反相高效液相色谱与离子交换色谱测定牛肉中肌肽含量的对比研究
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摘要
目的:考察反相高效液相色谱(RP-HPLC)紫外检测与离子交换色谱(IEC)电导检测2种方法对牛肉中肌肽检测的一致程度。方法:RP-HPLC色谱条件:Agilent TC-C_(18)色谱柱(5μm,4.6 mm×250 mm);流动相为乙腈-水(15∶85);柱温35℃;流速1.00 mL·min~(-1);进样体积25μL;检测波长210 nm。IEC色谱条件:Dionex AminoPac~(TM) PA-10(2 mm×50 mm)保护柱和Dionex AminoPac~(TM) PA-10(2 mm×250 mm)分析柱分离,金电极检测,直流安倍电位,检测波形为"Amino Acids(Reference)"。以70%超纯水和30%醋酸钠溶液(100 mmol)为淋洗液;Dionex AminoPac~(TM)PA-10柱流速为0.25 mL·min~(-1),进样体积均为25μL。分别对2种方法的检测下限、精密度、加样回收率等进行考察,并对2种方法测得牛肉样品的结果进行显著性检验(F检验和t检验)。结果:RP-HPLC法线性范围为0.20～5.00μg·mL~(-1) (R2=0.999 7),检测下限和定量下限分别为0.032和0.11μg·mL~(-1),方法精密度(RSD)为1.2%(n=11),样品回收率为97.7%;IEC法峰形对称,线性范围为0.20～8.00μg·mL~(-1) (R~2=0.999 0),检测下限和定量下限分别为0.005 0和0.017μg·mL~(-1),方法精密度(RSD)为1.2%(n=11),样品回收率为92.2%。对测定结果的F检验(方法精密度)表明两者无显著性差异,但t检验(方法系统误差)表明两者有显著差异。结论:2种方法均能较好地对样品中的肌肽进行定性和定量分析,但两者存在系统误差,原因需进一步探索。
Objective:To investigate the consistency of detecting carnosine in beef byse high performance chromatography(RP-HPLC)and ion exchange chromatography(IEC).M reevtherosdesd pha:RP-HPLC separation was accomplished on Agilent TC-C_(18)(5 μm,4.6 mm×250 mm)column with mobile phase of acetonitrile-water(15∶ 85)by flow rate of 1.00 mL·min~(-1) and the detection wavelength was 210 nm at 35 ℃. IEC separation was acTcomplished on Dionex AminoPac~(TM) PA-10(2 mm×50 mm)guard column and Dionex AminoPacM PA-10(2 mm×250 mm)analytical column with mobile phase of 70% ultrapure water-30% sodium acetate solution(100 mmol)by flow rate of 0.25 mL·min~(-1) and the detection wavelength was amino acids(reference)with amperometric detection on an a gold electrode.The detection limit,precision and sample recovery rate of the two methoRdse swuelrtes investigated,and the experiment results in beef samples were tested for significance(F-test and t-test).:RP-HPLC had good linearity in the range of 0.20-5.00 μg·mL~(-1)(R2 was 0.999 7),mits of detection and quantification were 0.032 μg·mL~(-1) and 0.11 μg·mL~(-1) the li respectively,the precision(RSD)was 1.2%(n=11),and the sample recovery rate was 97.7%.The shape of IEC was symmetrical and had good linearity in the range of 0.20-8.00 μg·mL~(-1)(R~2=0.999 0),the limits of detection and quantification were 0.005 0 μg·mL~(-1) and 0.017 μg·mL~(-1) respectively.The precision(RSD)was 1.2%(n=11),and the recovery was 92.2%.The F-test(precision of the methods)results showed there was no significant difference between the two methods.However,the t-test(systematic error of the methods)results showed there was significant difference between the two methods.Conclusion:The two methods can be used to qualitatively and quantitatively analyze carnosine in samples.However,there is systematic error between the two methods and the reasons need to be further explored.
Objective:To investigate the consistency of detecting carnosine in beef byse high performance chromatography(RP-HPLC)and ion exchange chromatography(IEC).M reevtherosdesd pha:RP-HPLC separation was accomplished on Agilent TC-C_(18)(5 μm,4.6 mm×250 mm)column with mobile phase of acetonitrile-water(15∶ 85)by flow rate of 1.00 mL·min~(-1) and the detection wavelength was 210 nm at 35 ℃. IEC separation was acTcomplished on Dionex AminoPac~(TM) PA-10(2 mm×50 mm)guard column and Dionex AminoPacM PA-10(2 mm×250 mm)analytical column with mobile phase of 70% ultrapure water-30% sodium acetate solution(100 mmol)by flow rate of 0.25 mL·min~(-1) and the detection wavelength was amino acids(reference)with amperometric detection on an a gold electrode.The detection limit,precision and sample recovery rate of the two methoRdse swuelrtes investigated,and the experiment results in beef samples were tested for significance(F-test and t-test).:RP-HPLC had good linearity in the range of 0.20-5.00 μg·mL~(-1)(R2 was 0.999 7),mits of detection and quantification were 0.032 μg·mL~(-1) and 0.11 μg·mL~(-1) the li respectively,the precision(RSD)was 1.2%(n=11),and the sample recovery rate was 97.7%.The shape of IEC was symmetrical and had good linearity in the range of 0.20-8.00 μg·mL~(-1)(R~2=0.999 0),the limits of detection and quantification were 0.005 0 μg·mL~(-1) and 0.017 μg·mL~(-1) respectively.The precision(RSD)was 1.2%(n=11),and the recovery was 92.2%.The F-test(precision of the methods)results showed there was no significant difference between the two methods.However,the t-test(systematic error of the methods)results showed there was significant difference between the two methods.Conclusion:The two methods can be used to qualitatively and quantitatively analyze carnosine in samples.However,there is systematic error between the two methods and the reasons need to be further explored.
引文
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[12]张君慧,张晖,王兴国,等.抗氧化活性肽的研究进展[J].中国粮油学报,2008,23(6):227ZHANG JH,ZHANG H,WANG XG,et al.Advancement of research on antioxidant peptides[J].J Chin Cereals Oils Assoc,2008,23(6):227
[13]杨菁,吴国强,白剑,等.邻苯二甲醛柱前衍生化高效液相色谱法测定组织中肌肽含量[J].中国生化药物杂志,2009,30(4):258YANG J,WU GQ,BAI J,et al.Determination of carnosine by reversed phased high performance fiquid chromatography with precolumn O-phthalaidehyde derivation[J].Chin J Biochem Pharm,2009,30(4):258
[14]韩木南,陈晓辉,纪晓影,等.HPLC法测定L-肌肽中的游离氨基酸的含量[J].沈阳药科大学学报,2010,27(4):303HAN MN,CHEN XH,JI XY,et al.Determination of free amino acids in L-carnosine by HPLC[J].J Shenyang Pharm Univ,2010,27(4):303
[15]王宝平,胡清宇,杨芳,等.高效毛细管电泳法测定肌肽含量方法的研究[J].世界中西医结合杂志,2011,6(12):1040WANG BP,HU QY,YANG F,et al.Study of high performance capillary electrophoresis in the determination of carnosine content[J].World J Integr Tradit West Med,2011,6(12):1040
[16]JOZANOVI?M,HAJDUKOVI?M,GALOVI?O,et al.Determination of anti-oxidative histidine dipeptides in poultry by microchip capillary electrophoresis with contactless conductivity detection[J].Food Chem,2017,221:1658
[17]GATTI R,ANDREATTA P,BOSCHETTI S.An high-performance liquid chromatographic method for the simultaneous analysis of acetylcarnitine taurinate,carnosine,asparagine and potassium aspartate and for the analysis of phosphoserine in alimentary supplements[J].J Chromatogr A,2013,1298(13):95
[18]周示玉,霍刚,邓学阳.高效液相色谱法测定化妆品中的肌肽含量[J].日用化学品科学,2017,40(4):23ZHOU SY,HUO G,DENG XY.Determination of carnosine in cosmetics by high performance liquid chromatography[J].Deterg Cosmet,2017,40(4):23
[19]曹永梅.营养型鸡精中肌肽,鹅肌肽含量的检测[J].食品工业,2013,34(2):183CAO YM.Determination of carnosine and anserine content in the nutritional of chicken[J].Food Ind,2013,34(2):183
[20]LIU Y,SU D,ZHANG L,et al.Endogenous L-carnosine level in diabetes rat cardiac muscle[J].Evid Based Complalt,2016,2016(6):6230825
[21]霍萌,朱秋劲.C18丙烯酰胺硅胶柱对肌肽分离能力的比较[J].分析试验室,2011,30(5):62HUO M,ZHU QJ.Comparison between the C18 column and the venusil HILIC for separation of 1-carnosine[J].Anal Lab,2011,30(5):62
[22]田颖刚,谢明勇,王维亚,等.乌骨鸡肌肉中肌肽的鉴定与测定[J].分析试验室,2007,26(1):5TIAN YG,XIE MY,WANG WY,et al.Identification and determination of carnosine from muscles of black-bone silky fowl[J].Anal Lab,2007,26(1):5
[23]ZHANG JH,LU Q,SHI WJ,et al.The induction apoptosis of HL-60 cells by low molecular weight compounds of taurine,ornithine and carnosine from new born calf liver[J].Chin J Appl Phys,2005,21(2):200
[24]LEE KT,SONG HS,PARK SM.Antioxidant effects of carnosine extracted from the eel Anguilla japonica[J].Fish Sci,2007,40(4):193
[25]YOUNG AB,SNYDER SH.A sensitive specific enzymaticfluorometric assay for homocarnosine[J].J Neurochem,2010,21(2):387
[2]BAUER K.Carnosine and homocarnosine,the forgotten,enigmatic peptides of the brain[J].Neurochem Res,2005,30(10):1339
[3]FRESTA CG,HOGARD ML,CARUSO G,et al.Monitoring carnosine uptake by RAW 264.7 macrophage cells using microchip electrophoresis with fluorescence detection[J].Anal Methods,2017,9(3):402
[4]樊凤艳,周虹,王字玲.肌肽抗全身性缺血/再灌注损伤的防治作用[J].医学分子生物学杂志,2007,4(2):178FAN FY,ZHOU H,WANG ZL.Protective effect of carnosine on ischemia-reperfusion injury[J].J Med Mol Biol,2007,4(2):178
[5]胡新旭,赵丽红,张勇,等.肌肽的研究进展[J].饲料工业,2013,34(7):59HU XX,ZHAO LH,ZHANG Y,et al.Research progress of carnosine[J].Feed Ind,2013,34(7):59
[6]周理红,许梓荣.肌肽的营养研究进展[J].中国饲料,2004(11):27ZHOU LH,XU ZR.Research of carnosine in animal nutrition[J].China Feed,2004(11):27
[7]孙志新,聂志强.肌肽在人体运动中作用的研究综述[J].安徽体育科技,2007,28(2):25SUN ZX,NIE ZQ.The summary of the function of carnosine in sports of the human body[J].J Anhui Sport Sci,2007,28(2):25
[8]BABIZHAYEV MA.Biological activities of the natural imidazolecontaining peptidomimetics n-acetylcarnosine,carcinine and L-carnosine in ophthalmic and skin care products[J].Life Sci,2006,78(20):2343
[9]邱玉朗,陈群,李林,等.肌肽对肉鸡生长,抗氧化性能及肉质的影响[J].饲料研究,2015(24):23QIU YL,CHEN Q,LI L,et al.Effects of carnosine on growth,antioxidant activity and meat quality of broilers[J].Feed Res,2015(24):23
[10]田莹,刘显军,边连全,等.肌肽与维生素E对育肥猪肉品质和抗氧化性能的影响[J].动物营养学报,2014,26(12):3723TIAN Y,LIU XJ,BIAN LQ,et al.Effects of carnosine and vitamin E on meat quality and antioxidant capacity of growing-finishing pigs[J].Acta Zoonutr Sin,2014,26(12):3723
[11]王国华,刘宝剑,魏东,等.肌肽对H9N2亚型猪流感病毒致小鼠肺部组织SOD,MDA,INOS和NO的影响[J].西北农业学报,2015,24(2):6WANG GH,LIU BJ,WEI D,et al.Effect of carnosine on SOD,MDA,INOS and NO in mice lung tissue infected with H9N2-SIV[J].Acta Agric Boreal-Occident Sin,2015,24(2):6
[12]张君慧,张晖,王兴国,等.抗氧化活性肽的研究进展[J].中国粮油学报,2008,23(6):227ZHANG JH,ZHANG H,WANG XG,et al.Advancement of research on antioxidant peptides[J].J Chin Cereals Oils Assoc,2008,23(6):227
[13]杨菁,吴国强,白剑,等.邻苯二甲醛柱前衍生化高效液相色谱法测定组织中肌肽含量[J].中国生化药物杂志,2009,30(4):258YANG J,WU GQ,BAI J,et al.Determination of carnosine by reversed phased high performance fiquid chromatography with precolumn O-phthalaidehyde derivation[J].Chin J Biochem Pharm,2009,30(4):258
[14]韩木南,陈晓辉,纪晓影,等.HPLC法测定L-肌肽中的游离氨基酸的含量[J].沈阳药科大学学报,2010,27(4):303HAN MN,CHEN XH,JI XY,et al.Determination of free amino acids in L-carnosine by HPLC[J].J Shenyang Pharm Univ,2010,27(4):303
[15]王宝平,胡清宇,杨芳,等.高效毛细管电泳法测定肌肽含量方法的研究[J].世界中西医结合杂志,2011,6(12):1040WANG BP,HU QY,YANG F,et al.Study of high performance capillary electrophoresis in the determination of carnosine content[J].World J Integr Tradit West Med,2011,6(12):1040
[16]JOZANOVI?M,HAJDUKOVI?M,GALOVI?O,et al.Determination of anti-oxidative histidine dipeptides in poultry by microchip capillary electrophoresis with contactless conductivity detection[J].Food Chem,2017,221:1658
[17]GATTI R,ANDREATTA P,BOSCHETTI S.An high-performance liquid chromatographic method for the simultaneous analysis of acetylcarnitine taurinate,carnosine,asparagine and potassium aspartate and for the analysis of phosphoserine in alimentary supplements[J].J Chromatogr A,2013,1298(13):95
[18]周示玉,霍刚,邓学阳.高效液相色谱法测定化妆品中的肌肽含量[J].日用化学品科学,2017,40(4):23ZHOU SY,HUO G,DENG XY.Determination of carnosine in cosmetics by high performance liquid chromatography[J].Deterg Cosmet,2017,40(4):23
[19]曹永梅.营养型鸡精中肌肽,鹅肌肽含量的检测[J].食品工业,2013,34(2):183CAO YM.Determination of carnosine and anserine content in the nutritional of chicken[J].Food Ind,2013,34(2):183
[20]LIU Y,SU D,ZHANG L,et al.Endogenous L-carnosine level in diabetes rat cardiac muscle[J].Evid Based Complalt,2016,2016(6):6230825
[21]霍萌,朱秋劲.C18丙烯酰胺硅胶柱对肌肽分离能力的比较[J].分析试验室,2011,30(5):62HUO M,ZHU QJ.Comparison between the C18 column and the venusil HILIC for separation of 1-carnosine[J].Anal Lab,2011,30(5):62
[22]田颖刚,谢明勇,王维亚,等.乌骨鸡肌肉中肌肽的鉴定与测定[J].分析试验室,2007,26(1):5TIAN YG,XIE MY,WANG WY,et al.Identification and determination of carnosine from muscles of black-bone silky fowl[J].Anal Lab,2007,26(1):5
[23]ZHANG JH,LU Q,SHI WJ,et al.The induction apoptosis of HL-60 cells by low molecular weight compounds of taurine,ornithine and carnosine from new born calf liver[J].Chin J Appl Phys,2005,21(2):200
[24]LEE KT,SONG HS,PARK SM.Antioxidant effects of carnosine extracted from the eel Anguilla japonica[J].Fish Sci,2007,40(4):193
[25]YOUNG AB,SNYDER SH.A sensitive specific enzymaticfluorometric assay for homocarnosine[J].J Neurochem,2010,21(2):387