Quantitation Of Amino Acids And Amines By Chromatography Pdf
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- Quantitation of Amino Acids and Amines by Chromatography, Volume 70
- Analytical Methods for Amino Acids
- Amino Acid Analysis
HPLC is the most popular method for analyzing amino acid components, which have currently gained attention due to the boom in health foods. This page discusses mainly the detection methods used to analyze amino acids.
In a relatively short time, the variety of AAA methods has evolved dramatically with more methods shifting to the use of mass spectrometry MS as a detection method. Another new aspect is miniaturization. However, most importantly, AAA in this day and age should be viewed in the context of Metabolomics as a part of Systems Biology. Amino Acid Analysis: Methods and Protocols presents a broad spectrum of all available methods allowing for readers to choose the method that most suits their particular laboratory set-up and analytical needs. In this volume, a reader can find chapters describing general as well as specific approaches to the sample preparation.
Quantitation of Amino Acids and Amines by Chromatography, Volume 70
A method was developed for simultaneous determination of 15 amino acids and 7 alkyl amines. The 22 derivatives were separated within 30 min including the equilibration time and detected by a fluorescence detector at an excitation wavelength of nm and emission wavelength of nm.
The analysis procedure was satisfactorily validated by the reproducibility, recovery, linearity and detection limit of the analytes. The relative standard deviations RSDs of retention time and peak area for individual amino acids and alkyl amines were consistently less than 0. The detection limit ranged from 0. The developed and validated method was successfully applied to the quantitative analysis of amino acids and alkyl amines in continental and marine aerosols in China.
Among the identified organic nitrogen compounds, 7 amino acids and 6 alkyl amines were detected in every aerosol sample. Glycine was the dominant amino acid, with the average of This work provided an accurate, sensitive and simple method to determine simultaneously amino acids and alkyl amines, and applied the proposed method to the first investigation of amino acids in Shanghai and amino acids and alkyl amines in Huaniao Island in China.
The finding of considerable amino acids and alkyl amines in continental and marine aerosols may exert significant implications on nitrogen cycling and atmospheric chemistry. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
The funder provided support in the form of salaries for authors [Xiongfeng Huang, Jing Lin, Xiaofei Qin, Congrui Deng], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.
Nitrogen controls both the diversities and quantity of living organisms in marine and terrestrial environments on Earth. The atmosphere can exert a substantial impact on the transport and transformation of nitrogen worldwide [ 1 ]. Increasing studies have suggested that organic nitrogen represents a ubiquitous and significant component of atmospheric nitrogen [ 2 , 3 ].
The contribution of organic nitrogen to the total nitrogen varied from site to site but the percentage was consistently around one third of the total nitrogen [ 2 , 4 , 5 ]. Considerable amounts of organic nitrogen have been found in different regions. For example, Miyazaki et al. To appropriately characterize their environmental effects, knowledge of the chemical forms of organic nitrogen is required. A variety of organic nitrogen compounds have been detected or hypothesized to exist in the atmosphere.
Of these compounds, amino acids and amines have probably been identified most extensively recently attributed to the following reasons [ 8 , 9 ]. First, as water-soluble organic carbon, amino acids and amines exerts the potential effect on the hygroscopic growth of particles and even affects visibility [ 10 — 13 ].
Second, considering the presence of basic nitrogen functional groups, amino acids and amines may contribute to the buffering capacity of particles [ 14 , 15 ]. Finally, amines present a potential toxic risk to human health because they are precursors of carcinogenic compounds such as nitroamines and nitramines [ 16 ]. Taken together, amino acids and amines appears to represent a considerable portion of atmospheric nitrogen and therefore might have significant effects on the environment and human health.
Facchini et al. Zhang and Anastasio [ 14 ] found considerable amounts of free and combined amino acids in PM 2. Finessi et al. Both amino acids and amines are important and ubiquitous components of organic nitrogen in the atmosphere.
However, most studies of organic nitrogen compounds in the atmosphere have focused on only amino acids or amines in individual region, and there are rare published reports of simultaneous measurements of amino acids and amines in both continental and marine atmosphere. This is largely attributed to the fact that few analytical techniques are sufficiently effective to determine atmospheric amino acids and amines simultaneously.
Although numerous measurement methods for amino acids have been reported, the effective separation and detection remains extremely challenging because of their high polarity and lack of strong chromospheres [ 24 , 25 ].
Quantitative analysis of amines is also difficult due to the similar limitations [ 26 — 29 ]. Therefore, studies on the simultaneous determination of atmospheric amino acids and alkyl amines are very rare. Even in the simultaneous determination of amino acids and amines, several disadvantages of the reported methods have been identified including co-elution of analytes, a limited number of organic nitrogen compounds, laborious derivatization procedures, long analysis time, matrix interferences and so on[ 24 , 30 ].
Increasing evidence has led to the recognition that there is a need for the simultaneous quantification of amino acids and alkyl amines at various concentrations presenting in the atmospheric matrix. The most common measurement methods for amino acids and alkyl amines include gas chromatography GC [ 25 , 30 ] and high performance liquid chromatography HPLC [ 24 , 30 ]. As amino acids and amines are basic and polar compounds, their separation and peak-shape performance on GC columns are often complicated for GC stationary-phase choices.
Derivatization can improve their GC separation and peak shape, but complete separation is seldom achieved. Another disadvantage of GC is time-consuming and laborious derivatization. Several HPLC analysis techniques have also been explored. Prior to analysis, amino acids and amines must also be derivatized. Proper selection of a chromophoric derivatization reagent for both amino acids and amines is an important consideration in HPLC analysis.
The derivatization of NBD-F, Dabsyl-Cl and FMOC-Cl all presents the interference effect of reagents unless the reagents are extracted prior to chromatographic analysis or the molar excess of reagents is carefully limited. Although the unique fluorescence properties of AQC-related compounds permit the analysis of derivatized samples without prior reagent removal yet with minimal reagent interference, the cost is relatively high and the derivatization procedure requires manual operation [ 30 ].
To the best of our knowledge, OPA can simultaneously react with amino acids and amines under fairly mild conditions to form highly fluorescent substances and the LC sampler allows auto-mixing between derivatizing reagents and analytes. In this study, we utilized OPA online derivatization in order to reduce labor, improve accuracy and save time. The goal of this study is to provide an effective measurement method for the simultaneous quantification of amino acids and alkyl amines in aerosols without laborious operation and excess time-consumption.
As far as we are aware, very limited information is available on the atmospheric amino acids and alkyl amines in China. Therefore, another goal of this study is to apply the proposed method to the quantitative analysis and provide an investigation of amino acids and alkyl amines in both continental and marine aerosols in China.
All chemicals were of reagent grade or better. The analyzed amino acids were as follows: alanine Ala , arginine Arg , aspartic acid Asp , histidine His , glutamic acid Glu , glycine Gly , isoleucine Ile , leucine Leu , methionine Met , phenylalanine Phe , serine Ser , threonine Thr , tyrosine Tyr , valine Val , and lysine Lys. The concentration of the amino acid standard solution, which was received from Sigma-Aldrich, was 2.
Briefly, the derivatization reaction was performed by sufficiently mixing of borate buffer 0. After derivatization, the mixture was injected into the column. It is noteworthy that the derivatizing reagents should be frozen and kept in the dark until use, and the borate buffer should be freshly prepared. Sampling intervals were generally taken as 24 h. Blank samples were collected by loading, carrying and installing the filter holder with the pump closed. Samples were ultrasonically extracted in methanol.
After concentration and filtration, the extract was directly analyzed. Extreme care was exercised to eliminate contamination. Each derivative was evaluated in terms of the reproducibility of retention time, peak area, and recovery studies.
The relative standard deviations RSDs of retention time for individual amino acids and alkyl amines were consistently below 0. Recovery was estimated through analysis by adding various known amounts of each amino acid and alkyl amine to quartz filters, and then performing pretreatment as aerosol samples. Blanks were also analyzed and all analytes were below the detection limit.
To verify the linearity of the response of different derivatives, standard solutions of 15 amino acids and 7 alkyl amines in the concentrations from 0. Linearity was evaluated by the determination coefficient of the least square regression R 2. The coefficient of regression exceeded 0. The results indicated that amino acids and alkyl amines showed good linearity spanning over three orders of magnitude under the proposed conditions.
Reproducibility, recovery, detection limit and linearity were assayed with satisfactory results Table 1. All of these results suggested that the proposed method was reproducible, sensitive, quantitative, and linear over a wide dynamic range. Given the fact that both amino acids and alkyl amines contain the—NH 2 functional group, amino acids and alkyl amines can simultaneously derivatize with OPA.
Additionally, the mild conditions of the derivatization reaction and the automixing function of the HPLC autosampler provide potential for online derivatization. Finally, fluorescence detection and experimental conditions offer sufficient sensitivity, selectivity, and linearity for quantifying variable levels of amino acid and alkyl amine in the atmospheric matrix.
Therefore, the proposed method combined advantages of simultaneous, simple and sensitive determination of amino acids and alkyl amines in complicated matrices in a short time.
Another potential highlight of the proposed method is the simultaneous determination of amino acids, amines and other NH 2 -containing compounds. The results mentioned above showed that the proposed method was capable of separating 15 amino acids and 7 alkyl amines within 30 min including equilibration time, as shown in Fig 1.
The retention time of the 15 amino acids and 7 alkyl amines ranged from 3. Twenty-two analytes were separated completely, except for Gly and Thr neither complete separation nor co-elution. Compared with the reported method in Table 2 , the proposed method offered the advantages of low analysis time, easy derivatization operation, good separation resolution, and high method accuracy. The proposed method was successfully applied in quantitative analysis of 15 amino acids and 7 alkyl amines in the continental and marine aerosols, as shown in Fig 2.
During our observations, 12 amino acids including Asp, Ser, Gly, Thr, Arg, Ala, Val, Met, Phe, Ile, Leu, and Lys and 6 alkyl amines including ethanolamine, methylamine, ethylamine, propylamine, butylamine, and pentylamine were identified and quantified in PM 2.
Among the detected organic nitrogen compounds, 7 amino acids and 6 alkyl amines were identified to be present in every sample from Shanghai, whereas 11 amino acids and 6 alkyl amines in every sample from Huaniao Island.
Moreover, 7 amino acids and 6 alkyl amines were both detected in every sample over both Shanghai and Huaniao Island. The concentrations of individual amino acids and alkyl amines detected are listed in Table 3. From Table 3 , the level of and between different amino acids fluctuated widely, often with over one order of magnitude.
For example, the concentrations of Thr in Shanghai ranged from 2. In our measurements, Gly was the most abundant amino acid among the amino acid species both in Shanghai and Huaniao Island. This fact might be attributed to the low reactivity of glycine [ 37 ]. The concentrations of Gly in PM 2.
Methylamine was the dominant alkyl amine, followed by ethanolamine, in both Shanghai and Huaniao Island. Levels of methylamine in PM 2. Considerable amino acids and alkyl amines have been detected in particulate matters at various locations.
For example, amino acids were found in PM 2. This study is consistent with previous studies, which also detected a marked predominance of Gly in aerosols in Venice [ 39 ]. Furthermore, a substantial contribution of Gly was observed in PM 2. Based on our available data, methylamine and ethanolamine appear to be ubiquitous and significant in the atmospheric environment. This is in agreement to the measurements of amines in continental and marine aerosols [ 14 , 17 , 40 ].
Analytical Methods for Amino Acids
HPCL determination of histamine, tyramine and amino acids in shrimp by-products. Biogenic amines in high concentration can affect the quality of foods and cause several diseases since they are present in products like seafood, wines, and other fermented products. Shrimp by-products are a rich source of protein hydrolysate that can be used as dietary supplements. Samples from shrimp by-products of dry protein powder, liquid protein hydrolysate and flour of shrimp head were analyzed to evaluate the histamine and tyramine contents. The mobile phase was a mixture of tetrahydrofuran, methanol and phosphate buffer at a flow rate of 0. The recovery values were
Amino Acid Analysis
Almut Hesse, Michael G. Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine HTyr and 4-fluorophenylalanine FPhe were used for calibration.
Metrics details. Nowadays, there is a growing interest in innovative and more efficient therapeutics—biopharmaceuticals, based on peptides or proteins.
A method was developed for simultaneous determination of 15 amino acids and 7 alkyl amines. The 22 derivatives were separated within 30 min including the equilibration time and detected by a fluorescence detector at an excitation wavelength of nm and emission wavelength of nm. The analysis procedure was satisfactorily validated by the reproducibility, recovery, linearity and detection limit of the analytes. The relative standard deviations RSDs of retention time and peak area for individual amino acids and alkyl amines were consistently less than 0. The detection limit ranged from 0. The developed and validated method was successfully applied to the quantitative analysis of amino acids and alkyl amines in continental and marine aerosols in China. Among the identified organic nitrogen compounds, 7 amino acids and 6 alkyl amines were detected in every aerosol sample.
Part 1. Amino Acids 1. Gas Chromatography 1. High Performance Liquid Chromatography 1. Part 2.
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