质谱测定虾青素酯中的脂肪酸-英文文献

Comparative Biochemistry and Physiology Part B 136(2003)317–3221096-4959/03/$-see front matter ᮊ2003Elsevier Inc.All rights reserved.doi:10.1016/S1096-4959(03)00209-4Fatty acids of astaxanthin esters in krill determined by mild massspectrometryShinichi Takaichi *,Kumi Matsui ,Masahisa Nakamura ,Mizuho Muramatsu ,a ,b bc Satoshi Hanada cBiological Laboratory,Nippon Medical School,Kosugi-cho,Nakahara,Kawasaki 211-0063,Japan aDepartment of Biology,School of Education,Waseda University,Nishiwaseda,Shinjuku 169-8050,Japan bNational Institute of Advanced Industrial Science and Technology (AIST ),Tsukuba 305-8566,JapancReceived 17April 2003;received in revised form 2July 2003;accepted 3July 2003AbstractKrill is a major source of astaxanthin,which has strong antioxidant activity.Fractions with astaxanthin monoesters and diesters of Antarctic krill Euphausia superba were isolated.Astaxanthin esters were separated by C18-HPLC depending on the number of carbons and double bonds of esterified fatty acid (s ).Small amounts of other lipids remained in the samples,but relative molecular masses of carotenoid esters could be measured by field desorption mass spectrometry without fragmentation and interference from contaminant lipids.The fatty acids were determined by calculation of difference between astaxanthin and astaxanthin esters.Only five kinds of fatty acids,dodecanoate,tetradecanoate,hexadecanoate,hexadecenoate and octadecenoate,were detected.Fast atom bombardment mass spectrom-etry and secondary ion mass spectrometry showed similar spectra.The fatty acid composition in astaxanthin esters was different from those in krill lipids.Therefore,determination of fatty acids in carotenoid esters by a combination of HPLC elution profile and mild mass spectrometry is found to be a useful tool.ᮊ2003Elsevier Inc.All rights reserved.Keywords:Astaxanthin;Astaxanthin ester;Carotenoid ester;Euphausia superba ;Fatty acid;Krill;Mass spectrometry1.IntroductionAntarctic krill are crustaceans with large bio-mass in the Southern Ocean and a good source of poly-unsaturated fatty acids,such as eicosapenta-enoate (C20:5)and docosahexaenoate (C22:6)(Bottino,1975;Phleger et al.,2002).Astaxanthin is a strong antioxidant (Palozza and Krinsky,1992),and it mainly distributes in the carapace of crustacean,the integuments of marine fish,the yeast Phaffia and green alga Haemato-*Corresponding author.Tel.:81-44-733-3584;fax:q 81-44-722-1231.E-mail address:takaichi@nms.ac.jp (S.Takaichi ).coccus sp.(Goodwin,1980,1984).Major carote-noids of krill are astaxanthin and its mono-and di-esters (Yamaguchi et al.,1983;Maoka et al.,1985;Foss et al.,1987).In krill,astaxanthin and its esters consist of three stereoisomers;(3R ,39R )-astaxanthin (60–70%)exists as a major compo-nent along with (3R ,39S ;meso )-astaxanthin (10–20%)and (3S ,39S )-astaxanthin (10–20%)(Maoka et al.,1985;Foss et al.,1987).The (3S ,39S )-form may be oxidized from (3R ,39R )-zeaxanthin,which is synthesized by plants in nature.The origin of the R -configuration of astaxanthin isomers in krill is still obscure.Although astaxanthin esters are known to be major carotenoids in marine animals,318S.Takaichi et al./Comparative Biochemistry and Physiology Part B 136(2003)317–322Fig.1.HPLC elution profile of carotenoids extracted from Ant-arctic krill Euphausia superba .See the text for details of the chromatographic conditions.A,M1–3and D1–5indicate the HPLC peaks,and the peak numbers are referred to in the text and figures.fatty acid compositions of astaxanthin esters in krill have not been investigated.In this study,astaxanthin esters in krill were analyzed by field desorption mass spectrometry (FD-MS ).The esterified fatty acids could be determined in spite of small amounts of other lipids remaining in the samples.2.Materials and methods 2.1.Samples and purificationAntarctic krill Euphausia superba was pur-chased in local markets.Carotenoids and lipids were extracted several times from the carapace of krill by acetone,and extracts were evaporated to dryness.The extract was dissolved in n -hexane and loaded on a column of Toyopearl DEAE-650M (Tosoh,Japan ),and the carotenoids and neutral lipids were eluted with n -hexane y acetone (8:2,v y v ),while polar lipids remained on the column (Takaichi et al.,2001).The carotenoid fraction was loaded on a column of silica gel 60(1.07734;Merck,Germany ),neutral lipids were eluted with n -hexane,a red band was eluted with n -hexane y acetone (9:1,v y v ),the next red band was with n -hexane y acetone (8:2,v y v )and a final red band with n -hexane y acetone (7:3,v y v ).Each red band was further purified by KC18TLC (Whatman,USA )developed with methanol and by silica gel TLC (1.13748;Merck )developed with n -hexane y acetone (8:2,v y v ),and finally each carotenoid ester was collected from HPLC described below.The HPLC system was equipped with a m -Bondapak C18column (8=100mm,RCM type;Waters,USA ),and eluted with methanol (2.0ml y min ).The absorption spectra of carotenoids were measured by an MCPD-3600photodiode array detector (Otsuka Electronics,Japan )attached to the HPLC apparatus (Takaichi and Ishidsu,1992).2.2.Analysis of carotenoid estersRelative molecular masses of carotenoids and carotenoid esters were measured by field desorp-tion mass spectrometry (FD-MS ),M-2500double-focusing gas chromatograph-mass spectrometer (Hitachi,Japan )equipped with a field-desorption apparatus (Takaichi,1993).Secondary ion mass spectrometry (SI-MS )and fast atom bombardment mass spectrometry (FAB-MS )were obtained fromthe M-2500and a JMS-HX y HX110A mass spec-trometer (JEOL,Japan ),respectively,using m -nitrobenzyl alcohol as a matrix.The fatty acid moieties of the carotenoid esters and krill lipids were converted to fatty acid methyl esters by treatment with 5%HCl in methanol solution at 608C for 20min (Takaichi and Ishidsu,1992).The fatty acid methyl esters were then analyzed by a gas chromatograph y mass spectrom-eter (M7200A GC y 3DQMS system;Hitachi )equipped with a DB-5ms capillary column (J&W Scientific,USA )coated with (5%-phenyl )-meth-ylpolysiloxane as described previously (Takaichi et al.,2001).3.Results3.1.Carotenoids and compositionFig.1shows a C18-HPLC elution profile of carotenoids extracted from Antarctic krill.Peaks A,M1–3and D1–5corresponded to the third,the second and the first red bands obtained from the silica gel column.The carotenoid in peak A was identified as astaxanthin based on the same retention time on HPLC and the same absorption spectrum with319S.Takaichi et al./Comparative Biochemistry and Physiology Part B 136(2003)317–322Fig.2.FD-MS spectra of astaxanthin monoesters in the peaks M1(a ),M2(b )and M3(c )in Fig.1.authentic astaxanthin (Roche,Switzerland )and the relative molecular mass of 596.All the HPLC peaks showed similar absorption spectra with astaxanthin in the HPLC eluent of methanol.By methanolysis of the carotenoid esters with HCl in methanol solution,carotenoid was liberated,and identified as astaxanthin as described above.From the retention times on HPLC,the second group (M1–3,Fig.1)and the third group (D1–5)were astaxanthin monoesters and astax-anthin diesters,respectively.The composition was astaxanthin (20%),astaxanthin monoesters (34%)and astaxanthin diesters (46%).3.2.Astaxanthin monoestersThe peaks M1,M2and M3were collected from HPLC.The major relative molecular mass of the peak M1analyzed by FD-MS was 778(Fig.2a ).Those of the peak M2were 806and 832(Fig.2b )and the peak M3were 834and 860(Fig.2c ).Recognized fragment peaks corresponding to carotenoid and fatty acid moieties were not foundin the low-mass region,and many mass peaks in Fig.2might be due to impurities.Similar mass spectra were obtained by SI-MS and FAB-MS (data not shown ).The relative molecular masses of carotenoid esters were easily determined under these mild mass spectrometry conditions in spite of the presence of lipids,while detection of the carotenoid moiety of the carotenoid esters was difficult by these methods due to the absence of fragmentation.Since SI-MS and FAB-MS require a matrix of m -nitrobenzyl alcohol,there are many mass peaks derived from the matrix,whereas FD-MS needs no matrixes and is more suitable for analysis of the relative molecular masses.The fatty acid moiety was calculated from the difference of the relative molecular masses of astaxanthin and astaxanthin esters.The mass of 778in peak M1(Fig.2a )corresponded to asta-xanthin dodecanoate (C12:0)monoester.Similarly,those of 806,832,834and 860corresponded to astaxanthin tetradecanoate (C14:0),hexadecenoate (C16:1),hexadecanoate (C16:0)and octa-decenoate (C18:1)monoesters,respectively.Two peaks on this HPLC system contained two astax-anthin monoesters.The peak M2contained astax-anthin C14:0and C16:1monoesters and the peak M3contained astaxanthin C16:0and C18:1mon-oesters.The HPLC peak areas of M1,M2and M3(Fig.1)were 17,29and 54%,respectively.The compositions of two astaxanthin monoesters in the peaks M2and M3were calculated from intensities of the mass peaks (Fig.2b and c ).The composition of astaxanthin monoesters were thus calculated to be 17,20,9,29and 25%of C12:0,C14:0,C16:1,C16:0and C18:1,respectively.The fatty acid moieties of the astaxanthin mono-ester fraction (M1–3)were converted into fatty acid methyl esters,and analyzed by GC.The major fatty acids were C16:0(76%)and C18:1(20%),and the trace amount of other fatty acids were also found.HPLC elution profile reflects esterified fatty acids of astaxanthin esters.When all of the fatty acid detected by GC described above was bound to astaxanthin as astaxanthin monoesters,HPLC elution profile was different from Fig. 1.This meant the astaxanthin monoester fraction (M1–3)contained other lipids,which could not been detected by FD-MS.3.3.Astaxanthin diestersThe major relative molecular masses in peak D5(Fig.1)were 1072,1098and 1124(Fig.3).In320S.Takaichi et al./Comparative Biochemistry and Physiology Part B136(2003)317–322Fig.3.FD-MS spectra of astaxanthin diesters in the peak D5 in Fig.1.the case of1072,the sum of the carbon number of fatty acids was32and the sum of double bond was zero.This corresponds to two C16:0.Similar-ly,for1098,the carbon number was34and the double bond was one,and the two fatty acids could be C16:0and C18:1.For1124,the carbon number was36and the double bond was two, corresponding to two C18:1.Consequently,peak D5contained astaxanthin C16:0and C18:1dies-ters.The major relative molecular masses in peak D4were1044,1070and1096.For1070,two pairs could be possible,C14:0and C18:1,and C16:0and C16:1diesters.Those of1044and1096 corresponded to C14:0and C16:0,and C16:1and C18:1diesters,respectively.In the peak D3,1016 corresponded to two C14:0,and C12:0and C16:0diesters,1042to C14:0and C16:1,and C12:0andC18:1diesters and1068to two C16:1diester.Inthe peak D2,988corresponded to C12:0and C14:0diester,and1014to C12:0and C16:1diester.Since the content of the peak D1was too small tocollect from HPLC,we could not measure therelative molecular masses,but this peak mightcontain astaxanthin C12:0diester from the reten-tion time on HPLC.The HPLC peak areas of D1,D2,D3,D4andD5(Fig.1)were3,12,33,28and24%,respec-tively.When the fatty acids of the astaxanthindiester fraction(D1–5)were analyzed by GC,they were C14:0(42%),C16:1(6%),C16:0(46%),C18:1(4%)and some minors.This com-position did not reflect the HPLC elution profile,and consequently,the astaxanthin diester fractionmight contain other lipids.Poly-unsaturated fattyacids were not found in astaxanthin esters basedon HPLC elution profile and FD-MS analysis.4.DiscussionThe presence of astaxanthin esters in animals, especially in marine,is well known(Goodwin, 1984).We found astaxanthin(20%),astaxanthin monoesters(34%)and astaxanthin diesters(46%) from Antarctic krill E.superba.Most carotenoids in krill have been reported as astaxanthin,and similar compositions have been reported for krill E.superba(Renstrøm and Liaaen-Jensen,1981; Yamaguchi et al.,1983;Maoka et al.,1985;Foss et al.,1987).Fatty acids of carotenoid esters have not been studied much in animals.We found that only five kinds of fatty acids,C12:0,C14:0,C16:0,C16:1 and C18:1,were esterified to astaxanthin esters inthe krill.Although poly-unsaturated fatty acidswere not found in astaxanthin esters,major fattyacids in the cellular lipids of krill was C14:0(17%),C16:0(26%),C16:1(16%),C18:1(8%), C20:5(26%)and C22:6(7%).Similar fatty acidcompositions have been reported(Bottino,1975;Phleger et al.,2002).Carotenoid esters occur in algae,fruits andpetals of higher plants,and animals and are notfound in bacteria(Goodwin,1980,1984).Acetate(C2:0)is common among algal carotenoids,e.g. fucoxanthin and peridinin.In siphonaxanthin ester of green-algae,2-trans-tetradecenoate(C14:1)is usually a specific and sole fatty acid esterified,but recently,other specific fatty acids have been found (Y oshii et al.,2002).Bacteria such as Rhodococ-cus,Halorhodospira and Heliorestis contain carot-enoid glucoside esters,whose fatty acids are only a few specific ones(Takaichi et al.,1990,2001, 2003;Niggli and Pfander,1999).In these,HPLC elution profiles showed few peaks corresponding to each carotenoid ester or carotenoid glucoside ester(Takaichi et al.,1990;Y oshii et al.,2002). Carotenoid esters of plants,such as lutein diesters from marigold petals,capsantin and capsorubin esters from red bell peppers and carotenoid esters in orange peels and concentrates(Zonta et al., 1987;Philip and Chen,1988;Wingerath et al., 1996),have many fatty acids.In these,HPLC elution profiles showed many carotenoid ester peaks.Contamination of carotenoid esters with otherlipids affects fatty acid composition analyzed byGC.Indeed,the fatty acid composition of theastaxanthin monoester fraction(M1–3,Fig.1)in321 S.Takaichi et al./Comparative Biochemistry and Physiology Part B136(2003)317–322krill determined by HPLC and FD-MS was differ-ent from that by GC in this study.In this study,we could determine some fattyacids in astaxanthin esters by FD-MS withoutfragmentation.FD-MS has been reported to showonly a molecular ion without fragmentation forcarotenoids,carotenoid glucoside and carotenoidglucoside esters(Takaichi,1993).SI-MS and FAB-MS showed similar spectra.Therefore,these meth-ods are useful tools for determination of fatty acidsin carotenoid esters containing small amounts oflipid impurities,and can be useful for carotenoidester analysis in animals and fruits,which containlarge amounts of lipids.Carbon numbers and sumof double bonds can be calculated for carotenoiddiesters,while determination of carbon number ofeach individual fatty acid is impossible by thismethod.Therefore,other spectroscopic methodsare necessary for fully understanding all the asta-xanthin esters.Major fatty acids among11different from asta-xanthin monoesters and diesters in shrimp Pan-dalus borealis was C16:1,C18:1,docosenoate (C22:1)and C22:6(Renstrøm and Liaaen-Jensen, 1981).In this case,the fatty acid composition ofthe purified astaxanthin esters was analyzed byGC.Major fatty acids in total phospholipids ineyes of P.borealis were C16:0,C18:1,C20:5andC22:6(Bell and Dick,1990).From the red crab langostilla Pleuroncodes plan-ipes,one astaxanthin monoester fraction and three astaxanthin diester fractions were purified from silica gel TLC(Coral-Hinostroza and Bjerkeng, 2002).Separation of astaxanthin diesters reflects the different esterified fatty acids,but the fatty acid composition analyzed by GC was similar to each other.Concerning biosynthesis of astaxanthin esters,an esterase must be present.This esterase canesterify both sides of astaxanthin to produce asta-xanthin monoesters and diesters,and only fivefatty acids appear to be substrates. 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