microbilogie

Chapter 9

Analysis of Molecular Species of Plant Glycolipidsby HPLC/APCI-MS

Ryo Yamauchi

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

IntroductionEdible plant glycolipids are thought to be nutrients in the human diet. Glycolipidsin higher plants consist mainly of steryl glycosides, glyceroglycolipids, and sphin-goglycolipids. These glycolipids are widely distributed, if not universal, in edibleplants (1,2). Plant glycolipid classes have been separated directly and quantified bynormal-phase high-performance liquid chromatography (HPLC) in previous studies(3,4). However, the molecular species of each glycolipid class were not fully charac-terized. Ripe fruit of the red bell pepper (Capsicum annuum L.) are used widely asvegetables and food additives, such as ground pepper (paprika) and oleoresin,which are good sources of carotenoid pigments. Red bell peppers also contain allthree of the above-mentioned glycolipid classes (5,6), and some micronutrientssuch as vitamins A, C, and E (7–9), but limited information is available on the con-tent and composition of such nutrients in fresh or processed products.

Atmospheric pressure chemical ionization mass spectrometry (APCI-MS) hasproven to be a very valuable technique for analysis of lipids from a variety ofclasses (10). This paper describes direct analyses of glycolipids from red bell pep-per using HPLC coupled with on-line APCI-MS. The glycolipid classes were firstseparated 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 (glucocere-broside, CMG, 5) (Fig. 9.1), and then the molecular species of each glycolipidwere 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 dis-tributor. The fruits were processed into pastes within the same day after harvestand stored at –30°C until analysis.

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Isolation of Glycolipid Classes

The lyophilized fruit pastes (100 g dry weight) of red bell pepper were extractedwith 600 mL chloroform/methanol (2:1, vol/vol) three times, and the total lipidswere 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 chro-matography (silica-gel BW-820MH, 70–200 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 wasr e m o v e d 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-

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



raphy (silica-gel 60 TLC, 0.25 mm thickness; Merck, Darmstadt, Germany) developedin 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, Rf0.14), were detected on the TLC plate. The bulk of the glycolipid fraction (0.79 g) wasthen separated by silica-gel column chromatography (2.5 × 30 cm). Each lipid classwas sequentially eluted by increasing the methanol concentration in mixtures of c h l o r o -form/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 sepa-rated into its molecular species by preparative HPLC. Reversed-phase HPLC wasdone 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 at205 nm. Proton nuclear magnetic resonance (1H NMR) spectra of molecular species ofCMG were recorded with a Varian Inova 400 FT-NMR spectrometer (Varian, PaloAlto, CA) with CDCl3/ C D3OD (2:1, vol/vol) as the solvent and tetramethylsilane asthe internal standard.

Analysis of Components in Glycolipids

The fatty acid (FA) and sugar compositions were determined by gas-liquid chro-matography (GLC) (12). The sterol composition was determined by GLC of thetrimethylsilyl derivatives after saponification (12,13).

HPLC/APCI-MS

HPLC was carried out using a Shimadzu LC-10AVv p pump equipped with aShimadzu SPD-10Av p ultraviolet/visible (UV/vis) detector (Shimadzu Co., Kyoto,Japan). Sample lipids were separated isocratically on a Luna 3µ C18(2) column (2.0 ×150 mm, Phenomenex, Torrance, CA) at 40°C. The mobile phase wasmethanol/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 main-tained at 0.2 mL/min. On-line UV detection at 205 nm was performed before MSdetection. APCI-MS was performed using a Shimadzu LCMS-QP8000α q u a d r u p o l emass spectrometer. The MS parameters were optimized by direct infusion of polyeth-yleneglycol standards into the source. An APCI probe voltage of 4.5 kV and a temper-ature of 400°C were used. Nebulizing gas (nitrogen) was delivered at a flow rate of 2.5L/min. The curved desolvation line (CDL) voltage was at –40 V with a temperature of250°C. The deflector voltage was maintained at +70 V for the analysis of ASG and SGor at +80 V for the analysis of MGDG, DGDG, and CMG. Ionization was performedin the positive-ion mode for all analyses and mass spectra were acquired in the massrange m / z 200–1000 at a scan rate of 3 s.



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. Eachglucoside contained campesterol and β-sitosterol as the sterol moieties, whose percent-ages, 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 compo-sition 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 α-linolenic acid (18:3, 8.3%).

HPLC analysis of ASG indicated at least seven molecular species, 1 a – g (Fig. 9.2).The APCI-MS mass spectrum of each peak exhibited the Na+ adduct ([M + Na]+) andfragment ions corresponding to campesterol at m / z 383.4 ([C2 8H4 7]+), 397.4( [ C2 8H4 5O ]+), and 215.2 ([C1 6H2 3]+), or β-sitosterol at m / z 397.4 ([C2 9H4 9]+), 411.4( [ C2 9H4 7O ]+), and 215.2 ([C1 6H2 3]+), and the fatty acyl moiety ([R2C O ]+) at m / z 2 3 9 . 2(16:0), 267.3 (18:0), 263.2 (18:2), or 261.2 (18:3). Thus, compounds 1 a – g were identi-fied as follows: 1 a, β-sitosteryl (6′-O-linolenoyl)-glucoside; 1 b, campesteryl (6′-O-linoleoyl)-glucoside; 1 c, β-sitosteryl (6′-O-linoleoyl)-glucoside; 1 d, campesteryl (6′-O-palmitoyl)-glucoside; 1 e, β-sitosteryl (6′-O-palmitoyl)-glucoside; 1 f, campesteryl (6′-O-stearoyl)-glucoside, and 1 g, β-sitosteryl (6′-O-stearoyl)-glucoside. The peak area per-centages of ASG 1 a – g in the total ion chromatogram in Figure 9.2 were calculated to be7.5, 11.2, 22.4, 13.1, 31.4, 5.1 and 9.2%, respectively, which corresponded to the per-centages calculated from the data of sterol and FA compositions described above.

HPLC analysis of SG showed two peaks, 2 a and 2 b (Fig. 9.3). The APCI-MSmass spectrum of each peak exhibited the Na+ adduct ([M + Na]+) and fragment ionscorresponding to the sterol moiety ([RC1 9H2 6O ]+, [RC1 9H2 8]+, and [C1 6H2 3]+). Thus,2 a was identified as campesteryl glucoside, having APCI-MS ions at m/z 585.4 ([M +N a ]+), 397.4 ([C2 8H4 5O ]+), 383.4 ([C2 8H4 7]+), and 215.2 ([C1 6H2 3]+); 2 b was β-sitosteryl glucoside (2 b), with APCI-MS ions at m / z 599.4 ([M + Na]+), 411.4( [ C2 9H4 7O ]+), 397.4 ([C2 9H4 9]+), and 215.2 ([C1 6H2 3]+). The peak area percentages ofSG 2 a and 2 b in the total ion chromatogram in Figure 9.3 were calculated to be 26.1and 73.9%, respectively, which corresponded to the sterol composition of SG deter-mined by GLC after saponification.

Molecular Species of Monogalactosyldiacylglycerol andD i g a l a c t o s y l d i a c y l g l y c e r o l

The FA compositions of MGDG (3) and DGDG (4) were determined by GLC to be asfollows: MGDG, 16:0 (3.6%), 18:0 (1.6%), oleic acid (18:1, 1.5%), 18:2 (8.6%), and18:3 (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 majorpeak, 3 a, and five minor peaks, 3 b – f, appeared in the total ion chromatogram ofMGDG (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 fromred bell pepper by HPLC/APCI-MS. Acylated steryl glucoside fraction (1) was separat-ed on a Luna C1 8 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 massspectra of peaks 1a–g are shown.



N a ]+) and fragment ions corresponding to diacylglycerol ([CH2( O C O R1) C H–

( O C O R2) C H2O H2]+ and [CH2( O C O R1) C H ( O C O R 2) C H 2]+), monoacylglycerol( [ C H2( O C O R1) C H ( O H ) C H2]+ and [CH2( O H ) C H ( O C O R2) C H2]+), and fatty acylmoieties ([R1CO]+ and [R2CO]+) (Figs. 9.4 and 9.5, Tables 9.1 and 9.2). Thus, themolecular species of MGDG were found to be 18:3/18:3 (3 a), 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 DGDGwere 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 calculatedfrom the total ion chromatograms indicated that the 18:3/18:3 species was predom-inant in MGDG, whereas the 18:3/18:3, 16:0/18:3, and 18:0/18:3 species were pre-dominant in DGDG (Tables 9.2 and 9.3).

The reversed-phase HPLC technique has been reported to allow rapid andreproducible separations of the main molecular species of the plant galactolipidsMGDG and DGDG (14,15). However, collection of the separated fractions andanalysis of the component FA by GLC was required to identify molecular species.The present on-line APCI-MS technique has enabled direct identification of themolecular species of these galactolipids without such processing, although it didnot give information on the sn positions of the two FA moieties, whether they werethe same or were two different FA.

Fig. 9.3. Total ion chromatogram and mass spectra of steryl glucoside from red bell pep-per by HPLC/APCI-MS. Steryl glucoside fraction (2) was separated on a Luna C1 8 c o l u m n(2 × 150 mm) developed with methanol/water (98:2, vol/vol) at 0.2 mL/min. The eluatewas monitored by total ions of APCI-MS. The mass spectra of peaks 2 a and 2 b are shown.



Molecular Species of Ceramide Monoglucoside

The CMG fraction (5) gave seven peaks, 5a–g, in the HPLC/APCI-MS total ionchromatogram (Fig. 9.6). Peaks 5 a – g were further separated by preparativereversed-phase HPLC, and their structures were identified by 1H NMR (16–19) as

Fig. 9.4. Total ion chromatogram and mass spectra of monogalactosyldiacylglycerolfrom red bell pepper by HPLC/APCI-MS. HPLC conditions were the same as describedin Figure 9.3. The mass spectra of peaks 3a–f are shown.



Fig. 9.5. Total ion chromatogram and mass spectra of digalactosyldiacylglycerol fromred bell pepper by HPLC/APCI-MS. HPLC conditions were the same as described inFigure 9.3. The mass spectra of peaks 4a–g 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- β-D-g l u c o p y r a n o s y l - 4 - h y d r o x y - 8 -sphingenine; 5 b, (4E, 8Z) -N- 2 ′- h y d r o x y p a l m i t o y l - 1 -O-β-D- g l u c o p y r a n o s y l - 4 , 8 - s p h i ng a-dienine; 5 c, (8Z)-N-2′-h y d r o x y p a l m i t o y l - 1 -O-β-D-glucopyranosyl-8-sphingenine; 5 d,( 8E) -N- 2 ′-h y d r o x y d o c o s a n o y l - 1 -O-β-D- g l u c o p y r a n o s y l - 4 - h y d r o x y - 8 - s p h i n g enine; 5 e,( 8E) -N- 2′- h y d r o x y t r i c o s a n o y l - 1 -O-β-D-glucopyranosyl-4-hydroxy-8-sphingenine; 5 f,( 8E) -N- 2′-h y d r o x y t e t r a c o s a n o y l - 1 -O-β-D- g l u c o p y r a n o s y l - 4 - h y d r o x y - 8 - s p h i n g e n i n e ;5 g, (8E) -N- 2′-h y d r o x y p e n t a c o s a n o y l - 1 -O-β-D-g l u c o p y r a n o s y l - 4 - h y d r o x y - 8 - s p h i n g e-nine (Fig. 9.7).

The APCI-MS mass spectra of CMG 5a–g exhibited the expected quasi-mole-cular ions ([M + H]+) and fragment ions corresponding to ceramide ([M –C6H9O5]+, [M – C6H11O6]+, and [M – C6H13O7]+), the sphingoid moiety ([R1CH–( O H ) C H ( N H2) C H2O H2]+, [R1C H ( O H ) C H ( N H2) C H2]+, [R1C H = C H ( N H2) C H2]+,and [sphingoid – (H2O )2]+), and the 2-hydroxy fatty acyl moiety ([R2C H ( O H ) –CONH3]+) (Fig. 9.6 and Table 9.3). The mass spectra of 5a and 5d–g showed thesame fragment ions at m/z 316.3 ([C14H27CH(OH)–CH(OH)CH(NH2)CH2OH2]+),298.3 ([C1 4H2 7C H ( O H ) C H ( O H ) C H ( N H2) C H2]+), 280.3 ([C1 4H2 7C H ( O H )CH=CH(NH2)CH2]+), and 262.3 ([sphingoid – (H2O)2]+), indicating the presenceof 4-hydroxy-8-sphingenine as the sphingoid moiety. On the other hand, the mainp e a k 5 b showed a different fragmentation pattern, which was characterized byintense fragment ions at m/z 696.5 ([M – OH]+), 516.5 (ceramide moiety, [M –C6H1 3O7]+), and 262.3 (sphingoid moiety, [C1 5H2 7C H = C H ( N H2) C H2]+), due tothe presence of 4,8-sphingadienine in the molecule. Whitaker (6) reported thatreversed-phase HPLC of cerebrosides isolated from bell pepper fruits gave onemajor peak in addition to four minor peaks. He deduced that the structure of themajor peak was (4E, 8Z) - 1 -O-β- g l u c o s y l -N- ( 2′- h y d r o x y p a l m i t o y l ) - 4 -t r a n s- 8 -c i s-sphingadienine, which corresponds to 5b in our chromatogram, and aside from theassignment of sphingoid cis/trans double bonds, he correctly deduced the struc-tures of 5c–f. Our analytical methods using APCI-MS have the advantage of directidentification of almost all of the molecular species of cerebrosides in the red bellpepper without the need for hydrolysis and derivatization. However, the 1H NMRanalysis was essential for the identification of the sphingoid c i s / t r a n s d o u b l ebonds.

Edible plant glycolipids are believed to play a role in the human diet as nutri-ents, but little is known about their processing and absorption in the digestive tractof mammals (20,21). Since molecular species containing α-linolenic acid (18:3)were the major species in MGDG and DGDG, these glycerogalactolipids would bean important source for this n-3 essential FA. To clarify the nutritional roles ofplant glycolipids, the average daily intake in the human body has been estimated tobe 140 mg ASG, 65 mg SG, 50 mg ceramide monohexoside, 90 mg MGDG, and220 mg DGDG (4). Fruits of red bell pepper appear to be a rich source of such gly-colipids in addition to the sources of carotenoid pigments and some other micronu-trients.



Fig. 9.6. Total ion chromatogram and mass spectra of ceramide monoglucoside fromred bell pepper by HPLC/APCI-MS. HPLC conditions are the same as described inFigure 9.3. Mass spectra of peaks 5a–g are shown.




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SummaryFive major glycolipid classes (ASG, SG, MGDG, DGDG, and CMG) from the fruitpaste of red bell pepper were separated by silica-gel column chromatography. Themolecular species of each glycolipid were separated and identified by reversed-phaseHPLC coupled with on-line APCI-MS. The molecular species of SG were β- s i t o s t e r y land campesteryl glucosides, and those of ASG were their fatty acid esters. The dili-nolenoyl species was predominant in MGDG in addition to small amounts of fiveother molecular species, whereas DGDG consisted of seven molecular species varyingin their degrees of unsaturation. The CMG class contained at least seven molecularspecies, which were characterized by 1H NMR. The combination of HPLC and APCI-MS is convenient and reliable for the separation and identification of the molecularspecies of plant glycolipids without any chemical modifications.

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Fig. 9.7. Structures of molecular species of ceramide monoglucoside (5a–g).



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19. Yamauchi, R., K. Aizawa, T. Inakuma, and K. Kato, Analysis of Molecular Species ofGlycolipids in Fruit Pastes of Red Bell Pepper (Capsicum annuum L.) by High-Performance Liquid Chromatography–Mass Spectrometry, J. Agric. Food Chem. 49:622–627 (2001).



20. Andersson, L., C. Bratt, K.C. Arnoldsson, B. Herslöf, N.U. Olsson, B. Sternby, et al.,Hydrolysis of Galactolipids by Human Pancreatic Lipolytic Enzymes and DuodenalContents, J. Lipid Res. 36: 1392–1400 (1995).

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microbilogie

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plant glycolipid classes

red bell peppers

glycolipid fraction

steryl glucoside sg

steryl glycosides

analysis of lipids

acylated steryl glucosideasg

phase hplcapcims