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Chapter 9

Analysis of Molecular Species of Plant Glycolipids by HPLC/APCI-MS


Ryo Yamauchi
Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan

Introduction
Edible plant glycolipids are thought to be nutrients in the human diet. Glycolipids in higher plants consist mainly of steryl glycosides, glyceroglycolipids, and sphingoglycolipids. These glycolipids are widely distributed, if not universal, in edible plants (1,2). Plant glycolipid classes have been separated directly and quantified by normal-phase high-performance liquid chromatography (HPLC) in previous studies (3,4). However, the molecular species of each glycolipid class were not fully characterized. Ripe fruit of the red bell pepper (Capsicum annuum L.) are used widely as vegetables and food additives, such as ground pepper (paprika) and oleoresin, which are good sources of carotenoid pigments. Red bell peppers also contain all three of the above-mentioned glycolipid classes (5,6), and some micronutrients such as vitamins A, C, and E (79), but limited information is available on the content and composition of such nutrients in fresh or processed products. Atmospheric pressure chemical ionization mass spectrometry (APCI-MS) has proven to be a very valuable technique for analysis of lipids from a variety of classes (10). This paper describes direct analyses of glycolipids from red bell pepper using HPLC coupled with on-line APCI-MS. The glycolipid classes were first separated by silica-gel column chromatography to obtain acylated steryl glucoside (ASG, 1), steryl glucoside (SG, 2), monogalactosyldiacylglycerol (MGDG, 3), digalactosyldiacylglycerol (DGDG, 4), and ceramide monoglucoside (glucocerebroside, CMG, 5) (Fig. 9.1), and then the molecular species of each glycolipid were separated and characterized by reversed-phase HPLC/APCI-MS.

Materials and Methods


Materials Fruit of the red bell pepper (C. annuum L. var. Capia) was supplied by a local distributor. The fruits were processed into pastes within the same day after harvest and stored at 30C until analysis.

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Fig. 9.1. Representative structures of acylated steryl glucoside (1), steryl glucoside (2), monogalactosyldiacylglycerol ( 3 ), digalactosyldiacylglycerol ( 4 ), and ceramide monoglucoside (5).

Isolation of Glycolipid Classes The lyophilized fruit pastes (100 g dry weight) of red bell pepper were extracted with 600 mL chloroform/methanol (2:1, vol/vol) three times, and the total lipids were obtained following the method of Folch et al. (11). The total lipids (3.26 g) were dissolved in 20 mL of chloroform and subjected to silica-gel column chromatography (silica-gel BW-820MH, 70200 mesh; Fuji Silysia Chemical Ltd., Kasugai, Japan; 4.5 ! 30 cm) with sequential elutions of chloroform (1 L), acetone (2 L), and methanol (1 L). Each solvent eluate was pooled, and the solvent was removed in vacuo to obtain neutral lipids (1.73 g) from the chloroform, glycolipids (0.79 g) from the acetone, and phospholipids (0.63 g) from the methanol, respectivel y . An aliquot of the glycolipid fraction was analyzed by silica-gel thin-layer chromatog-

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raphy (silica-gel 60 TLC, 0.25 mm thickness; Merck, Darmstadt, Germany) developed in chloroform/methanol (85:15, vol/vol). Six major spots, capsanthin (Rf 0.85), ASG (1, Rf 0.75), MGDG (3, Rf 0.60), SG (2, Rf 0.43), CMG (5, Rf 0.31), and DGDG (4, Rf 0.14), were detected on the TLC plate. The bulk of the glycolipid fraction (0.79 g) was then separated by silica-gel column chromatography (2.5 ! 30 cm). Each lipid class was sequentially eluted by increasing the methanol concentration in mixtures of chloroform/methanol: a red pigment capsanthin (35 mg) was eluted with chloroform/ methanol (99:1, vol/vol); ASG (43 mg), chloroform/methanol (98:2, vol/vol); MDDG (138 mg), chloroform/methanol (95:5, vol/vol); SG (171 mg), chloroform/methanol (90:10, vol/vol); CMG (76 mg), chloroform/methanol (85:15, vol/vol); and DGDG (141 mg), chloroform/methanol (80:20, vol/vol). The CMG fraction was further separated into its molecular species by preparative HPLC. Reversed-phase HPLC was done with an Inertsil Prep-ODS column (1.0 ! 25 cm; GL Sciences, Tokyo, Japan) developed with methanol at a flow rate of 5 mL/min and the eluate was monitored at 205 nm. Proton nuclear magnetic resonance (1H NMR) spectra of molecular species of CMG were recorded with a Varian Inova 400 FT-NMR spectrometer (Varian, Palo Alto, CA) with CDCl3/CD3OD (2:1, vol/vol) as the solvent and tetramethylsilane as the internal standard. Analysis of Components in Glycolipids The fatty acid (FA) and sugar compositions were determined by gas-liquid chromatography (GLC) (12). The sterol composition was determined by GLC of the trimethylsilyl derivatives after saponification (12,13). HPLC/APCI-MS HPLC was carried out using a Shimadzu LC-10AVvp pump equipped with a Shimadzu SPD-10Av p ultraviolet/visible (UV/vis) detector (Shimadzu Co., Kyoto, Japan). Sample lipids were separated isocratically on a Luna 3 C18(2) column (2.0 ! 150 mm, Phenomenex, Torrance, CA) at 40C. The mobile phase was methanol/ethanol (3:2, vol/vol) for the analysis of ASG or methanol/water (98:2, vol/vol) for the analyses of SG, MGDG, DGDG, and CMG, with the flow rate maintained at 0.2 mL/min. On-line UV detection at 205 nm was performed before MS detection. APCI-MS was performed using a Shimadzu LCMS-QP8000" quadrupole mass spectrometer. The MS parameters were optimized by direct infusion of polyethyleneglycol standards into the source. An APCI probe voltage of 4.5 kV and a temperature of 400C were used. Nebulizing gas (nitrogen) was delivered at a flow rate of 2.5 L/min. The curved desolvation line (CDL) voltage was at 40 V with a temperature of 250C. The deflector voltage was maintained at +70 V for the analysis of ASG and SG or at +80 V for the analysis of MGDG, DGDG, and CMG. Ionization was performed in the positive-ion mode for all analyses and mass spectra were acquired in the mass range m / z 2001000 at a scan rate of 3 s.

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Results and Discussion


Molecular Species of Acylated Steryl Glucoside and Steryl Glucoside The components of ASG (1) and SG (2) were analyzed by GLC after hydrolysis. Each glucoside contained campesterol and #-sitosterol as the sterol moieties, whose percentages, respectively, were 24.3 and 75.7% for ASG, and 27.7 and 72.3% for SG. Glucose was the only sugar detected in both compounds. Furthermore, the FA composition of ASG was determined to be palmitic acid (16:0, 49.9%), stearic acid (18:0, 8.7%), linoleic acid (18:2, 33.1%), and "-linolenic acid (18:3, 8.3%). HPLC analysis of ASG indicated at least seven molecular species, 1ag (Fig. 9.2). The APCI-MS mass spectrum of each peak exhibited the Na+ adduct ([M + Na]+) and fragment ions corresponding to campesterol at m / z 383.4 ([C 28 H47 ]+ ), 397.4 ([C28H45O]+), and 215.2 ([C16H23]+), or #-sitosterol at m/z 397.4 ([C29H49]+), 411.4 ([C29H47O]+), and 215.2 ([C16H23]+), and the fatty acyl moiety ([R2CO]+) at m/z 239.2 (16:0), 267.3 (18:0), 263.2 (18:2), or 261.2 (18:3). Thus, compounds 1ag were identified as follows: 1 a, #-sitosteryl (6$-O-linolenoyl)-glucoside; 1b, campesteryl (6$-Olinoleoyl)-glucoside; 1c, #-sitosteryl (6$-O-linoleoyl)-glucoside; 1 d, campesteryl (6$-Opalmitoyl)-glucoside; 1e, #-sitosteryl (6$-O-palmitoyl)-glucoside; 1f, campesteryl (6$-Ostearoyl)-glucoside, and 1g, #-sitosteryl (6$-O-stearoyl)-glucoside. The peak area percentages of ASG 1ag in the total ion chromatogram in Figure 9.2 were calculated to be 7.5, 11.2, 22.4, 13.1, 31.4, 5.1 and 9.2%, respectively, which corresponded to the percentages calculated from the data of sterol and FA compositions described above. HPLC analysis of SG showed two peaks, 2a and 2b (Fig. 9.3). The APCI-MS mass spectrum of each peak exhibited the Na+ adduct ([M + Na]+) and fragment ions corresponding to the sterol moiety ([RC19H26O]+, [RC19H28]+, and [C16H23]+). Thus, 2a was identified as campesteryl glucoside, having APCI-MS ions at m/z 585.4 ([M + Na]+), 397.4 ([C28H45O]+), 383.4 ([C28H47]+), and 215.2 ([C16H23]+); 2b was #sitosteryl glucoside (2 b), with APCI-MS ions at m/z 599.4 ([M + Na]+), 411.4 ([C29H47O]+), 397.4 ([C29H49]+), and 215.2 ([C16H23]+). The peak area percentages of SG 2a and 2b in the total ion chromatogram in Figure 9.3 were calculated to be 26.1 and 73.9%, respectively, which corresponded to the sterol composition of SG determined by GLC after saponification. Molecular Species of Monogalactosyldiacylglycerol and Digalactosyldiacylglycerol The FA compositions of MGDG (3) and DGDG (4) were determined by GLC to be as follows: MGDG, 16:0 (3.6%), 18:0 (1.6%), oleic acid (18:1, 1.5%), 18:2 (8.6%), and 18:3 (86.0%); DGDG, 16:0 (13.9%), 18:0 (7.8%), 18:1 (1.5%), 18:2 (12.5%), 18:3 (64.3%). MGDG and DGDG were analyzed by reversed-phase HPLC. One major peak, 3 a, and five minor peaks, 3 b f, appeared in the total ion chromatogram of MGDG (Fig. 9.4), whereas seven peaks, 4 a g, appeared in that of DGDG (Fig. 9.5). The APCI-MS mass spectrum of each peak exhibited the Na+ adduct ([M +

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Fig. 9.2. Total ion chromatogram and mass spectra of acylated steryl glucoside from red bell pepper by HPLC/APCI-MS. Acylated steryl glucoside fraction (1) was separated on a Luna C18 column (2 ! 150 mm) developed with methanol/ethanol (3:2, vol/vol) at 0.2 mL/min. The eluate was monitored by total ions of APCI-MS. The mass spectra of peaks 1ag are shown.

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Fig. 9.3. Total ion chromatogram and mass spectra of steryl glucoside from red bell pepper by HPLC/APCI-MS. Steryl glucoside fraction (2) was separated on a Luna C18 column (2 ! 150 mm) developed with methanol/water (98:2, vol/vol) at 0.2 mL/min. The eluate was monitored by total ions of APCI-MS. The mass spectra of peaks 2 a and 2 b are shown.

Na] +) and fragment ions corresponding to diacylglycerol ([CH2 (OCOR 1)CH (OCOR2)CH 2OH2]+ and [CH2(OCOR1)CH(OCOR 2)CH 2]+), monoacylglycerol ([CH2(OCOR1)CH(OH)CH2]+ and [CH2(OH)CH(OCOR2)CH2]+), and fatty acyl moieties ([R1CO]+ and [R2CO]+) (Figs. 9.4 and 9.5, Tables 9.1 and 9.2). Thus, the molecular species of MGDG were found to be 18:3/18:3 (3a), 18:2/18:3 (3 b ), 16:0/18:3 (3c), 18:1/18:3 (3d), 16:0/18:2 (3e), and 18:0/18:3 (3f); those of DGDG were 18:3/18:3 (4a), 18:2/18:3 (4b), 16:0/18:3 (4c), 18:1/18:3 (4d), 16:0/18:2 (4e), 18:0/18:3 (4f), and 18:0/18:2 (4g). The molecular species compositions calculated from the total ion chromatograms indicated that the 18:3/18:3 species was predominant in MGDG, whereas the 18:3/18:3, 16:0/18:3, and 18:0/18:3 species were predominant in DGDG (Tables 9.2 and 9.3). The reversed-phase HPLC technique has been reported to allow rapid and reproducible separations of the main molecular species of the plant galactolipids MGDG and DGDG (14,15). However, collection of the separated fractions and analysis of the component FA by GLC was required to identify molecular species. The present on-line APCI-MS technique has enabled direct identification of the molecular species of these galactolipids without such processing, although it did not give information on the sn positions of the two FA moieties, whether they were the same or were two different FA.

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Fig. 9.4. Total ion chromatogram and mass spectra of monogalactosyldiacylglycerol from red bell pepper by HPLC/APCI-MS. HPLC conditions were the same as described in Figure 9.3. The mass spectra of peaks 3af are shown.

Molecular Species of Ceramide Monoglucoside The CMG fraction (5) gave seven peaks, 5ag, in the HPLC/APCI-MS total ion chromatogram (Fig. 9.6). Peaks 5 a g were further separated by preparative reversed-phase HPLC, and their structures were identified by 1H NMR (1619) as

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Fig. 9.5. Total ion chromatogram and mass spectra of digalactosyldiacylglycerol from red bell pepper by HPLC/APCI-MS. HPLC conditions were the same as described in Figure 9.3. The mass spectra of peaks 4ag are shown.

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follows: 5 a, (8E)-N- 2$- h y d r o x y p a l m i t o y l - 1 -O -#-D -glucopyranosyl-4-hydroxy-8sphingenine; 5b, (4E,8Z)-N-2$-hydroxypalmitoyl-1-O-#-D-glucopyranosyl-4,8-sphingadienine; 5c, (8Z)-N -2$-hydroxypalmitoyl-1-O-#-D-glucopyranosyl-8-sphingenine; 5d, (8E) -N- 2$-hydroxydocosanoyl-1-O -#-D-glucopyranosyl-4-hydroxy-8-sphingenine; 5e, ( 8E) -N -2$- h y d r o x y t r i c o s a n o y l - 1 -O -#-D-glucopyranosyl-4-hydroxy-8-sphingenine; 5f, ( 8E) -N- 2$-hydroxytetracosanoyl-1-O -#-D-glucopyranosyl-4-hydroxy-8-sphingenine; 5 g, (8E) -N- 2$-hydroxypentacosanoyl-1-O-#-D-glucopyranosyl-4-hydroxy-8-sphingenine (Fig. 9.7). The APCI-MS mass spectra of CMG 5ag exhibited the expected quasi-molecular ions ([M + H]+ ) and fragment ions corresponding to ceramide ([M C6H9O5]+, [M C6H11O6]+, and [M C6H13O7]+), the sphingoid moiety ([R1CH (OH)CH(NH2)CH2OH2]+, [R1CH(OH)CH(NH2)CH2]+, [R1CH=CH(NH2)CH2]+, and [sphingoid (H2O)2]+), and the 2-hydroxy fatty acyl moiety ([R2CH(OH) CONH3]+) (Fig. 9.6 and Table 9.3). The mass spectra of 5a and 5dg showed the same fragment ions at m/z 316.3 ([C14H27CH(OH)CH(OH)CH(NH2)CH2OH2]+), 298.3 ([C 1 4H 2 7 C H ( O H ) C H ( O H ) C H ( N H 2 ) C H 2 ] + ), 280.3 ([C 1 4 H 2 7 C H ( O H ) CH=CH(NH2)CH2]+), and 262.3 ([sphingoid (H2O)2]+), indicating the presence of 4-hydroxy-8-sphingenine as the sphingoid moiety. On the other hand, the main peak 5b showed a different fragmentation pattern, which was characterized by intense fragment ions at m/z 696.5 ([M OH]+), 516.5 (ceramide moiety, [M C6H13O7]+), and 262.3 (sphingoid moiety, [C15H27CH=CH(NH2)CH2]+), due to the presence of 4,8-sphingadienine in the molecule. Whitaker (6) reported that reversed-phase HPLC of cerebrosides isolated from bell pepper fruits gave one major peak in addition to four minor peaks. He deduced that the structure of the major peak was (4E, 8Z) - 1 -O-# -glucosyl-N- ( 2$-hydroxypalmitoyl)-4-trans- 8 -cis sphingadienine, which corresponds to 5b in our chromatogram, and aside from the assignment of sphingoid cis/trans double bonds, he correctly deduced the structures of 5cf. Our analytical methods using APCI-MS have the advantage of direct identification of almost all of the molecular species of cerebrosides in the red bell pepper without the need for hydrolysis and derivatization. However, the 1H NMR analysis was essential for the identification of the sphingoid cis/trans double bonds. Edible plant glycolipids are believed to play a role in the human diet as nutrients, but little is known about their processing and absorption in the digestive tract of mammals (20,21). Since molecular species containing "-linolenic acid (18:3) were the major species in MGDG and DGDG, these glycerogalactolipids would be an important source for this n-3 essential FA. To clarify the nutritional roles of plant glycolipids, the average daily intake in the human body has been estimated to be 140 mg ASG, 65 mg SG, 50 mg ceramide monohexoside, 90 mg MGDG, and 220 mg DGDG (4). Fruits of red bell pepper appear to be a rich source of such glycolipids in addition to the sources of carotenoid pigments and some other micronutrients.

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Fig. 9.6. Total ion chromatogram and mass spectra of ceramide monoglucoside from red bell pepper by HPLC/APCI-MS. HPLC conditions are the same as described in Figure 9.3. Mass spectra of peaks 5ag are shown.

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Fig. 9.7. Structures of molecular species of ceramide monoglucoside (5ag).

Summary
Five major glycolipid classes (ASG, SG, MGDG, DGDG, and CMG) from the fruit paste of red bell pepper were separated by silica-gel column chromatography. The molecular species of each glycolipid were separated and identified by reversed-phase HPLC coupled with on-line APCI-MS. The molecular species of SG were #-sitosteryl and campesteryl glucosides, and those of ASG were their fatty acid esters. The dilinolenoyl species was predominant in MGDG in addition to small amounts of five other molecular species, whereas DGDG consisted of seven molecular species varying in their degrees of unsaturation. The CMG class contained at least seven molecular species, which were characterized by 1H NMR. The combination of HPLC and APCIMS is convenient and reliable for the separation and identification of the molecular species of plant glycolipids without any chemical modifications. References
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