DC.) (Gramineae), citrus [143], and some melons [144], contain C-glycosides of flavones as their main flavonoids. The peels of citrus fruits contain large quantities of polymethoxylated flavones, such as, tangeretin, nobiletin, and sinensetin [67, 143].
The flavonols have absorption maxima at 250-270 nm (UV Band II) and 350-380 nm (UV band I), while the flavones have absorption maxima at 250-270 nm (band II) and 330-350 nm (band I) (Table 2) [43, 67]. For example, the UV spectra of quercetin 3-O-galactoside (flavonol) and sinnengetin (flavone) are shown in Figure 4A and luteolin and luteolin 7-O-glucoside are shown in and Figure 7. The position of the UV absorption bands and the shifts caused by oxygenation, methylation, or glycosylation, are important. There are several general rules concerning shifts caused by substitution that can be used for identification. Shift reagents [43], such as sodium acetate (NAOAc) and aluminum chloride (AlCl3) have been used postcolumn to deduce the oxygenation pattern of the flavone skeleton [98]. This is not necessary for most of the common food flavonoids.
3.2.1.1. Identification of Non-glycosylated Flavonols and Flavones (Aglycones)
Compared to the ubiquitous glycosylated forms of the flavones and flavonols, the aglycones are present in far fewer plant materials and at much lower concentrations. Since relatively more aglycone standards are available, however, their identification and quantification in plant materials is easier than it is for any other group of phenolic compounds. In addition, complete data is available on the retention times and UV and MS spectra for most aglycones. Thus, most aglycones in most plant materials can be positively identified.
LC-MS Profiling and Quantification of Food Phenolic Components ... 25
225 250 275 300 325 350 375 400 425 nm
Norm .
0 50 100 150 200 250 300 350
*DAD1, 32.247 (359 m AU, - ) Ref=0.001 & 50.954 of STDGF081.D
3
A
*DAD1, 29.342 (75.9 m AU, - ) Ref=0.002 & 50.956 of STDGF086.D
*DAD1, 19.768 (88.9 m AU,Apx) Ref=0.001 & 50.955 of STDGF140.D
*DAD1, 27.586 (97.2 m AU, - ) Ref=0.006 & 50.953 of STDGF133.D
2 1
4
nm
225 250 275 300 325 350 375 400 425
Norm .
0 50 100 150 200 250 300
*DAD1, 26.395 (279 m AU, - ) Ref=1.756 & 36.689 of LCOLU337.D
*DAD1, 30.022 (342 m AU, - ) Ref=1.769 & 54.582 of LCOLU184.D
*DAD1, 25.459 (60.4 m AU, - ) Ref=0.006 & 43.106 of 07-10062.D
*DAD1, 26.239 (113 m AU, - ) Ref=0.006 & 43.106 of 07-10062.D
B 1
2 3
4
Figure 7. UV spectra of: A) 1=kaempferol, 2=kaempferol 3-O-glucoside, 3=luteolin, and 4=luteolin 7-O-glucoside; B) 1=ellagic acid, 2=quercetin, 3=morin, and 4=bobinetin.
Mabry et al [43] summarized the relationship between the UV spectra and degree of oxygenation of the aglycones making it possible to deduce the structural skeleton based on UV data alone. For example, increased oxygenation of the B-ring produces a bathochromic (longer wavelength) shift of λmax of band I, no shift of λmax of band II, and an added peak at a wavelength below band II. The additional peak can be used to distinguish common isomeric flavones and flavonols. For example, kaempferol has one hydroxyl function at the C4' position of the B-ring and one peak (band II ) with λmax around 266 nm. Luteotin has two hydroxyl groups at the C3' and C4' positions of the B-ring and an additional peak (λmax 254 nm) just below the band II peak (λmax 264 nm) (Figure 7A). Increasing hydroxylation of the A-ring produces a notable bathochromic shift for the maximum of band II and a smaller shift for Band I. Hydroxylation at C5 has a significant effect on both Band I (a 3-10 nm shift) and band II (a 6-17 nm shift) while O-methylation at C5 produces a 5-15 nm hypsochromic (shorter wavelength) shift in both Bands I and II. Addition of an O-methyl group at C3 and C4' produces 12-17 and 3-10 nm hypsochromic shifts in Bands I and II, respectively.
Oxygenation at other positions (such as C7, C3', C6, or C5') has little or no effect on the UV spectra.
There are additional publications that provide UV data for more than 50 flavonols and flavones [43,144-147] and for HPLC separation of common aglycones [147]. These sources can provide assistance on the assignment of the hydroxy functions in the flavonoid skeleton.
There are more than 200 biflavonyls found in plants [35] and a limited number of flavones and flavonols also contain one or more alkyl groups, such as prenyl [CH2CH=C(CH3)2, add 68], 1,1-dimethylprop-2-en-1-yl (add 68), 3-hydroxy-3-methylbutyl (add 86), geranyl [CH2CH=C(CH3)CH2CH2CH=C(CH3)2, add 134], lavandulyl {CH2CH[C(CH3)=CH2]CH2CH=C(CH3)2, add 136}, 3-methylsuccinoyl (HO-CH(COOH)-CH2-CHO, add 114), 5-hydroxy-2-isopropenyl-5-methylhexyl (add 154), 4-hydroxyphenylmethyl (add 104), 4-hydroxyphenylethyl (add 120), and prenyloxy (add 84),
Long-Ze Lin and James M. Harnly
DAD1 C , Sig=270,8 Ref=450,100 (D:\QUANT-2\QUANT372.D)
Ledum leaf extract
DAD1 A, Sig=350,4 Ref=450,100 (D:\QUANT-2\QUANT378.D)
Cranberry extract
DAD1 A, Sig=350,4 Ref=450,100 (D:\QUANT-2\QUANT381.D)
Fuji apple peel extract 1
Figure 8. Chromatograms (270 nm) recorded with a Zorbax-XSD-C18 column (Agilent Corp) of A) ledum leaves, B) cranberries, and C) Fuji apple skins. Peaks were identified as 1=chlorogenic acid, 2=(+)-catechin, 3=(-)-epicatechin, 4=rutin, 5=quercetin-3-O-galactose, 6=quercetin-3-O-glucoside, 7=quercetin-3-O-xylopyranoside, 8=quercetin-3-O-arabinopyranoside,
9=quercetin-3-O-arabinofuranoside, 10=quercetin 3-O-rhamnoside, 11=quercetin-3-O-6”-acetylglucoside, and 12=quercetin-3-O-acetylglucoside.
methylenedioxy (add 44). These functional groups are found most commonly at the C6 or C8 (or both) positions, and occasionally at other positions on the flavonoid skeleton [35]. Thus, it is worth remembering that the mass of these subsitutions, which might be attached to the flavonoid skeleton and add the related mass contribution, although they don’t occur frequently.
3.2.1.2. Identification of O-monoglycosylated Flavonols and Flavones
Most of the naturally existing O-glycosylated flavonoids are mono or disaccharides with linkages at the C3, C7, C3', or C4' positions (when present on the aglycone). The monosaccharides are usually one of several isomeric pentoses, hexoses, oxyhexoses, or rhamnoses (the only deoxyhexosyls to form flavonoid glycosides) [35, 51, 67]. These flavonol glycosides can be easily distinguished by their UV and mass spectra. The saccharide type can be easily deduced from the mass difference between the molecular ion and aglycone ion (Table 5). For example, a mass difference of 132 amu is indicative of a pentosyl, a difference of 146 amu is indicative of a rhamnosyl, 162 amu for a hexosyl, and 178 amu for an oxyhexosyl.
UV spectra and retention time are, in general, the primary basis for assignment of the linkage positions of saccharides to the aglycone. As Mabry et. al. summarized [43] and we have observed [67], 3-O-Glycosides and 4'-O-Glycosides produced 12-17 and 3-12 nm
LC-MS Profiling and Quantification of Food Phenolic Components ... 27 hypsochromic shifts, respectively, in UV band I. Attachment of a saccharide in another position (such as the C7, C3', C6, or C5' position) has little or no effect on the UV spectra.
As an example, consider the following results obtained using the standardized profiling method. Quercetin 3-O-glucoside has a retention time (tR) of 27.40 min, a UV absorbance maximum (λmax) at 256 nm, a shoulder at 266 nm (266sh), and a second λmax at 354 nm (a hypsochromic shift of 16 nm). It elutes earlier than quercetin 4'-O-glucoside (tR 32.95 min, λmax 256, 266sh, 364 nm, a hypsochromic shift of 8 nm). Luteolin 7-O-glucoside (tR 27.44 min, λmax 254, 266, 348 nm, no hypsochromic shift) elutes earlier than its 4'-O-glucoside (tR 30.32 min, λmax 254, 268, 336 nm, a hypsochromic shift of 12 nm). In both cases, the UV shifts and the retention times accurately indicate the position of the saccharide linkages. Thus, the UV shifts and elution order can be used to accurately identify these isomeric flavonoid glycosides.
DAD1 A, Sig=350,4 Ref=700,100 (LIN\ETEST429.D)
2 Cashew apple extract 14
20 22 24 26 28 30 32 34 m in
DAD1 A, Sig=350,4 Ref=700,100 (LIN\ETEST435.D)
Cashew apple extract + Cranberry extract
m in
DAD1 A, Sig=350,4 Ref=700,100 (LIN\ETEST430.D)
a
Figure 9. Chromatograms (350 nm) of the extracts of: A) cashew apples, B) cashew apples and cranberries (combined), and C) cranberries. Peaks were identified as: a=quercetin 6”-acetylgalactoside, 2=myricetin galactoside, 3=myricetin glucoside, 4=myricetin
3-O-xylopyranoside, 5=myricetin 3-O-arabinopyranoside, 6=myricetin 3-O-arabinofuranoside, 7=myricetin rhamnoside, 8=quercetin galactoside, 9=quercetin glucoside, 10=quercetin
3-O-xylopyranoside, 11=quercetin 3-O-arabinopyranoside, 12=quercetin 3-O-arabinofuranoside, 13=kaempferol 3-O-glucoside, and 14=quercetin 3-O-rhamnoside.
Changes in elution times have also been observed to be a function of the type of saccharide when linked at the same position. For example, as the saccharides at the C3 position of quercetin change, the retention times also change. The elution order, as observed using the standardized profiling method, from earliest to latest, is galactoside, glucoside, xyloside, arabinopyranoside, arabinofuranoside, rhamnoside, and finally the glucuronide.
There are no standards available for any quercetin 3-O-pentosides. Instead, we found that
Long-Ze Lin and James M. Harnly 28
cranberries can serve as a reference plant material in which the three quercetin 3-O- pentoside isomers have been positively identified [67, 93, 148].
Initially, chromatograms were acquired for the cranberry extract using the same column (Agilent Zorbax-XSD C18 column) and gradient reported in the literature to confirm the elution order and relative peak intensities of the glycosides in the extract. Then, chromatograms were acquired for the extract using the standardized phenolic profiling method with both the Agilent column and the Waters Symmetry column. The three 3-O-pentoside isomers and two hexoside isomers were identified on both columns and their retention times and UV spectra and MS spectra recorded using the standardized profiling method. Based on these data, it was possible to use the standardized method to identify these isomers in other plant materials such as cashew apples, Fuji apple skins, and ledum leaves as shown in Figures 8 and 9. For the best comparison, the extracts of cranberries, Fuji apple skins, ledum leaves, and cashew apples were screened in rapid sequence and spiked with the cranberry extract. In this manner, identification of two arabinosides in Fuji apple skins and most of the quercetin 3-O-glycosides in cashew apples and ledum leaves was made for the first time [67,93]. The ledum leaves (Ledum glandulosum L.) (Ericaceae), are used to make a native American Indian tea that is drunk in some areas of the US.
The elution order identified for the quercetin 3-O-monoglycosides should apply to the glycosides of other flavonols, such as kaempferol, myricetin and isorhamnetin. These flavonol 3-O-monoglycosides are widely distributed in many other fruits (e.g. plums, peaches, pears, persimmons, loquats, and grapes), vegetables (e.g. dill, fennel, onions, lettuce, and common beans), and other plant materials.
Positive identification of the flavonol aglycones, following hydroysis of the extract, greatly simplifies identification of the glycosides in plant materials. Using tea as an example, the determination that kaempferol, quercetin and myricetin were the only tetrahydroxy-, pentahydroxy- and hexahydroxy-flavones in the hydrolyzed tea extract made it possible to confirm that the 13 tea flavonol 3-O-glycosides that provided PI/NI aglycone ions ([A+H]+/[A-H]-) at m/z 303/301, should be formed from quercetin. Similarly, the 27 tea glycosides with [A+H]+/[A-H]- at m/z 287/285 could only be the 3-O-glycosides of kaempferol and the 4 glycosides with [A+H]+/[A-H]- at m/z 319/317 could only be myricetin 3-O-glyocosides [120].
3.2.1.3. Identification of O-di-, tri- and Polyglycosylated Flavonoids
As mentioned previously, more than 50 disaccharides and 30 trisaccharides are found as all the O-monoglycosides [35,39], O,O-diglycosides, or more complex glycosides, and each of the saccharide can form number of the glycosides with diffirent flavonol or flavone [35,39]. Therefore, when a polyglycosylated flavonoid has the same sugar type (such as pentosyl) and sequence as a known flavonoid glycoside, a first approximation is that the unknown might also has the same glycosyls and linkage positions in its saccharide as the known flavonoid glycoside. This approximation is based on general biogenetic pathways in plant metabolism that lead to formation of flavonoid glycosides with common features.
They are found in many foods, such as the brassicas [149, 150], spinach [133-135], snow pea pods [142], green beans [151], quinoa seeds (Chenopodium quinoa wild) [152]
(Centrospemeae), leeks (Allium porrum L.), papaya fruit, pineapple, pomegranates, teas [120], and Ginkgo biloba leaves [119].
LC-MS Profiling and Quantification of Food Phenolic Components ... 29 The identification of flavonol diglycosides and other polyglycosides is based on the analysis of retention times and UV and MS data as described for the monoglycosides in the previous section. Thus, many glycosides can be identified with respect to their components, the flavonol and the saccharide. The remaining problem is to identify the isomers, i.e. the linkage positions and the exact sugars. The best approach to solving this problem is to obtain appropriate standards or reference compounds. Since standards are not available for many of the less common flavonoids, a wide variety of plant materials were screened using our standardized profiling method to find the key glycosides with suitable substitutions of the glycosides and elution orders. Groups of isomeric quercetin diglycosides were found in some foods.
DAD1 A, Sig=350,4 Ref=700,100 (LIN\PLANT267.D)
Red pearl onion A
1 2
DAD1 A, Sig=350,4 Ref=700,100 (LIN\PLANT268.D)
Red onion B
DAD1 A, Sig=350,4 Ref=700,100 (LIN\PLANT269.D)
Yellow onion C
DAD1 A, Sig=350,4 Ref=700,100 (LIN\PLANT270.D)
D Shallot
Figure 10. Chromatograms (350 nm) of the extracts (250 mg/5.0 ml solvent/50 µl injected) of A) red pearl onions, B) red onions, C) yellow onions, and D) shallots. Peaks were identified as 1=quercetin 3,7,4'-O,O,O-triglucoside, 2=quercetin 7,4'-O,O-diglucoside, 3=quercetin 3,4'-O,O-diglucoside, 4=isorhamnetin 3,4'-O,O-diglucoside, 5=quercetin 3-O-galactoside, 6=quercetin 3-O-glucoside, 7=quercetin 3-O-diglucoside, 8=isorhamnetin 3-O-glucoside, 10=quercetin 4'-O-glucoside, 12=isorhamnetin 4'-O-glucoside, 16=quercetin. The remaining peaks were not identified.
Red pearl onions, shallots (Allium ascalonicum L.) (Liliaceae), and several other onions (A. cepa L. and varieties) were found to contain quercetin 7,4'-O,O-diglucosides (tR 16.66 min, λmax 256, 266sh, 366 nm, a hypsochromic shift of 6 nm), 3,4'-O,O-diglucosides (tR 18.76 min, λmax 256sh, 266, 344 nm, a hypsochromic shift of 28 nm), quercetin 3,3',4'-O,O,O-triglucoside (tR 6.23 min, λmax 256, 266sh, 346 nm, a hypsochromic shift of 28 nm), and isorhamnetin 7,4'- and 3,4'-O,O-diglucoside (tR 18. 90 min and 20.70 min) [153,154] (Figure
Long-Ze Lin and James M. Harnly 30
10). Arugula [Eruca vesicaria (L.) Maton] (Crucifeae) contained quercetin 3,7-O,O-diglucoside (tR 10.03 min, λmax 256, 266sh, 354 min, a hypsochromic shift of 18 nm) and green lettuce contained quercetin 3,7-O,O-diglucoside malonate (tR 13.42 min, λmax 256, 266sh, 354 min), which were converted to the parent quercetin 3,7-O,O-diglucoside [150].
The elution order and UV shifts of these quercetin diglucosides and triglucosides can be used to identify the same flavonol glucosides in other foods and plant materials.
The strength of the sugar-O-aglycone bond is position dependent in the order C7>C4'>C5'=C3'>C3>C5 [51]. With MS, the removal of saccharides will follow the reverse order, i.e. the saccharide at the C5 position is lost first and the 7 spostion is lost last. This cleavage order is helpful in the deduction of the positions of two different monosaccharides on the aglycone. For example, a quercetin 3,7-diglycoside formed from rhamnose and glucose has a molecular ion [M+H]+ = m/z 611 and fragments at m/z 449 and 303. According to the cleavage order listed above, the difference of 162 amu (glucosyl) between PI molecular ion of m/z 611 and the fragment of m/z 449 was from the loss of the gluocosyl at the C3 position and the mass difference of 146 amu (rhamnosyl) between the fragment of m/z 449 and the aglycone (m/z 303) indicates the rhamnosyl is at the C7 position. Thus, this glycoside was identified as quercetin 7-O-rhamnoside-3-O-glucoside.
In the following paragraphs, four flavonol polyglycosides are used to explain the determination of the sugar sequence and the assignment of the structures. First, a Ginkgo biloba leaf flavonoid (tR 19.14 min, λmax 254, 266sh, 354 nm, [M+H]+ = m/z 757, [A+H]- = m/z 303) has two fragments at m/z 611 and m/z 465. The aglycone ion is formed by the loss of its 3 sugars. These data were used to deduce the sugar sequence. The retention time and UV and MS data suggested that this glycoside was formed from a pentahydroxyflavone (MW=302) and glycosylation at 3 positions by two rhamnosyls and one hexosyl (i.e, dirhamnosyllhexosyl, Table 5). As shown in Figure 11, the PI fragment ion at m/z 611 (Y2) is formed by the loss of the third glycosyl (757-146=611), a rhamnosyl from the glycoside, and the fragment ion at m/z 465 (Y1) is formed by loss of the secondary glycosyl (611-146=465), a rhamnosyl from first fragment, and finally the fragment at m/z 303 (Y0 = [A+H]+) is formed by the loss of the last hexosyl (465-162=303) to form the aglycone. The aglycone was confirmed to be quercetin since it is the only pentohydroxyflavone in the hydrolyzed extract.
Thus, this glycoside is a quercetin 3-O-dirhamnosylhexoside and was finally identified as quercetin 3-O-2",6"-dirhamnosylglucoside since this flavonoid was previously reported in Ginkgo biloba and the only flavonol triglycoside detected in the extract that matched the recorded retention time and UV and MS data of the peak [119].
Two polyglycosylated flavonols were detected in three lentils. The retention time (13.37 min) of the first, an unknown triglycoside was much less than that (21.77 min) of kaempferol 3-O-sophorotrioside ([M+H]+ m/z 773) found in sugar snap and snow peas [142] The retention time was 7 min shorter than that of kaempferol 3-O-triglucoside in sugar bean pods suggesting that this triglycoside might have saccharides at the C3, C7, or C4' position. The unknown triglycoside had the same UV absorption spectra as kaempferol 3-O-sophorotrioside which indicates glycosylation at the C3 and C7 positions [43,67,142]. MS data showed a parent ion peak at m/z 757 ([M+H]+), with fragments at 611 (loss of the rhamnosyl of the glycoside, the third sugar at the C3 position), 449 (loss of the hexosyl of the glycoside, the second sugar at the C3 position), and 287 ([A+H]+), formed by loss of the hexosyl of the
LC-MS Profiling and Quantification of Food Phenolic Components ... 31
O
O HO
OH OH
OH
O O O
rhamnosyl
OH O rhamnosyl
OH
2"
Y2
Y1
Y0
6"
m/z
300 400 500 600 700
0 5 10 15
[M+H]+ Y2+
Y0+
757.1 611.0
303.2
355.1 465.2
411.2
Y1+
Figure 11. Mass spectrum (PI100) of quercetin 3-O-2",6"-dirhamnosylglucoside in Ginkgo leaf extract and the related fragmentation scheme.
glycoside, the first sugar at the C7 position). Thus, the analyzed glycoside was provisionally identified as kaempferol 3-O-rhamnosylhexoside-7-O-hexoside. The compound was then positively identified as kaempferol 3-O- rutinoside-7-O-galactoside which has been reported in lentils and has matching retention times and UV and MS data [142].
The retention times and UV spectra of the other unkown lentil glycoside also matched those of kaempferol suggesting that theis tetraglycoside also had saccharides at the C3 and C7 positions. As shown in Figure 12, kaempferol O-tetraglycoside lost its first glycosyl, a hexosyl of the trisaccharides at the C3 or C7 position, to form the fragment at m/z 741 (Y3, 903-162=741). The glycoside lost a second glycosyl, a rhamnosyl of the disaccharide at the C3 or C7 position, to form the fragment at m/z 595 (Y2, 741-146=595), followed by loss of its third glycosyl, the hexosyl, connected to the C3 position to form the fragment at m/z 433 (Y1, 597-162=433), and finally, lost its last glycosyl, a rhamnosyl at the C7 position to form its aglycone ion at m/z 287 (Y0, i.e., [A+H]+, 433-146=287). Thus, this flavonoid should be kaempferol 3-O-hexosyl-rhamnosyl-hexoside-7-O-rhamnoside or one of its isomers. Since kaempferol 3-O-rutinoside-7-O-rhamnoside has been reported in lentils [35, 142], the unknown compound is most likely kaempferol 3-O-hexosylrutinoside-7-O-rhamnoside, and less likely to be kaempferol 3-O-rutinoside-7-O-hexosylrhamnoside, for biogenetic reasons.
Long-Ze Lin and James M. Harnly 32
Recently, two lentil kaempferol tetraglycosides were isolated as a mixture and their structures were elucidated by NMR [152]. They were identified as kaempferol 3-O-beta- glucopyranosyl(1→2)-O-[alpha-rhamnopyranosyl(1→6)]-beta-galactopyranoside-7-O-alpha-rhamnopyranoside and kaempferol
Recently, two lentil kaempferol tetraglycosides were isolated as a mixture and their structures were elucidated by NMR [152]. They were identified as kaempferol 3-O-beta- glucopyranosyl(1→2)-O-[alpha-rhamnopyranosyl(1→6)]-beta-galactopyranoside-7-O-alpha-rhamnopyranoside and kaempferol