基于过甲酸氧化法PITC-HPLC对18种氨基酸的测定研究

发布时间：2019-02-13 17:29

【摘要】：蛋白质是生物体结构和生命活动的物质基础,而氨基酸是组成蛋白质的基本单位,因此,氨基酸影响着蛋白质功能的发挥,是机体不可或缺的成分。近年来,随着社会经济的快速发展和生活节奏的加快,快餐饮食逐渐流行,营养的不均衡导致的慢性病问题逐渐凸显。由此,人们愈来愈多地消费各种氨基酸保健品或氨基酸类药物以满足人体均衡的营养需求以及对疾病的治疗。该类产品中必需氨基酸的含量是否充足、种类是否齐全、比例是否适当以及能否被人体充分吸收利用等是评价产品营养价值高低的主要标准。因此,建立一套快速、准确地测定该类食品中氨基酸的方法对上述工作的开展具有重要的现实意义。目前常用高效液相色谱法(HPLC)检测氨基酸,但是由于大部分氨基酸缺少生色基团,紫外吸收弱,因此需对氨基酸进行衍生。为此,本文以异硫氰酸苯酯(PITC)作为衍生试剂,建立了一套高效液相色谱分析方法,并基于过甲酸氧化对传统的PITC衍生方法进行改进,拓宽PITC衍生法对氨基酸的检测范围。建立了以PITC为柱前衍生试剂的HPLC法测定氨基酸口服液中的氨基酸。通过对PITC衍生剂量、衍生时间、衍生温度等条件进行了优化,选择合适的衍生条件,同时在HPLC分离过程中考察了流动相缓冲液浓度、pH、柱温等对氨基酸分离的影响,并对该方法进行了系统性试验。结果表明,18种氨基酸能够实现完全分离,各分离度(Rs)均大于1.5。在稳定性实验中,胱氨酸在该衍生条件下不稳定,易降解,在16 h内其峰面积下降94%。为弥补PITC-HPLC对胱氨酸测定的不足,通过采用过甲酸对氨基酸样品进行预处理,使胱氨酸转换成磺基丙氨酸,然后与PITC进行衍生。由于过甲酸不稳定、易挥发,本文简易制备了氧化剂过甲酸,并以活性氧含量为指标,考察了制备过程中反应时间、反应温度、过甲酸浓度等对制备的影响。通过正交试验,筛选出过甲酸的最佳制备条件。结果表明,过甲酸能将胱氨酸与蛋氨酸分别氧化成磺基丙氨酸与蛋氨酸砜,且氧化产物与PITC衍生后能稳定存在。采用上述方法对复方蛋氨酸胆碱片、胱氨酸片、氨基酸口服液中氨基酸进行了测定,结果表明该方法具有很好的适用性,可用于对18种氨基酸的准确测定。
[Abstract]:Protein is the material basis of biological structure and life activity, and amino acid is the basic unit of protein. Therefore, amino acid affects the function of protein and is an indispensable component of organism. In recent years, with the rapid development of social economy and the accelerated pace of life, the fast food diet is becoming more and more popular, and the chronic diseases caused by the imbalance of nutrition gradually become prominent. As a result, more and more people consume various amino acid health products or amino acid drugs to meet the balanced nutritional needs of the human body and the treatment of diseases. Whether the content of essential amino acids in this kind of products is sufficient, whether the types are complete, whether the proportion is appropriate and whether it can be fully absorbed and utilized by the human body are the main criteria for evaluating the nutritional value of the products. Therefore, it is of great practical significance to establish a rapid and accurate method for the determination of amino acids in this kind of food. At present, high performance liquid chromatography (HPLC) is commonly used to detect amino acids, but because most amino acids lack chromophore groups and UV absorption is weak, amino acids need to be derived. In this paper, a set of high performance liquid chromatography (HPLC) method was established using phenylisothiocyanate (PITC) as derivative reagent, and the traditional PITC derivatization method was improved based on performic acid oxidation to widen the detection range of amino acids by PITC derivatization. A HPLC method for the determination of amino acids in oral solution of amino acids using PITC as precolumn derivatization reagent was established. The effects of buffer concentration of mobile phase and column temperature of pH, on the separation of amino acids were investigated in the process of HPLC separation by optimizing the conditions of PITC derivatization dose, derivatization time and derivative temperature, and selecting the appropriate derivatization conditions, and the effects of the concentration of mobile phase buffer and the column temperature of pH, on the separation of amino acids were investigated. The method is tested systematically. The results showed that 18 amino acids could be separated completely, and the (Rs) of each degree of separation was more than 1.5. In the stability experiment, cystine was unstable and easy to degrade under the derivatization condition, and its peak area decreased 94. 4% within 16 h. In order to make up for the deficiency of PITC-HPLC in the determination of cystine, the amino acid sample was pretreated with performic acid to convert cystine to sulfoalanine and then derivatized with PITC. Because performic acid is unstable and volatile, the oxidant performic acid is simply prepared in this paper. The effects of reaction time, reaction temperature and concentration of performic acid on the preparation of performic acid are investigated with the content of reactive oxygen as the index. The optimum preparation conditions of performic acid were obtained by orthogonal test. The results show that performic acid can oxidize cystine and methionine into sulfoalanine and methionine sulfone respectively and the oxidation products can exist stably after derivation with PITC. This method was used to determine amino acids in compound methionine choline tablets, cystine tablets and amino acid oral liquids. The results showed that the method was suitable for the accurate determination of 18 kinds of amino acids.
【学位授予单位】：南昌航空大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.72;TS207.3

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