反相高效液相色谱测定食品中酚类抗氧化剂_英文_

PHENOLIC ANTIOXIDANTS DETERMINATION IN FOOD　Received date :2010-10-20;revision received date :2010-12-20　E -mail :lubingx in @njnu .edu .cnITEMS USING REVERSED -PHASE HPLCChen X ij in 1,Zhang Zhicai2(1.Colleg e of U rban Constructio n &Safety Eng ineer ing ,N anjing U niver sity of T echnolog y ,N anjing ,210009,P.R.China;2.Scho ol of F oo d Science and Bio techno lo gy ,Jiang su U niver sity ,Zhenjiang ,212013,P.R.China)Abstract :T he determ inatio n of synthetic phenolic ant iox idants (SPA s)including pro pyl g allate (P G ),ter tiar y but yl hy dr oquino ne (T BHQ ),butylated hy dr o xy anisole (BHA )a nd but ylated hy dr ox y toluene (BHT )in foo d item s is repor ted using hig h per for mance liquid chr omato gr aphy (HP LC ).A C 18column is used as the sta tio nar y pha se,acet onitr ile and wat er:A cet ic acid (1%)is used as t he mobile phase of g radient elutio n and the U V det ect or is set at 280nm.U nder the abov e conditions,fo ur ant iox idents is complet ely separ ated w it hin 8min.T he limit of detect ion ,linear r ange ,and r epr o ducibility o f HP LC ar e ev aluated .Isolation par ameter s o f SP A s fr om differ ent ty pes o f foo d items (cooking o il,mar gar ine and butt er,and cheese)ar e optimized.SPA s are ex tra ct ed fro m fo od it ems thro ugh ex tr actio n with methanol/acet onitr ile (1∶1,in vo lume ),vo rtex ,ult raso nic tr eatment and pr ecipitation in a freezer (2h).F or co oking o il marg ar ine,but ter and cheese at 50and 200mg /L ,r eco ver ies o f SPA s are 93.3%—108.3%(PG ),85.3%—108.3%(T BHQ ),96.7%—101.2%(BHA ),and 73.9%—94.6%(BHT ).T he metho d is applied to the determina tio n o f SPA s in 38foo d it ems (16co oking oils,8marg ar ine,6butter and 6cheese samples).T he lev els of SPA s in posit ive samples are all belo w the leg al limit s of China .Key words :phenolic antio xidants (P A s );hig h per fo rmance liquid chro mato gr aphy (HPL C );gr adient elution ;fo odCLC number :X792 Document code :A Article ID :1005-1120(2011)02-0218-06INTRODUCTIONNaturalandartificialantiox idantsplayimportant r oles in inhibiting o il ox idatio n reactio n of food.It is w ell kno wn that too m uch ox idatio n reaction will cause adverse influence ,pr oducing unpleasantodorsandharmfulcompoundsincluding aldehydes,ketones and or ganic acids.Since natural antio xidants are generally unstable,fo odproducerspreferartificialantio xidantsincluding pro py l gallate (PG ),ter tiary butyl hydr oquinone (TBHQ),butylated hy drox yanisole (BHA )and butylated hydr oxy toluene (BHT ).Their str uctures are show n in Fig.1.Adding sy nthetic phenolic antiox idants (SPAs )to fat,co oking oil can inhibit ox idation reaction inproduction and deposit pr ocess,thus ther e havebeen mo re than 50y ears for the use of SPAs in food industry [1-2].How ever,there ex ist problems in the use of SPAs that BHA and BHT are revealed to be har mful to liver and cause cancer[3-4].So,the use o f SPAs in fo od is strictlyregulated,though different countries have their different perm itted lim its.For ex ample,the use of T BHQ is allo w ed in USA,but not in som e Europeancountries.Generally,SPAconcentration of 100—200ug /g in oil and fat is allow ed,and this is the direct reason for the determination o f SPAs in the food industry quality control pro cess [5].For the analysis of antioxidants ,sev er al different techniques have been used ,including thin -lay erchromatog raphy(TCL )[6],gasF ig.1　Chemical str uctur es o f studied SPA schro mato graphy(GC)[7],capillary electropho resis (CE)[8]and stripping voltammetry[9],yet all of the metho d above may be interfered by the samples. With its advantag es of high precision and sensitivity,the high perform ance liquid chro mato graphy(HPLC)became the main technique for the analy sis o f SPAs in meat juice, dehydr ated soups,meat gravy,dehy drated m eat, dehydr ated pet food,baked food,palm oil, po tatoes and co rn chips,popcor n,cheese, breakfast cereals,drink pow der mix ture and livers[10].In the related analysis,the recov er ies o f spiked PG,T BHQ and BHA are above90%, tho ug h the reco very of BHT is o nly75%—90%, w hich do es not m eet the analy sis requirem ents.Sample preparation is the ex perim ent step before the HPLC separatio n,and it is a key point fo r the ex periment.T he solid phase extractio n metho d is mainly used in antiox idant co ncentratio n and separatio n w hich is based o n so lubility principle.T he liquid-liquid extractio n metho d is w idely used in multi-com po nent so lution separation of SPAs in fo od.So,the exper im ent is to set up a simple method for the analy sis of SPA s.1　MATERIALS AND METHODSThere are38food samples including16kinds ofco oking oil,16kinds o f bread spr ead and6 kinds of bread cream.T he16kinds o f bread spread include6kinds of butter and10kinds of cheese.PG(97%),TBHQ(97%),BHA(98%)and BHT(99%)are pur chased from Aldr ich Co.(WI, U SA),and MeOH and ACN(HPLC grade)are pur chased from Merck(Dar mstadt,Germany). Glacial acetic acid and isopropano l are A.R.g rade.W ater is tw o tim es distilled w ater.SPAs stock standard solution are prepar ed in M eOH/ACN(1∶1,in vo lum e)w ith500mg/ml as the co ncentratio n,after being shakened to a clear so lution,sealed by aluminum foil,and sto red at4°C for o ne month.Before used in the HPLC analysis,the sto ck standard solutions are diluted with1∶1(in volume)MeOH/ACN to be suitable concentr ations.T he instrumentation used in the sample preparation pro cess includes ultr asound bath(up 5200H)and v ortex vibrator(VORT EX-6,Beijing Chuangbo Bio-Tech Co.Ltd.).The UV absor ption spectra are perfo rmed using UV-visible spectro meter(Varian,Cary50).A sample(10.0g)is ex tracted w ith M eOH/ ACN in a corked flask(100.0ml)for15min by shaking under high spread,and then is centrifuged at3500r/m in for10min.The super natant is collected and co oled in the refrigerato r for1h.T he clear liquid obtained is injected directly into the HPLC sy stem.The extractio n pro cess is show n in Fig2.F ig.2　Scheme fo r deter mination of SP AsT he HPLC analyses are carried out on a Varian HPLC sy stem(Ag ilent HPLC120), consisting of an auto sampler,a240Pro Star pum p,a320Pro Star U V detecto r o perated byCP -SCANVIEW version 6so ftw are .HPLC separ ation is carr ied out o n a particle size 250mm ×4.0mm and Lichrospher column 5um.T he mobile phase is com po sed o f water (1%H AC)as mobile phase A and acetonitrile (1%ACN )as mobile phase B.The analytical separation is perform ed using gradient elution.A seg mented gr adient of m obile phase B is increased fro m 30%to 90%in 5min ,follow ed by ramping of m obile phase B to 100%in 4min and is held co nstantly fo r 1—2min.Then the mobile phase is filtered and deg assed.T he U V detectio n w av elength is set at 280nm.The flow rate is m aintained at 1.5ml/min.After each experiment,the sy stem is eluted w ith w ater /MeOH (20∶80,in volum e )fo r 30m in .2　RESULTS AND DISCUSSION2.1　SPAs UV absorption spectraThe max imum absor ption appear s at 275,295,290and 280nm for PG,T BHQ,BHA and BHTrespectively.So the HPLCdetectio nw avelength is set at 280nm .2.2　HPLC conditionWhen the mobile phase is chosen as M eOH/ACN (1∶1,in volume )w ith flo w rate of 1.0ml /m in,BHA and BH T are separ ated w ell,but no t for PG and TBHQ.With mobile phase A as M eOH /ACN (1∶1,in vo lum e ),B as w ater /acetic acid (99∶1,in volume )of ratio A ∶B =98∶2(in vo lum e)and flo wr ate of 0.5ml/m in,PG and TBHQ still could not be separated.So,the g radient elution reported by Razali et al [11]is used w ith little adjustment,by using M eOH/ACN (1∶1,in volume)and w ater /HAC(99∶1,in vo lum e )as mobile phase ;or ACN and M eOH /ACN (1∶1,in volume )as m obile phase .The acidified water is used to pro hibit ionization of hydrox yl in phenol co mpounds[12].T he tw o mobile phases both can g iv e w ell separatio n.The second one is faster,so it is cho sen in the experiment.Within o nly 8m in,PG ,TBHQ ,BHA and BHT are separated in sequence ,w hich is in acco rdance w ith their polarity,as show n in Fig. 3.2.3　Linearity and detection limitsT he SPA standard mix ture solution (0.1—1.0mg /l)are injected into HPLC to o btain its sensitivity .T he detection limits are obtained withF ig .3　Chro mato gr ams fo r gr adient elutio n o f SP A -fr ee palm o lein sample spiked with 50mg ・l -1each SPA a t flowr ate of 1.5ml ・m in -1sig nal no ise ratio3.T he detectio n limit for PG is 0.3mg/l.The calibration curres are linear over the r ang e of1.0—300mg/l fo r all the fo ur SPA s.2.4　Reproducibility studyThe intra-day repeatability of the peak area is exam ined by injecting50m g/l SPAs m ix tur e so lution5tim es into the HPLC system.Relativ e standard dev iation(RSD)for retention tim es and peak areas are0.5%—1.6%and1.1%—3.8%, respectiv ely.T he same metho d is used to o btain the repro ducibility after5day s,and it is fo und that RSD for retention times and peak areas ar e all below4%.2.5　Optimization of extraction condition2.5.1　Recov ery o f coo king oil sampleSample preparation is a very important pr ocess befor e HPLC analysis.The aim of the sam ple preparation pro cess is to increase the analy sis sensitivity by remov ing interfering matrix co mpo nents and particulates and concentrating the analyses.PG,TBHQ,BHA and BHT disso lved in isopropano l are added in M alaysia refined palm oil w hich does not co ntain SPAs.The final co ncentratio ns for these SPAs are50and200m g/ l for each.T he solutions are mixed w ell to hav e all the SPAs dissolved.Razali et al[11]used extraction w ith methanol metho d to analyze the sam e palm o il.But extaction w ith m ethanol does no t suit well w ith the ex traction of SPAs,especially for the extraction o f BHT.T his is w hy the paper studies extraction w ith MeOH/ACN(1∶1,in v olume). The shortcoming of ex traction with methanol is that the reco veries of PG and TBHQ are high,as the ex traction of fat and pro tein are achieved sim ultaneously,but the recov ery of BHT is low because of its low polarity[13].According to repo rt,ACN is a suitable ex tr actant.T hus,in the ex periment,MeOH/ACN is used.After extractio n and ultrasound bath,the ex tr acts are sav ed in refrigerator for2h.T he recoveries are o bviously increased by using MeOH/ACN(1∶1,in v olum e)as extractant.T he same effect is achieved by using ultrasound/v ortex technique,especially for the extractio n of BHT.T he final results sho w that the reco veries for the four SPAs are94.6%—108.3%.2.5.2　Recoveries of bread spread and cheese sam plesT he recovery ex perim ent of bread spread and cheese is done w ith the same recovery m ethod described above,and the recovery of BHT from the bread spread is fo und to be very low.So, increase the time for ultrasound bath from15min to25min,vor tex tim e from15s(1200r/min)to 5min4m in(1400r/min)or1min(1600r/ m in),thus the r ecoveries of the fo ur SPAs from bread and cheese can be increased.T he recov eries of bread spread and cheese sam ples spiked w ith SPAs200mg/l are listed in T able1,found with the optimized ex traction conditio n.T able1show s that the recov ery of BHT recov ery is sim ilar w ith the values found by other research groups(for ex ample,Rafecas et al[14]found the value to be87%w ith ACN/ propanol as ex tractant,and Karo vicˇov & Sˇimko[10]fo und the value to be74%w ith ACN as extractant).Our exper im ent show s that w ith M eOH/ACN(1∶1,in vo lum e)as ex tratant and after ultrasound bath and v ortex v ibration,the extractio n rate can be increased.Also, refrigerato r storing can help to precipitate fat m aterial,and all of these techniques can simplify the sam ples w hich can increase the HPLC column life term.Table1　C omparison of recoveries of SPAs in f ood products(n=3)SPA s50mg/lOil Cheese Br ead spr ead200mg/lO il Cheese Bread spr eadP G107.999.7108.1104.293.2104.1 T BHQ104.186.1107.4103.692.894.8 BHA98.4102.197.696.297.196.82.5.3　Sam ple analysisThe details of the foods containing SPAs are listed in Table2.T he sam ples ar e analyzed w ith the optim ized extraction condition.T he SPA peaks are identified by retention time,and this metho d is proved by spiked sam ple.The deter mination is achieved by ex ternal standard metho d using linear regression.Sixteen coo king oils are analyzed,and the results show that most samples do not contain SPAs ex cept for tw o samples containing88.9mg/ kg BHT and20.2ng/kg TBHQ,as show n in Table 2.Among16bread spread sam ples,7 sam ples are fo und to contain BHT(14.4—175.0 mg/kg)and8samples are found to contain BHA (5.2—103.9m g/kg),as sho wn in Table2.All the foo d items containing SPAs are from M alay sia,w hich contain BHA or BHT,or both. No SPA is found in imported bread spread sam ples.No PG or TBHQ is found in any bread spread sam ples.No SPA is found in6cheese samples.The values of SPA amount fo und are below200m g/kg w hich is the M alaysia maxim um permitted levels.T he fo od pr oduer m ar ks the related SPAs containing food abo ut the antiox idant,but not about the kinds and amounts.The results also show that ther e is a hig h absorption at 3.5(R.T.)in each the chrom ato grams o f the cheese samples except for1 cheese sample,and the peak may corr espond to preserv atives,such as sor bic acid or benzoic acid. This is proved by injection o f sorbic acid and benzoic acid samples dissolv ed in M eOH/ACN(1∶1,in volume)into the HPLC system under the same condition,and the r esults show that the peaks of sorbic acid and benzoic acid ar e at 3.5 min(R.T.),w hich m eans that most cheese samples contain preservatives.Table2　Levels of SPAs f ound in SPA-positive f ood itemsSample P G BHA BHT T BHQ T o talRefined palm olein n.d.n.d.n.d.20.620.6 n.d.n.d.89.2n.d.89.2But ter n.d.n.d.36.8n.d.36.8 n.d.25.837.4n.d.63.2 n.d.n.d.15.4n.d.15.4 n.d.45.740.6n.d.86.3A r tificial butt er n.d.103.870.2n.d.174 n.d.n.d.142.8n.d.142.8 n.d.66.554.3n.d.120.8 n.d.71.552.1n.d.123.6 n.d.63.854.8n.d.118.6 n.d.44.672.5n.d.117.1 n.d. 5.3151.7n.d.1573　CONCLUSIONAnalysis of SPAs is an im por tant task in executing foo d safety law s.U sing liquid-liquid extraction w ith M eOH/ACN as ex tr actant can increase the recoveries of SPAs fro m oil,bread spread and cheese samples.U ltrasound bath and vor tex vibrator can increase r ecoveries.Our extraction method uses less or ganic solvent co mpar ed w ith previous studies[7,14].12.5%o f the oil samples contained SPAs,w hile no cheese sam ples contain SPAs.68.7%bread spread sam ples contain SPAs(14.4—75.0ng/kg) though the am o-unt is bellow the permitted lev el of200mg/kg.References:[1]　Fo rmanek Z,K er ry J P,Hig gins F M,et al.Additio n o f sy nt het ic a nd nat ur al ant iox idants to a-toco pher yl acetat e supplemented beef pa tties:Effects of antio xidants and pa ckag ing o n lipidox idatio n[J].M eat Science,2001,58:337-341. [2]　M cCa rthy T L,K er ry J P,K err y J F,et al.Evalua tion of t he antio xidant po tential of natur alfoo d/plant ex tr acts as compar ed with syntheticant iox idants and vitamin E in r aw and coo ked por kpatties[J].M eat Science,2001,57:45-52.[3]　G rice H C.Safety ev aluation o f buty lat edhy dr oto luene(BHT)in t he liver,lung andg astr ointestinal tr act[J].Fo od and ChemicalT ox ico lo gy,1986,24:1127-1130.[4]　W ichi H P.Enchanced t umpr development bybutylated hydro x yaniso le(BHA)fro m theper spectiv e of effect o n fo resto mach and oesophag ealsquamo us epithelium[J].F oo d and ChemicalT ox ico lo gy,1988,26:717-723.[5]　N o guera-O rt J F,V illanuev a-Caman~as R M,R amis-R amos G.D ir ect injectio n o f edible o ils asmicro emulsio ns in a micellar mobile phase applied tot he liquid chr oma tog raphic deter mination ofsy nt het ic a ntiox idants[J].A naly tica Chimica A ct a,1999,387:127-134.[6]　R agazzi E,V er onese G.Quantitativ e analysis ofphenolic compounds after thin-lay er chr omat og raphyseparat ion[J].Jo urnal o f Chro mato gr aphy,1973,77:369-375.[7]　G onz lez M,G alleg o M,V alc rcel M.Gaschr omato gr aphic flo w metho d fo r thepr eco ncentr atio n and simultaneous determ inatio n ofantiox idant and pr eserv ativ e additiv es in fatt y foo ds[J].Jo ur nal o f Chro matog r aphy A,1999,848:529-536.[8]　Boy ce M C,Spickett E E.Separa tio n of food g r adeantiox idants(synthetic and nat ur al)using mix edmicellar electr okinet ic capillar y chr oma tog raphy[J].Jour nal of A gr icultural and Fo od Chemist ry,1999,47:1970-1975.[9]　Guanghan L,Y u W,L eiming Y,et al.Deter minatio n of ascor bic acid in fr uits andveg etables by str ipping vo lt ammetr y on a g lassycar bon elect ro de[J].F ood Chemistry,1994,51:237-239.[10]K ar ov icˇov J,Sˇimko P.Review:deter mination of sy nthetic antiox idants in foo d by high-per for manceliquid chr omato gr aphy[J].Jour nal ofChr omat og raphy A,2000,882:271-281.[11]R azali I,N or haya H,N o rasima h A S.Deter minatio n of ant iox idants in palm oil pro ducts byhigh perfo rm ance liquid chro mato gr aphy[J].Elaeis,1997,9:25-33.[12]L pez M,M ar t nezF,Del V alle C,et al.A na ly sisof pheno lic constit uents of bio lo gical inter est in r edwines by high-per for mance liquid chr om atog ra phy[J].Jo urnal of Chro mato gr aphy A,2001,922:359-363.[13]P ag e B D,Charbo nneau C F.L iquidchr omat og raphic determinatio n o f seven antiox idantsin dr y fo od[J].Journal of the A sso ciatio n o f OfficialAnalyt ical Chemists,1989,72:259-265.[14]R afecas M,Guar diola F,Iller a M,et al.L iquidchr omat og raphic determinatio n o f pheno licant iox idants in baker y pro ducts[J].Jo ur nal ofChr omat og raphy A,1998,822:305-309.反相高效液相色谱测定食品中酚类抗氧化剂陈锡进1　张志才2(1.南京工业大学城市建设与安全工程学院,南京,210009,中国;2.江苏大学食品与生物工程学院,镇江,212013,中国)摘要:描述了利用高效液相色谱(HPL C)测定食品中没食子酸丙酯(PG),叔丁基对苯二酚(T BHQ),叔丁基羟基茴香醚(BHA)和二丁基羟基甲苯(BHT)4种合成酚类抗氧化剂(SP As)的方法。