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

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-C₁₈色谱柱（5 μm，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25 μL；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^TMPA-10柱流速为0.25 mL·min^-1，进样体积均为25 μL。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0.20~5.00 μg·mL^-1（R²=0.999 7），检测下限和定量下限分别为0.032和0.11 μg·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0.20~8.00 μg·mL^-1（R²=0.999 0），检测下限和定量下限分别为0.005 0和0.017 μg·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 by reversed phase high performance chromatography (RP-HPLC) and ion exchange chromatography (IEC).Methods:RP-HPLC separation was accomplished on Agilent TC-C₁₈ (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 accomplished on Dionex AminoPac^TM PA-10 (2 mm×50 mm) guard column and Dionex AminoPac^TM 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 methods were investigated, and the experiment results in beef samples were tested for significance (F-test and t-test).Results:RP-HPLC had good linearity in the range of 0.20-5.00 μg·mL^-1 (R² was 0.999 7), the limits of detection and quantification were 0.032 μg·mL^-1 and 0.11 μg·mL^-1 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 (R²=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.

[1] OUALI A,HERRERA-MENDEZ CH,COULIS G,et al.Revisiting the conversion of muscle into meat and the underlying mechanisms[J].Meat Sci,2006,74(1):44
[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):178 FAN 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):59 HU 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):27 ZHOU 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):25 SUN 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 imidazole-containing 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):23 QIU 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):3723 TIAN 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):6 WANG 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):227 ZHANG 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):258 YANG 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):303 HAN 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):1040 WANG 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] JOZANOVIĆ M, HAJDUKOVIĆ M, GALOVIĆ 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):23 ZHOU 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):183 CAO 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):62 HUO 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):5 TIAN 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 enzymatic-fluorometric assay for homocarnosine[J].J Neurochem,2010,21(2):387