高效液相色谱法检测罗非鱼血浆中L-肉碱的方法研究
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摘要
以罗非鱼为对象测定血浆中的肉碱含量,就流动相、柱温对色谱峰的影响等方面对样品处理方法及衍生化反应的条件进行研究,建立相应的高效液相色谱法,Kromasil Sil色谱柱(250mm×4.6mm,5μm),流动相为乙腈—柠檬酸缓冲液(90∶10,体积比)。每1000mL流动相中加入0.25mL三乙胺,流速1.2mL/min,柱温35℃,检测波长260nm,进样量为20μL。选取10%的对溴苯乙酰基溴为衍生试剂,70℃水浴90min。方法学上考察结果表明,该方法专属性高,其线性回归方程和相关系数分别为y=0.08376x+0.09663(n=6,r~2=0.9995),最低检测限为1μmol/L,日内和日间变异系数分别为1.79%~5.56%、9.83%~11.81%,平均回收率为95.83%,12h峰面积变化相对标准偏差为6.58%,可将其作为检测L-肉碱的分析方法。
The effects of mobile phase,column temperature to chromatographic peak,method of sample handling and derivation reaction conditions on L-carnitine levels of plasma in GIFT tilapia Oreochromis niloticus with body weight of(82.71±1.28)g,and the corresponding HPLC method was established under conditions of Kromasil Sil chromatographic column(250 mm×4.6 mm,5 microns),and mobile phase consisting of acetonitrile-citric acid buffer solution(90∶10,V/V),0.25 mL per 1 Lliquid phase joining triethylamine,flow rate of 1.2 mL/min,35 ℃ of column temperature,detection wavelength of 260 nm and 20μL of sample.The 10% of bromobenzene acetyl bromide was used as derivative reagent at 70 ℃water bath for 90 minutes.Methodological investigation revealed that the corresponding HPLC method has high specificity,with the equation of linear regression and the correlation coefficient of y=0.08376 x+0.09663(n=6,r2=0.9995),the minimum detection limit of 1μmol/L,the intra-day and inter-day variation coefficients of 1.79%—5.56% and 9.83%—11.81%,respectively,the average recovery of 95.83%,and the 12 hours peak area relative standard deviation of 6.58%.The findings indicate that the established HPLC method can be used as an assay for detection of L-carnitine in fish.
The effects of mobile phase,column temperature to chromatographic peak,method of sample handling and derivation reaction conditions on L-carnitine levels of plasma in GIFT tilapia Oreochromis niloticus with body weight of(82.71±1.28)g,and the corresponding HPLC method was established under conditions of Kromasil Sil chromatographic column(250 mm×4.6 mm,5 microns),and mobile phase consisting of acetonitrile-citric acid buffer solution(90∶10,V/V),0.25 mL per 1 Lliquid phase joining triethylamine,flow rate of 1.2 mL/min,35 ℃ of column temperature,detection wavelength of 260 nm and 20μL of sample.The 10% of bromobenzene acetyl bromide was used as derivative reagent at 70 ℃water bath for 90 minutes.Methodological investigation revealed that the corresponding HPLC method has high specificity,with the equation of linear regression and the correlation coefficient of y=0.08376 x+0.09663(n=6,r2=0.9995),the minimum detection limit of 1μmol/L,the intra-day and inter-day variation coefficients of 1.79%—5.56% and 9.83%—11.81%,respectively,the average recovery of 95.83%,and the 12 hours peak area relative standard deviation of 6.58%.The findings indicate that the established HPLC method can be used as an assay for detection of L-carnitine in fish.
引文
[1]王聪.章鱼中肉碱、章鱼肉碱检测方法研究[D].青岛:中国海洋大学,2011.
[2]王永侠,占秀安,郑严严.L-肉碱及其在水产养殖中的应用研究进展[J].广东饲料,2008,17(5):39-40.
[3]孙志浩,王雷.L-肉碱制备和应用研究的概况[J].食品与发酵工业,1996,212(2):64-68.
[4]祝虹,吉承平.运用Linux系统打造NAT防火墙的技术研究与实现[J].电子设计工程,2013,21(3):17-19.
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[8]Marquis N,Fritz I.Enzymological determintation of free carnitine concentration in rat tissues[J].J Lipid Res,1964,5(2):184.
[9]Cederblad G,Lindstedt S.A method for the determination of carnitine in the picomol range[J].Clin Chim Acta,1972,37(3):235-243.
[10]李红艳,陈小珍,张慧.液相色谱—荧光法测定婴幼儿奶粉中右旋肉碱[J].分析试验室,2013,32(7):1-4.
[11]李晨钟,张素华,邱鸿鑫,等.放射酶分析法检测红细胞胰岛素降解酶活性[J].重庆医科大学学报,1998,23(1):1-3.
[12]Jochen Schafer,Heinz Reichmann.A spectrophotometric method for the determination of free and esterified carnitine[J].Clinica Chimica Act A,1989,182(1):87-94.
[13]邹雨佳,聂实践,李东.L-肉碱的检测方法[J].生物工程进展,1999,19(1):72-75.
[14]Minkler P,Ingalls S,Kormos L,et al.Determination of carnitine butyrobetaine,and betain as 4-bromaphenacyl ester derivatives by high-performance liquid chromatography[J].J Chromatogr,1984,336(2):271-283.
[15]焦莉莉,杨文双,蔺小红,等.健康成人与肝脏疾病患者血浆肉毒碱的检测[J].检验医学,2007,22(5):528-530.
[16]梅长林,徐洪实,顾书华,等.血液透析病人血浆游离肉碱测定及临床意义[J].中华医学检验杂志,1998,21(6):34-37.
[17]蔡燕娜.串联质谱检测正常儿童干血滤纸片中游离肉碱和酰基肉碱含量[D].广州:广州医学院,2011.
[18]修娴.非肥胖型PCOS患者血清中左旋肉毒碱浓度和代谢指标的检测及意义[D].长沙:中南大学,2009.
[19]孙庆宝,刘小传,李克.柱前衍生—正相高效液相色谱法测定兔血清中游离肉毒碱[J].中国组织工程研究与临床康复,2007,11(49):9929-9932.
[20]林淼,赵志辉,雷萍.反相高效液相色谱法测定饲料及饲料添加剂中L-肉碱含量[J].理化检验:化学分册,2009,45(11):1268-1270.
[21]徐连明,王振中,毕宇安,等.HPLC法测定减肥胶囊中左旋肉碱的含量[J].海峡药学,2009,21(11):46-47.
[22]梁冬生,常碧影.柱前衍生高效液相色谱法测定氨基酸[J].色谱,1993,11(1):140-143.
[23]祝伟霞,杨冀州,刘亚风,等.保健品中左旋肉碱的反相高效液相色谱法测定[J].分析测试学报,2008,27(10):1124-1127.
[24]Engalls S T,Minkler P E,Hoppel C L,et al.Derivatization of carboxylic acids by reaction with 4′-bromophenacyl trifluorome thanesulfonate prior to determination by high-performance liquid chromatography[J].J Chromatography,1984(299):365-376.
[25]甘宾宾,黎少豪.HPLC法测定保健食品中左旋肉碱含量的研究[J].中国卫生检验杂志,2010,20(7):1688-1689.
[26]Musser R E,Goodband R D,Tokach M D,et al.Effects of L-carnitine fed during gestation and lactation on sow and litter performance[J].Professional Animal Scientist,1998,14(1):28-35.
[27]赵亚明,李任,王得新,等.高效液相色谱法检测血浆左旋肉碱方法的建立[J].中国临床神经科学,2006,14(5):528-532.
[28]宋涛.高效液相色谱在生命科学中的应用[R].济南:中国出版年鉴社,1998.
[29]Nezu J,Tamai I.Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium iondependent carnitine transporter[J].Nature Genet,1999(21):91-94.
[2]王永侠,占秀安,郑严严.L-肉碱及其在水产养殖中的应用研究进展[J].广东饲料,2008,17(5):39-40.
[3]孙志浩,王雷.L-肉碱制备和应用研究的概况[J].食品与发酵工业,1996,212(2):64-68.
[4]祝虹,吉承平.运用Linux系统打造NAT防火墙的技术研究与实现[J].电子设计工程,2013,21(3):17-19.
[5]廖晖,史雯静,何晓峰,等.Blended Learning在运动系统疾病课程教学中的应用研究[J].中国医学教育技术,2013(1):11-14.
[6]Travassos L R,Celuta O.Microbiological assay of carnitine[J].Anal Biochem,1974,58(2):485-499.
[7]Friedman.Determination of carnitine in biological materials[J].Archives of Biochemistry and Biophysics,1958,75(1):24-31.
[8]Marquis N,Fritz I.Enzymological determintation of free carnitine concentration in rat tissues[J].J Lipid Res,1964,5(2):184.
[9]Cederblad G,Lindstedt S.A method for the determination of carnitine in the picomol range[J].Clin Chim Acta,1972,37(3):235-243.
[10]李红艳,陈小珍,张慧.液相色谱—荧光法测定婴幼儿奶粉中右旋肉碱[J].分析试验室,2013,32(7):1-4.
[11]李晨钟,张素华,邱鸿鑫,等.放射酶分析法检测红细胞胰岛素降解酶活性[J].重庆医科大学学报,1998,23(1):1-3.
[12]Jochen Schafer,Heinz Reichmann.A spectrophotometric method for the determination of free and esterified carnitine[J].Clinica Chimica Act A,1989,182(1):87-94.
[13]邹雨佳,聂实践,李东.L-肉碱的检测方法[J].生物工程进展,1999,19(1):72-75.
[14]Minkler P,Ingalls S,Kormos L,et al.Determination of carnitine butyrobetaine,and betain as 4-bromaphenacyl ester derivatives by high-performance liquid chromatography[J].J Chromatogr,1984,336(2):271-283.
[15]焦莉莉,杨文双,蔺小红,等.健康成人与肝脏疾病患者血浆肉毒碱的检测[J].检验医学,2007,22(5):528-530.
[16]梅长林,徐洪实,顾书华,等.血液透析病人血浆游离肉碱测定及临床意义[J].中华医学检验杂志,1998,21(6):34-37.
[17]蔡燕娜.串联质谱检测正常儿童干血滤纸片中游离肉碱和酰基肉碱含量[D].广州:广州医学院,2011.
[18]修娴.非肥胖型PCOS患者血清中左旋肉毒碱浓度和代谢指标的检测及意义[D].长沙:中南大学,2009.
[19]孙庆宝,刘小传,李克.柱前衍生—正相高效液相色谱法测定兔血清中游离肉毒碱[J].中国组织工程研究与临床康复,2007,11(49):9929-9932.
[20]林淼,赵志辉,雷萍.反相高效液相色谱法测定饲料及饲料添加剂中L-肉碱含量[J].理化检验:化学分册,2009,45(11):1268-1270.
[21]徐连明,王振中,毕宇安,等.HPLC法测定减肥胶囊中左旋肉碱的含量[J].海峡药学,2009,21(11):46-47.
[22]梁冬生,常碧影.柱前衍生高效液相色谱法测定氨基酸[J].色谱,1993,11(1):140-143.
[23]祝伟霞,杨冀州,刘亚风,等.保健品中左旋肉碱的反相高效液相色谱法测定[J].分析测试学报,2008,27(10):1124-1127.
[24]Engalls S T,Minkler P E,Hoppel C L,et al.Derivatization of carboxylic acids by reaction with 4′-bromophenacyl trifluorome thanesulfonate prior to determination by high-performance liquid chromatography[J].J Chromatography,1984(299):365-376.
[25]甘宾宾,黎少豪.HPLC法测定保健食品中左旋肉碱含量的研究[J].中国卫生检验杂志,2010,20(7):1688-1689.
[26]Musser R E,Goodband R D,Tokach M D,et al.Effects of L-carnitine fed during gestation and lactation on sow and litter performance[J].Professional Animal Scientist,1998,14(1):28-35.
[27]赵亚明,李任,王得新,等.高效液相色谱法检测血浆左旋肉碱方法的建立[J].中国临床神经科学,2006,14(5):528-532.
[28]宋涛.高效液相色谱在生命科学中的应用[R].济南:中国出版年鉴社,1998.
[29]Nezu J,Tamai I.Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium iondependent carnitine transporter[J].Nature Genet,1999(21):91-94.