摘要
目的建立同时测定大鼠血清中19种氨基酸含量的异硫氰酸苯酯(phenyl isothiocyanate,PITC)柱前衍生化-超高效液相色谱法,并进行验证及初步应用。方法采用ACQUITY UPLC BEH C_(18)色谱柱(2.1 mm×100 mm,1.7μm),以0.1 mol/L醋酸钠缓冲液-乙腈为流动相,梯度洗脱,体积流量0.3 mL/min,柱温52℃,检测波长254 nm。在优化色谱条件和供试品制备方法的基础上对方法进行专属性、线性、精密性、稳定性和准确性验证,并采用建立的方法对大鼠血清中19种氨基酸的含量进行测定。结果大鼠血清中19种氨基酸在10 min内分离良好,空白衍生化溶剂和内标化合物对氨基酸分析无干扰;各氨基酸在相应浓度范围内的线性关系良好(r≥0.9980),检测限为0.0046~0.1865μg/mL,定量限为0.0139~0.5596μg/mL;精密性和稳定性试验的RSD均小于4%;平均加标回收率为90%~110%,RSD均小于5%。大鼠血清中氨基酸含量在4.837~110.233μg/mL之间。结论建立的方法灵敏度高,简便易行,适用于大鼠血清中氨基酸的分析。
Objective To develop an ultra performance liquid chromatography(UPLC)method for simultaneous determination of 19 amino acids in rat serum by employing the pre-column derivatization of phenyl isothiocyanate(PITC),verify and preliminarily apply the method.Methods The analysis was performed on an ACQUITY UPLC~?BEH C_(18)column(2.1mm×100 mm,1.7μm)by using 0.1 mol/L sodium acetate buffer solution-acetonitrile as mobile phase at the flow rate of 0.3 mL/min in gradient elution.The column temperature was fixed at 52℃,and the detection wavelength was 254 nm.The specificity,linearity,precision,stability and accuracy of the method were verified based on the optimized chromatographic conditions and preparation method of sample solution,and the contents of 19 amino acids in rat serum were determined by using the developed method.Results All 19 amino acids in rat serum were separated well within 10 min,and the blank derivatization solvent and internal standard compounds had no interference with the analysis of amino acids.All the components exhibited good linear correlation in the given range(r≥0.9980),the limit of detection was 0.0046-0.1865μg/mL,and the limit of quantitation was 0.0139-0.5596μg/mL.The RSDs of precision and stability tests were all less than 4%.The average spike recoveries ranged from 90%to 110%and the RSDs were less than 5%.The contents of amino acids in rat serum ranged from 4.837 to 110.233μg/mL.Conclusion The developed method is sensitive,simple and feasible,which is suitable for the analysis of amino acids in rat serum.
作者
张瑞虎
李瑞
张潇予
董雪媛
柴欣
王跃飞
ZHANG Ruihu;LI rui;ZHANG Xiaoyu;DONG Xueyuan;CHAI Xin;WANG Yuefei(Institute of Traditional Chinese Medicine,Tianjin University of Traditional Chinese Medicine,State Key Laboratory of Component-based Chinese Medicine,Tianjin Key Laboratory of Chemistry and Analysis of Traditional Chinese Medicine,Tianjin 301617,China)
出处
《中国生物制品学杂志》
CAS
CSCD
2024年第6期723-730,共8页
Chinese Journal of Biologicals
基金
国家自然科学基金面上项目(81873192)
天津市研究生科研创新项目(2021YJSB288,2021YJSS186)
天津中医药大学研究生科研创新项目(YJSKC-20211004,YJSKC-20212017)。
关键词
血清
异硫氰酸苯酯
柱前衍生化法
超高效液相色谱
氨基酸
Serum
Phenyl isothiocyanate(PITC)
Pre-column derivatization
Ultra-high performance liquid chromatogra-phy(UPLC)
Amino acids
