基于過甲酸氧化法PITC-HPLC對(duì)18種氨基酸的測(cè)定研究
發(fā)布時(shí)間：2019-02-13 17:29
【摘要】：蛋白質(zhì)是生物體結(jié)構(gòu)和生命活動(dòng)的物質(zhì)基礎(chǔ),而氨基酸是組成蛋白質(zhì)的基本單位,因此,氨基酸影響著蛋白質(zhì)功能的發(fā)揮,是機(jī)體不可或缺的成分。近年來(lái),隨著社會(huì)經(jīng)濟(jì)的快速發(fā)展和生活節(jié)奏的加快,快餐飲食逐漸流行,營(yíng)養(yǎng)的不均衡導(dǎo)致的慢性病問題逐漸凸顯。由此,人們愈來(lái)愈多地消費(fèi)各種氨基酸保健品或氨基酸類藥物以滿足人體均衡的營(yíng)養(yǎng)需求以及對(duì)疾病的治療。該類產(chǎn)品中必需氨基酸的含量是否充足、種類是否齊全、比例是否適當(dāng)以及能否被人體充分吸收利用等是評(píng)價(jià)產(chǎn)品營(yíng)養(yǎng)價(jià)值高低的主要標(biāo)準(zhǔn)。因此,建立一套快速、準(zhǔn)確地測(cè)定該類食品中氨基酸的方法對(duì)上述工作的開展具有重要的現(xiàn)實(shí)意義。目前常用高效液相色譜法(HPLC)檢測(cè)氨基酸,但是由于大部分氨基酸缺少生色基團(tuán),紫外吸收弱,因此需對(duì)氨基酸進(jìn)行衍生。為此,本文以異硫氰酸苯酯(PITC)作為衍生試劑,建立了一套高效液相色譜分析方法,并基于過甲酸氧化對(duì)傳統(tǒng)的PITC衍生方法進(jìn)行改進(jìn),拓寬PITC衍生法對(duì)氨基酸的檢測(cè)范圍。建立了以PITC為柱前衍生試劑的HPLC法測(cè)定氨基酸口服液中的氨基酸。通過對(duì)PITC衍生劑量、衍生時(shí)間、衍生溫度等條件進(jìn)行了優(yōu)化,選擇合適的衍生條件,同時(shí)在HPLC分離過程中考察了流動(dòng)相緩沖液濃度、pH、柱溫等對(duì)氨基酸分離的影響,并對(duì)該方法進(jìn)行了系統(tǒng)性試驗(yàn)。結(jié)果表明,18種氨基酸能夠?qū)崿F(xiàn)完全分離,各分離度(Rs)均大于1.5。在穩(wěn)定性實(shí)驗(yàn)中,胱氨酸在該衍生條件下不穩(wěn)定,易降解,在16 h內(nèi)其峰面積下降94%。為彌補(bǔ)PITC-HPLC對(duì)胱氨酸測(cè)定的不足,通過采用過甲酸對(duì)氨基酸樣品進(jìn)行預(yù)處理,使胱氨酸轉(zhuǎn)換成磺基丙氨酸,然后與PITC進(jìn)行衍生。由于過甲酸不穩(wěn)定、易揮發(fā),本文簡(jiǎn)易制備了氧化劑過甲酸,并以活性氧含量為指標(biāo),考察了制備過程中反應(yīng)時(shí)間、反應(yīng)溫度、過甲酸濃度等對(duì)制備的影響。通過正交試驗(yàn),篩選出過甲酸的最佳制備條件。結(jié)果表明,過甲酸能將胱氨酸與蛋氨酸分別氧化成磺基丙氨酸與蛋氨酸砜,且氧化產(chǎn)物與PITC衍生后能穩(wěn)定存在。采用上述方法對(duì)復(fù)方蛋氨酸膽堿片、胱氨酸片、氨基酸口服液中氨基酸進(jìn)行了測(cè)定,結(jié)果表明該方法具有很好的適用性,可用于對(duì)18種氨基酸的準(zhǔn)確測(cè)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.
【學(xué)位授予單位】：南昌航空大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O657.72;TS207.3
本文編號(hào)：2421784
[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.
【學(xué)位授予單位】：南昌航空大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O657.72;TS207.3
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