2,4-二硝基氟苯柱前衍生HPLC-UV法测定海稻米中的γ-氨基丁酸含量
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摘要
为建立海水稻活性成分γ-氨基丁酸(GABA)的高效液相色谱-紫外检测(HPLC-UV)方法。以2,4-二硝基氟苯(FDNB)为衍生化试剂,采用高效液相色谱法测定了精白米、玉米、海稻米、发芽海稻米24 h、发芽海稻米48 h、发芽海稻米72 h的γ-氨基丁酸(GABA)含量,研究了FDNB的投量,醋酸铵溶液-乙腈作为流动相以及样品的pH对GABA衍生物保留时间的影响,并考察了该方法的精密度、重复性和稳定性及加标回收率。结果表明,发芽海稻米24 h中GABA的含量最高,为34.35μg/mg,玉米、未发芽海稻米、发芽海稻米48 h、发芽海稻米72 h中GABA的含量分别为为3.74、3.63、17.03、9.54μg/mg,而精白米中GABA含量几乎为零; FDNB投量为0.3 m L时,可确保反应完全;用0.5%醋酸铵水溶液∶乙腈(V∶V)=85∶15作流动相克服了泵压不稳定的现象;用硼酸盐缓冲液对衍生化后的样品进行稀释(1、5、10、15、20、40倍)后,样品的pH在9.0左右,GABA衍生物的保留时间几乎不漂移,在15.8 min左右;该方法精密度高、重复性和稳定性好,相对标准偏差值分别是0.08%、0.66%和0.60%;样品在24 h内稳定,回收率在99.29%～101.35%。结论本实验方法简便、结果可靠,可用于高含量GABA功能性食品的质量控制。
2,4-dinitrofluorobenzene( FDNB) was used as the derivatization reagent in this study to establish a method for the determination of γ-Aminobutyric acid( GABA) of polished rice,corn,sea rice,germinated sea rice for 24 h,germinated sea rice for 48 h,germinated sea rice for 72 h.The effect on the retention time of GABA derivatives of FDNB volume,ammonium acetate solution-acetonitrile as mobile phase and the pH value of the sample were studied using an HPLC-UV analyzer.The precision,repeatability and stability of the method and the recovery rate of the standard were also investigated,the result was that the content of GABA in the germinated sea rice was the highest which was 34.35 μg/mg,and the content of GABA in corn,ungerminated sea rice,germinated sea rice for 48 h and germinated sea for 72 h were 3.74,3.63,17.03 and 9.54 μg/mg, respectively. while the GABA content in polished rice was almost zero; When the FDNB dosage was up to 0.3 m L,the reaction is guaranteed to be complete; Using 0.5% ammonium acetate aqueous solution∶ acetonitrile( V∶ V) = 85∶ 15 as the mobile phase to overcome the instability of pump pressure; After diluted 1,5,10,15,20 and 40 time by borate buffer the pH value of samples was aroud 9.0 which ensure that the retention time of GABA derivatives hardly drifts.The precision,repeatability and stability of the method was high and the relative standard deviation values were 0.08%,0.66% and 0.60%,respectively.The sample was stable within 24 h and the recovery rate was 99.29% ～ 101.35%. The method was simple and reliable,it could be used for quality control of high content GABA functional foods.germination( p < 0.05). Sea rice is a plant resource with high GABAcontent,and the time of germination was shown to had a significant effect on the content of GABA. The sea rice with high content of GABA may be potentially applied in functional food. The simple and reliable method can be used for the quality control of functional food with high GABA content.
2,4-dinitrofluorobenzene( FDNB) was used as the derivatization reagent in this study to establish a method for the determination of γ-Aminobutyric acid( GABA) of polished rice,corn,sea rice,germinated sea rice for 24 h,germinated sea rice for 48 h,germinated sea rice for 72 h.The effect on the retention time of GABA derivatives of FDNB volume,ammonium acetate solution-acetonitrile as mobile phase and the pH value of the sample were studied using an HPLC-UV analyzer.The precision,repeatability and stability of the method and the recovery rate of the standard were also investigated,the result was that the content of GABA in the germinated sea rice was the highest which was 34.35 μg/mg,and the content of GABA in corn,ungerminated sea rice,germinated sea rice for 48 h and germinated sea for 72 h were 3.74,3.63,17.03 and 9.54 μg/mg, respectively. while the GABA content in polished rice was almost zero; When the FDNB dosage was up to 0.3 m L,the reaction is guaranteed to be complete; Using 0.5% ammonium acetate aqueous solution∶ acetonitrile( V∶ V) = 85∶ 15 as the mobile phase to overcome the instability of pump pressure; After diluted 1,5,10,15,20 and 40 time by borate buffer the pH value of samples was aroud 9.0 which ensure that the retention time of GABA derivatives hardly drifts.The precision,repeatability and stability of the method was high and the relative standard deviation values were 0.08%,0.66% and 0.60%,respectively.The sample was stable within 24 h and the recovery rate was 99.29% ～ 101.35%. The method was simple and reliable,it could be used for quality control of high content GABA functional foods.germination( p < 0.05). Sea rice is a plant resource with high GABAcontent,and the time of germination was shown to had a significant effect on the content of GABA. The sea rice with high content of GABA may be potentially applied in functional food. The simple and reliable method can be used for the quality control of functional food with high GABA content.
引文
[1]祝一文,赵方贵,成云峰,等.‘海稻86’耐盐碱胁迫生理机制的初步研究[J].青岛农业大学学报:自然科学版,2018,35(1):32-39.
[2]杨军,王海凤,郭涛.海稻86的籼粳性鉴定[J].山东农业科学2016,48(10):24-26.
[3]杨胜远,陆兆新,吕风霞,等.γ-氨基丁酸的生理功能和研究开发进展[J].食品科学,2005,26(9):546-551.
[4]Zheng Xiongxi,Ramamoorthy S,Hui Shen,et al. GABA transmission in the nucleus accumbens is altered after withdrawal from repeated cocaine[J].J Neurosci,2003,23(8):3498-3505.
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[6]陈恩成,张名位,彭超英,等.比色法快速测定糙米中γ-氨基丁酸含量研究[J].中国粮油学报,2006,21(1):125-128.
[7]张晖,徐永,姚惠源.纸层析法定量测定米胚芽中的γ-氨基丁酸[J].无锡轻工大学学报,2004,23(2):101-103.
[8]任红波.氨基酸自动分析仪快速测定糙米中的γ-氨基丁酸[J].杂粮作物,2003,23(4):246-247.
[9]刘兴勇,樊建麟,师江,等.玛咖中γ-氨基丁酸测定及其含量影响分析[J].现代食品科技,2017,33(2):205-209.
[10]何瑾,屈凡伟,张峻,等.超高效液相色谱/质谱法测定血液中γ-氨基丁酸[J].分析试验室,2015,34(9):1022-1025.
[11]程威威,吴跃,林亲录,等.HPLC法测定发芽糙米中氨基丁酸中不同衍生方式的比较研究[J].中国农学通报,2014,30(9):279-284.
[12]张晖,吴胜芳,姚惠源.OPA柱前自动衍生-紫外检测米胚芽中的γ-氨基丁酸的HPLC法研究[J].食品与发酵工业,2003,29(10):50-52.
[13]Amir Hayat,Taj Muhammad Jahangir,Muhammad Yar Khuhawar,et al.HPLC determination of gamma amino butyric acid(GABA)and some biogenic amines(BAs)in controlled,germinated,and fermented brown rice by pre-column derivatization[J].Journal of Cereal Science,2015,64:56-62.
[14]刘玲君,凌森,陈亮,等.高效液相色谱法检测含乳饮料中γ-氨基丁酸的含量[J].食品工业,2016(9):262-264.
[15]Nana Li,Yang Liu,Yue Zhao,et al. Simultaneous HPLC determination of amino acids in tea infusion coupled to precolumn derivatization with 2,4-dinitrofluorobenzene[J]. Food Anal Methods,2016,9:1307-1314.
[16]赵国群,王维.比色法快速测定γ-氨基丁酸含量[J].食品研究与开发,2015(10):85-89.
[17]樊星,陈红,高雪峰,等.溴甲酚绿比色法检测γ-氨基丁酸含量[J].现代商贸工业,2017(27):197-198.
[18]姜卫荣,蒋士新,孙永梅.右美托咪定对缺血缺氧性脑损伤新生大鼠的神经保护作用及机制[J].中国老年学,2017,37(10).
[19]刘兴勇,樊建麟,师江,等.玛咖中γ-氨基丁酸测定及其含量影响分析[J].现代食品科技,2017(2):205-209.
[20]赵海波.HPLC法测定发芽糙米中γ-氨基丁酸含量试验[J].农业科技与装备,2017(3):50-51.
[21]陈雪,刘明,汪丽萍,等.柱前在线衍生-HPLC法测定发芽糙米中γ-氨基丁酸含量[J].粮油食品科技,2017,25(3):48-53.
[22]周婷,程威威,林亲录,等.丹磺酰氯柱前衍生发芽糙米中γ-氨基丁酸的HPLC分析[J].食品科技,2014(1):282-286.
[23]张凡,周远征,赵远,等.HPLC异硫氰酸苯酯柱前衍生化测定黄柏及其不同炮制品中17种氨基酸的含量[J].中华中医药学刊,2017(9):2259-2262.
[24]张琳,尤进茂,单亦初,等.氨基酸的咔唑-9-乙基氯甲酸酯柱前衍生高效液相色谱分析[J].分析测试学报,2002,21(5):16-19.
[25]张志清,徐杰,丛军,等.高效液相色谱法测定发芽麦粒中γ-氨基丁酸(GABA)含量[J].中国粮油学报,2015,30(11):135-139.
[26]Zhang Z,Jie X,Cong J,et al. Determination ofγ-aminobutyric acid(GABA)in germinated wheat seed by HPLC[J].Journal of the Chinese Cereals&Oils Association,2015.
[27]Ding J,Ulanov A V,Dong M,et al.Enhancement of gamaaminobutyric acid(GABA)and other health-related metabolites in germinated red rice(Oryza sativa L.)by ultrasonication[J].Ultrasonics Sonochemistry,2017,40(Pt A).
[28]Zhang Q,Xiang J,Zhang L,et al. Optimizing soaking and germination conditions to improve gamma-aminobutyric acid content in japonica and indica germinated brown rice[J].Journal of Functional Foods,2014,10(10):283-291.
[2]杨军,王海凤,郭涛.海稻86的籼粳性鉴定[J].山东农业科学2016,48(10):24-26.
[3]杨胜远,陆兆新,吕风霞,等.γ-氨基丁酸的生理功能和研究开发进展[J].食品科学,2005,26(9):546-551.
[4]Zheng Xiongxi,Ramamoorthy S,Hui Shen,et al. GABA transmission in the nucleus accumbens is altered after withdrawal from repeated cocaine[J].J Neurosci,2003,23(8):3498-3505.
[5]张晖,姚惠源,姜元荣.富含γ-氨基丁酸保健食品的研究与开发[J].食品与发酵工业,2002(9):69-72.
[6]陈恩成,张名位,彭超英,等.比色法快速测定糙米中γ-氨基丁酸含量研究[J].中国粮油学报,2006,21(1):125-128.
[7]张晖,徐永,姚惠源.纸层析法定量测定米胚芽中的γ-氨基丁酸[J].无锡轻工大学学报,2004,23(2):101-103.
[8]任红波.氨基酸自动分析仪快速测定糙米中的γ-氨基丁酸[J].杂粮作物,2003,23(4):246-247.
[9]刘兴勇,樊建麟,师江,等.玛咖中γ-氨基丁酸测定及其含量影响分析[J].现代食品科技,2017,33(2):205-209.
[10]何瑾,屈凡伟,张峻,等.超高效液相色谱/质谱法测定血液中γ-氨基丁酸[J].分析试验室,2015,34(9):1022-1025.
[11]程威威,吴跃,林亲录,等.HPLC法测定发芽糙米中氨基丁酸中不同衍生方式的比较研究[J].中国农学通报,2014,30(9):279-284.
[12]张晖,吴胜芳,姚惠源.OPA柱前自动衍生-紫外检测米胚芽中的γ-氨基丁酸的HPLC法研究[J].食品与发酵工业,2003,29(10):50-52.
[13]Amir Hayat,Taj Muhammad Jahangir,Muhammad Yar Khuhawar,et al.HPLC determination of gamma amino butyric acid(GABA)and some biogenic amines(BAs)in controlled,germinated,and fermented brown rice by pre-column derivatization[J].Journal of Cereal Science,2015,64:56-62.
[14]刘玲君,凌森,陈亮,等.高效液相色谱法检测含乳饮料中γ-氨基丁酸的含量[J].食品工业,2016(9):262-264.
[15]Nana Li,Yang Liu,Yue Zhao,et al. Simultaneous HPLC determination of amino acids in tea infusion coupled to precolumn derivatization with 2,4-dinitrofluorobenzene[J]. Food Anal Methods,2016,9:1307-1314.
[16]赵国群,王维.比色法快速测定γ-氨基丁酸含量[J].食品研究与开发,2015(10):85-89.
[17]樊星,陈红,高雪峰,等.溴甲酚绿比色法检测γ-氨基丁酸含量[J].现代商贸工业,2017(27):197-198.
[18]姜卫荣,蒋士新,孙永梅.右美托咪定对缺血缺氧性脑损伤新生大鼠的神经保护作用及机制[J].中国老年学,2017,37(10).
[19]刘兴勇,樊建麟,师江,等.玛咖中γ-氨基丁酸测定及其含量影响分析[J].现代食品科技,2017(2):205-209.
[20]赵海波.HPLC法测定发芽糙米中γ-氨基丁酸含量试验[J].农业科技与装备,2017(3):50-51.
[21]陈雪,刘明,汪丽萍,等.柱前在线衍生-HPLC法测定发芽糙米中γ-氨基丁酸含量[J].粮油食品科技,2017,25(3):48-53.
[22]周婷,程威威,林亲录,等.丹磺酰氯柱前衍生发芽糙米中γ-氨基丁酸的HPLC分析[J].食品科技,2014(1):282-286.
[23]张凡,周远征,赵远,等.HPLC异硫氰酸苯酯柱前衍生化测定黄柏及其不同炮制品中17种氨基酸的含量[J].中华中医药学刊,2017(9):2259-2262.
[24]张琳,尤进茂,单亦初,等.氨基酸的咔唑-9-乙基氯甲酸酯柱前衍生高效液相色谱分析[J].分析测试学报,2002,21(5):16-19.
[25]张志清,徐杰,丛军,等.高效液相色谱法测定发芽麦粒中γ-氨基丁酸(GABA)含量[J].中国粮油学报,2015,30(11):135-139.
[26]Zhang Z,Jie X,Cong J,et al. Determination ofγ-aminobutyric acid(GABA)in germinated wheat seed by HPLC[J].Journal of the Chinese Cereals&Oils Association,2015.
[27]Ding J,Ulanov A V,Dong M,et al.Enhancement of gamaaminobutyric acid(GABA)and other health-related metabolites in germinated red rice(Oryza sativa L.)by ultrasonication[J].Ultrasonics Sonochemistry,2017,40(Pt A).
[28]Zhang Q,Xiang J,Zhang L,et al. Optimizing soaking and germination conditions to improve gamma-aminobutyric acid content in japonica and indica germinated brown rice[J].Journal of Functional Foods,2014,10(10):283-291.