RP-HPLC Determination of Amino Acids with Precolumn Derivatization
摘 要
向氨基酸溶液中加入对甲氧基苯磺酰氯(MOBS-Cl)试剂,在55 ℃水浴中加热35 min使氨基酸衍生化,所得含有氨基酸衍生物的溶液用于反相高效液相色谱分析。选用ODS C18柱(4.6 mm×150 mm,5.0 μm)为固定相,以不同体积比的15 mmol·L-1磷酸二氢钠溶液和乙腈为流动相进行梯度淋洗,以235 nm作为检测波长,提出了测定氨基酸的柱前衍生-反相高效液相色谱法。17种氨基酸衍生物的峰面积与其浓度均在0.01~5.0 mmol·L-1范围内呈线性关系,检出限(3S/N)在0.001 0~0.005 0 mmol·L-1之间。取5.0,25.0,50.0 g·L-1氨基酸标准溶液从衍生化操作开始分别重复测定5次,根据所测得峰面积值计算其相对标准偏差均小于5.0%。
Abstract
Solution of amino acid was mixed with the derivatizing reagent, 4-methoxybenzenesulfonyl chloride (MOBS-Cl), and derivatized for 35 min at 55 ℃ in a water bath. The solution containing the derivative of amino acid was used for RP-HPLC analysis, using ODS C18 column (4.6 mm×150 mm, 5.0 μm) as stationary phase and mixtures of 15 mmol·L-1 NaH2PO4 solution and acetonitrile in different ratios as mobile phase in the gradient elution. UV-detection at the wavelength of 235 nm was adopted in the determination. Linear relationships between values of peak area and concentration of the 17 amino acids were obtained in the same range from 0.01 to 5.0 mmol·L-1. Detection limits (3S/N) found for this method were in the range of 0.001 0-0.005 0 nmol·L-1. Precision of the method was tested with 5.0, 25.0, 50.0 g·L-1 of the standard amino acid solutions for 5 determinations including the precolumn derivatization process, values of RSD′s calculated from data of peak area were less than 5.0%.
中图分类号 O657.7
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收稿日期 2010/9/18
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备注周桂友(1981-),男,山东临沂人,硕士,主要从事理化检验工作。
引用该论文: ZHOU Gui-you,HOU Yan-fang,DONG Xin-ji. RP-HPLC Determination of Amino Acids with Precolumn Derivatization[J]. Physical Testing and Chemical Analysis part B:Chemical Analysis, 2011, 47(8): 889~893
周桂友,侯艳芳,董新吉. 柱前衍生-反相高效液相色谱法测定氨基酸[J]. 理化检验-化学分册, 2011, 47(8): 889~893
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【9】屠春燕,赵珺,葛佳璐,等.新型衍生试剂柱前衍生氨基酸的高效液相色谱分析[J],分析测试学报, 2008,27(7):681-685.
【2】TANG Tao, SHI Tian-yu, QIAN Kun, et al. Determination of biogenic amines in beer with pre-column derivatization by high performance liquid chromatography[J], Journal of Chromatography B, 2009,877:507-512.
【3】COBLE P G, TIMPERMAN A T. Fluorescence detection of proteins and amino acids in capillary electrophoresis using a post-column sheath flow reactor[J]. Journal of Chromatography A, 1998,829:309-315.
【4】HE Li, LEE J R, NGUYEN D Q A, et al. Quantitative analysis of valiolamine through pre-column derivatization with phenylisocyanate using high-performance liquid chromatography with UV detection: Selection of reagent, identification of derivative and optimization of derivatization conditions[J]. Journal of Pharmaceutical and Biomedical Analysis, 2009,49:957-963.
【5】ZAHOU E, JRNVALL H, BERGMAN T. Amino acid analysis by capillary electrophoresis after phenylthiocarbamylation[J]. Analytical Biochemistry, 2000,281:115-122.
【6】MARASCHIELLO C, MIRANDA E, MILLAN E, et al. Phenylisothiocyanate and dansyl chloride precolumn derivatizations for the high performance liquid chromatography analysis of the antitumoral agent ES-285 in dog plasma[J]. Journal of Chromatography B, 2003,791:1-11.
【7】SHEN Zuo-jun, SUN Zi-min, WU Lin, et al. Rapid method for the determination of amino acids in serum by capillary electrophoresis[J]. Journal of Chromatography A, 2002,979:227-232.
【8】瞿其曙,汤晓庆,胡效亚,等.柱前衍生法在氨基酸分析测定中的应用[J],化学进展, 2006,18(6):104-108.
【9】屠春燕,赵珺,葛佳璐,等.新型衍生试剂柱前衍生氨基酸的高效液相色谱分析[J],分析测试学报, 2008,27(7):681-685.
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