食品中叶黄素、玉米黄质和β-隐黄质同时检测方法的建立
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摘要
本文采用高效液相色谱法建立了食品中叶黄素、玉米黄质、β-隐黄质同时检测的方法,最终确定最优前处理条件为:试样中加入30 mL含0.1%的2,6-二叔丁基-4-甲基苯酚(BHT)乙醇溶液和10 mL 60%氢氧化钾水溶液,置于恒温水浴箱内50℃振荡皂化15 min,处理样液用100 mL正己烷∶乙醚∶环己烷(2∶2∶1)混合液萃取。结果显示,实验建立的方法提取效果优于国标方法和超声提取方法,可以实现叶黄素的快速温和提取,提取效果较佳。以小米和大米为样品进行方法学验证,结果显示,叶黄素、玉米黄质、β-隐黄质分别在0.2342～9.3689、0.3004～12.0167、0.3734～14.9371μg/mL范围内线性关系良好,平行测定6次,叶黄素、玉米黄质的相对标准偏差(RSD)分别为1.41%、4.98%,小米中未检测到β-隐黄质,加标回收率均在85.6%～98.8%,表明该方法重现行良好、准确可行。该方法的建立可以弥补相关标准的不足,解决实际工作中存在的问题,为产品开发,食物评价和监管提供技术方法。
In this study,a method for the simultaneous analysis and detection of lutein,zeaxanthin and β-cryptoxanthin in food was developed using high-performance liquid chromatography(HPLC). The optimal pretreatment conditions were adding with 30 mL 0.1% 2,6-di-tertbutyl-4-methyl phenol(BHT)ethanol solution and 10 mL 60% potassium hydroxide solution for samples. Then the samples were saponificated in a water bath for 15 min at a constant temperature of 50 ℃ by thermostatic waterbath,followed by extraction using 100 mL solvent mixture of n-hexane,ethyl ether and cyclohexane(2∶2∶1). The results showed that this method was superior to the national standard and ultrasonic extraction,because it could be applied rapidly and the mild extraction of lutein provided an enhanced extraction effect. A millet and rice sample was used for methodological verification and the results showed that the linear relations for lutein,zeaxanthin and β-cryptoxanthin were good in the range of 0.2342～9.3689,0.3004～12.0167 and 0.3734～14.9371 μg/mL,respectively. Six parallel experiments were conducted and the relative standard deviations of lutein and zeaxanthin were 1.41% and 4.98%,respectively,and their spiked recoveries were between 85.6% and 98.8%,thus indicating that this method had sound repeatability and reliability characteristics. The establishment of this method can make up for the current deficiency of relevant standards. Furthermore,it provides solutions to various problems in practical work,as well as technical assistant for product development,food evaluation and supervision in the food production industry.
In this study,a method for the simultaneous analysis and detection of lutein,zeaxanthin and β-cryptoxanthin in food was developed using high-performance liquid chromatography(HPLC). The optimal pretreatment conditions were adding with 30 mL 0.1% 2,6-di-tertbutyl-4-methyl phenol(BHT)ethanol solution and 10 mL 60% potassium hydroxide solution for samples. Then the samples were saponificated in a water bath for 15 min at a constant temperature of 50 ℃ by thermostatic waterbath,followed by extraction using 100 mL solvent mixture of n-hexane,ethyl ether and cyclohexane(2∶2∶1). The results showed that this method was superior to the national standard and ultrasonic extraction,because it could be applied rapidly and the mild extraction of lutein provided an enhanced extraction effect. A millet and rice sample was used for methodological verification and the results showed that the linear relations for lutein,zeaxanthin and β-cryptoxanthin were good in the range of 0.2342～9.3689,0.3004～12.0167 and 0.3734～14.9371 μg/mL,respectively. Six parallel experiments were conducted and the relative standard deviations of lutein and zeaxanthin were 1.41% and 4.98%,respectively,and their spiked recoveries were between 85.6% and 98.8%,thus indicating that this method had sound repeatability and reliability characteristics. The establishment of this method can make up for the current deficiency of relevant standards. Furthermore,it provides solutions to various problems in practical work,as well as technical assistant for product development,food evaluation and supervision in the food production industry.
引文
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[3]余召辉,赵宗仁,李贺,等.叶黄素的药理作用[J].吉林医药学院学报,2017,38(1):58-60.
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[5]Tang L,Zhang Y,Jiang Y,et al.Dietary wolfberry ameliorates retinal structure abnormalities in db/db mice at the early stage of diabetes[J].Experimental Biology and Medicine,2011,236(9):1051-1063.
[6]Kim Se,Hyeuk Lee,Jung Min,et al.Proposed cytotoxic mechanisms of the saffron carotenoids crocin and crocetin on cancer cell lines[J].Biochemistry and Cell Biology-Biochimie ET Biologie Cellulaire,2014,92:105-111.
[7]Hofe Carolyn R,Feng Limin,Zephyr Dominique.Fruit and vegetable intake,as reflected by serum carotenoid concentrations,predicts reduced probability of polychlorinated biphenyl -associated risk for type 2 diabetes:National health and nutrition examination survey 2003～2004[J].Nutrition Research,2014,34:285-293.
[8]Heo J Y,Kim S,Kang J H,et al.Determination of lutein from green tea and green tea by-products using accelerated solvent extraction and UPLC[J].Journal of Food Science,2014,79(5):C816-C821.
[9]李涛,张慧,张志忠,等.β-隐黄质的特性及其应用[J].中国食品添加剂,2011(4):156-158.
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[13]康翠欣,朱静璇,汪芳芳,等.高效液相色谱法快速测定婴幼儿乳粉中叶黄素的含量[J].食品科技,2017(11):318-321,327.
[14]Mellado-Ortega E,Hornero-Méndez D.Isolation and identification of lutein esters,including their regioisomers,in tritordeum(×Tritordeum Ascherson et Graebner)grains:Evidence for a preferential xanthophyll acyltransferase activity[J].Food Chemistry,2012,135(3):1344-1352.
[15]程红艳,陈军辉,赵恒强,等.反相高效液相色谱-电喷雾质谱法测定浒苔中的叶黄素[J].食品科学,2010(18):206-211.
[16]时有明,孙艳琳,魏生贤,等.万寿菊花渣中残余叶黄素酯的红外光谱分析[J].光谱实验室,2010,27(4):1286-1289.
[17]Chen X,Wu J,Zhou S,et al.Application of near-infrared reflectance spectroscopy to evaluate the lutein and β-carotene in Chinese kale[J].Journal of Food Composition and Analysis,2009,22(2):148-153.
[18]Abrahamsson V,Rodriguez-Meizoso I,Turner C.Determination of carotenoids in microalgae using supercritical fluid extraction and chromatography[J].Journal of Chromatography A,2012,1250:63-68.
[19]陈青,刘志敏.超临界流体色谱的研究进展[J].分析化学,2004,32(8):1104-1109.
[20]Sivathanu B,Palaniswamy S.Purification and characterization of carotenoids from green algae Chlorococcum humicola by HPLC-NMR and LC-MS-APCI[J].Biomedicine & Preventive Nutrition,2012,2(4):276-282.
[21]Dachtler M,Glaser T,Kohler K,et al.Combined HPLC-MS and HPLC-NMR on-line coupling for the separation and determination of lutein and zeaxanthin stereoisomers in spinach and in retina[J].Analytical Chemistry,2001,73(3):667-674.
[22]中华人民共和国国家卫生和计划生育委员会.GB 5009.248-2016食品安全国家标准食品中叶黄素的测定.2016.
[23]Fratianni A,Mignogna R,Niro S,et al.Determination of lutein from fruit and vegetables through an alkaline hydrolysis extraction method and HPLC analysis[J].Journal of Food Science,2015,80(12):C2686-C2691.
[24]Huang X Y,Liu Y W,Di D L,et al.An improved LC-DAD method for simultaneous determination of lutein,β-carotene and lycopene in tomato and its products[J].Chromatographia,2010,71(3-4):■,et al.Determination of lutein by high-performance thin-layer chromatography using densitometry and screening of major dietary carotenoids in food supplements[J].Journal of Chromatography A,2012,1231:59-65.
[26]Aruna G,Mamatha B S,Baskaran V.Lutein content of selected Indian vegetables and vegetable oils determined by HPLC[J].Journal of food composition and analysis,2009,22(7-8):632-636.
[27]李大婧,刘春菊,肖亚冬,等.叶黄素及其顺式异构体的快速检测[J].食品科学,2016,37(4):206-211.
[2]Bian Q,Gao S,Zhou J,et al.Lutein and zeaxanthin supplementation reduces photooxidative damage and modulates the expression of inflammation-related genes in retinal pigment epithelial cells[J].Free Radical Biology and Medicine,2012,53(6):1298-1307.
[3]余召辉,赵宗仁,李贺,等.叶黄素的药理作用[J].吉林医药学院学报,2017,38(1):58-60.
[4]Wang C C,Chang S C,B Stephen Inbaraj,et al.Isolation of carotenoids,flavonoids and polysaccharides from Lycium barbarum L.and evaluation of antioxidant activity[J].Food Chemistry,2010,120:184-192.
[5]Tang L,Zhang Y,Jiang Y,et al.Dietary wolfberry ameliorates retinal structure abnormalities in db/db mice at the early stage of diabetes[J].Experimental Biology and Medicine,2011,236(9):1051-1063.
[6]Kim Se,Hyeuk Lee,Jung Min,et al.Proposed cytotoxic mechanisms of the saffron carotenoids crocin and crocetin on cancer cell lines[J].Biochemistry and Cell Biology-Biochimie ET Biologie Cellulaire,2014,92:105-111.
[7]Hofe Carolyn R,Feng Limin,Zephyr Dominique.Fruit and vegetable intake,as reflected by serum carotenoid concentrations,predicts reduced probability of polychlorinated biphenyl -associated risk for type 2 diabetes:National health and nutrition examination survey 2003～2004[J].Nutrition Research,2014,34:285-293.
[8]Heo J Y,Kim S,Kang J H,et al.Determination of lutein from green tea and green tea by-products using accelerated solvent extraction and UPLC[J].Journal of Food Science,2014,79(5):C816-C821.
[9]李涛,张慧,张志忠,等.β-隐黄质的特性及其应用[J].中国食品添加剂,2011(4):156-158.
[10]FAO/WHO press compendium of food additive specifications[C].Food & Agriculture Org,1990,52(9):105-106.
[11]Helrick K.Official methods of analysis[M].AOAC,1990.
[12]官咏仪,林海丹,邱志超,等.高效液相色谱法测定保健品中的叶黄素含量[J].食品安全质量检测学报,2017(10):4039-4043.
[13]康翠欣,朱静璇,汪芳芳,等.高效液相色谱法快速测定婴幼儿乳粉中叶黄素的含量[J].食品科技,2017(11):318-321,327.
[14]Mellado-Ortega E,Hornero-Méndez D.Isolation and identification of lutein esters,including their regioisomers,in tritordeum(×Tritordeum Ascherson et Graebner)grains:Evidence for a preferential xanthophyll acyltransferase activity[J].Food Chemistry,2012,135(3):1344-1352.
[15]程红艳,陈军辉,赵恒强,等.反相高效液相色谱-电喷雾质谱法测定浒苔中的叶黄素[J].食品科学,2010(18):206-211.
[16]时有明,孙艳琳,魏生贤,等.万寿菊花渣中残余叶黄素酯的红外光谱分析[J].光谱实验室,2010,27(4):1286-1289.
[17]Chen X,Wu J,Zhou S,et al.Application of near-infrared reflectance spectroscopy to evaluate the lutein and β-carotene in Chinese kale[J].Journal of Food Composition and Analysis,2009,22(2):148-153.
[18]Abrahamsson V,Rodriguez-Meizoso I,Turner C.Determination of carotenoids in microalgae using supercritical fluid extraction and chromatography[J].Journal of Chromatography A,2012,1250:63-68.
[19]陈青,刘志敏.超临界流体色谱的研究进展[J].分析化学,2004,32(8):1104-1109.
[20]Sivathanu B,Palaniswamy S.Purification and characterization of carotenoids from green algae Chlorococcum humicola by HPLC-NMR and LC-MS-APCI[J].Biomedicine & Preventive Nutrition,2012,2(4):276-282.
[21]Dachtler M,Glaser T,Kohler K,et al.Combined HPLC-MS and HPLC-NMR on-line coupling for the separation and determination of lutein and zeaxanthin stereoisomers in spinach and in retina[J].Analytical Chemistry,2001,73(3):667-674.
[22]中华人民共和国国家卫生和计划生育委员会.GB 5009.248-2016食品安全国家标准食品中叶黄素的测定.2016.
[23]Fratianni A,Mignogna R,Niro S,et al.Determination of lutein from fruit and vegetables through an alkaline hydrolysis extraction method and HPLC analysis[J].Journal of Food Science,2015,80(12):C2686-C2691.
[24]Huang X Y,Liu Y W,Di D L,et al.An improved LC-DAD method for simultaneous determination of lutein,β-carotene and lycopene in tomato and its products[J].Chromatographia,2010,71(3-4):■,et al.Determination of lutein by high-performance thin-layer chromatography using densitometry and screening of major dietary carotenoids in food supplements[J].Journal of Chromatography A,2012,1231:59-65.
[26]Aruna G,Mamatha B S,Baskaran V.Lutein content of selected Indian vegetables and vegetable oils determined by HPLC[J].Journal of food composition and analysis,2009,22(7-8):632-636.
[27]李大婧,刘春菊,肖亚冬,等.叶黄素及其顺式异构体的快速检测[J].食品科学,2016,37(4):206-211.