柱前衍生化液相色谱法测定牛血清中游离氨基酸含量  

Determination of free amino acid content in bovine serum by precolumn derivatization-high performance liquid chromatography

作　　者：贾鹏禹[1] 孙蕊[1] 寇芳[1] 李文杰[1] 修建成 武瑞[1] JIA Peng-yu;SUN Rui;KOU Fang;LI Wen-jie;XIU Jian-cheng;WU Rui(Heilongjiang Bayi Agricultural University,Daqing 163319,Heilongjiang Province,China)

机构地区：[1]黑龙江八一农垦大学,黑龙江大庆163319

出　　处：《中国生物制品学杂志》2018年第8期869-873,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金项目(31472249);大庆市指导性科技计划项目(zd-2017-34);黑龙江八一农垦大学"校内培育课题资助计划"项目(XZR2015-15)

摘　　要：目的建立牛血清中18种游离氨基酸含量的柱前衍生化液相色谱测定方法,并进行验证。方法采用乙腈沉淀方法去除血清样品中的蛋白,以异硫氰酸苯酯(phenylisothiocyanate,PITC)在三乙胺碱性条件下衍生游离氨基酸,采用氨基酸分析柱(Sepax AAA,250 mm×4.6 mm ID)及乙腈-醋酸钠缓冲体系梯度洗脱分离18种目标组分,紫外检测器波长为254 nm。对建立的方法进行重现性、准确性和精密性验证,并确定方法的线性范围及检测限。结果18种游离氨基酸在60 min内完成分离,各目标组分在0.5~2.5μmol/L浓度范围内呈良好的线性关系,方法检测限在0.019~0.163μmol/L之间,相对标准偏差(RSD)在0.42%~2.28%之间。准确性和精密性试验中检测各组分的平均回收率在93.10%~101.61%之间,RSD在0.19%~3.82%之间。结论采用PITC柱前衍生化液相色谱法可实现牛血清中18种游离氨基酸的含量测定,该方法简便易行,重现性、准确性及精密性良好,可为动物营养及生理研究提供检测手段。Objective To develop a pre-column derivatization-high performance liquid chromatography method for simultaneous determination of contents of 18 kinds of free amino acids in bovine serum. Methods Samples were pretreated by acetonitrile to remove the proteins. The free amino acids were derived with phenyl isothiocyanate（PITC）under triethylamine alkaline condition. Separation was performed on an Sepax AAA column（250 mm × 4. 6 mm ID）using acetonitrile-sodium acetate buffer solution as mobile phase in gradient mode,and the detection wavelength was set at 254 nm. The developed method was verified for reproducibility,accaurcy and precision,and determined for linear range and detection limit. Results The separation of 18 free amino acids was completed within 60 min,and the linear ranges of various components were 0. 5 - 2. 5 μmol/L,while the detection limits were 0. 019 - 0. 163 μmol/L,with RSDs of 0. 42 - 2. 28%. The mean recovery rates in accuracy and precision tests were 93. 10% - 101. 61%,with a RSD of 0. 19% - 3. 82%. Conclusion Totally 18 kinds of free amino acids in bovine serum were determined by pre-column derivatization method with PITC. The method was simple,reproducible,accurate and precise,which provided a tool for study on animal nutrition and physiology.

关 键 词：柱前衍生化 液相色谱法 牛血清 游离氨基酸 

分 类 号：O657.72[理学—分析化学]