Determination of neurotoxin 3-N-oxalyl-2,3-diaminopropionic acid and non-protein amino acids in Lathyrus sativus by precolumn derivatization with 1-fluoro-2,4-dinitrobenzene

A rapid and simple method is presented for determining neuro-excitatory nonprotein amino acid 3-N-oxalyl-2,3-diaminopropionic acid (beta-ODAP) and non-protein amino acids in Lathyrus sativus. Seed and foliage extracts of Lathyrus sativus were treated with 1-fluoro-2,4-dinitrobenzene (FDNB) and a reversed-phase high-performance liquid chromatography method (RP HPLC) for the separation of the derivatives in the pmol range is reported. The RP HPLC method and a colorimetric method were compared for measuring ODAP.     

CE methods for the determination of non-protein amino acids in foods

In addition to the 20 amino acids universally distributed as protein constituents in living organisms, there are other amino acids of non-protein origin that can be found in foods. The determination of these non-protein amino acids is interesting since they can be indicative of the quality and safety of foods. This work presents for the first time an updated and comprehensive review devoted to show the possibilities of capillary electrophoresis for the determination of non-protein amino acids in food samples. The results reported have been classified according to the chemical structure of the non-protein amino acid studied. Separation conditions as well as detection systems used have been detailed since most of these amino acidic compounds do not possess chromophore groups detectable by conventional UV-Vis detection, being in this case necessary a previous derivatization step. Finally, the application of microchip electrophoresis to the determination of non-protein amino acids in foodstuffs is also included in this review.     

HPLC Analysis of Amino Acids with Ion Exchange Chromatography and O-Phthalaldehyde Post-Column Derivatization: Applications to the Assay of Endogenous Free Amino Acids and Their Transport in Human Placental Villus

Abstract

A method using high performance liquid chromatography (HPLC) for the analysis of primary amino acids in human placenta is described. This method involves separation of primary amino acids by high performance ion-exchange chromatography followed by post column derivatization using O-phlthalaldehyde (OPA) and 2-mercaptoethanol and fluorescence (excitation 340 nm and emission 410 nm) detection of derivatives. Waters 840 HPLC Amino Acid System was used for this purpose.

For analysis, villus tissue was extracted with acetonitrile, and the recovered amino acids were reconstituted in a sodium diluent (pH 2.2). The complete profile of the primary amino acids in the sample could be constructed in about 90 minutes. Up to 44 samples can be analyzed without special attention. Using this method, essential amino acids (threonine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine) and nonessential amino acids (aspartic acid, serine, glutamic acid, glycine, alanine, arginine) were detected and quantified in human placental villus in pmol quantities. Plots of peak heights (or areas) were linear for several amino acids. The same method was also used for (a) the assay of free primary amino acids in umbilical bloods, (b) the efflux of amino acids from isolated human placental villus, and (c) to study the uptake of α-aminoisobutyric acid (AIB), a non-metabolizable amino acid, by the isolated placental villus.     

HPLC Determination of Amino Acids by Pre-Column Fluorescence Derivatization with Carbazole-9-yl-Acetyl Chloride

Atpresentseparationsandquantitativedeterminationsofaminoacidsbymeansofnewfluorescencereagentsforpre-columnorpost-columnderivatizationinRP-HPLCarestillanactivefiled,developmentshavingbeensummarizedbySnyder'.MostaminoacidsdonotshowUVabsorptionin220-254urn,henceinordertoincreasedetectionsensitivityandimproveselectivity,generallyderivatizationreagentsareemployed.Phenylisothiocyanate(PITC)',OPAand3,5-dinitrobenzoylchloride3arewellknownderivatizationreagefltsforthedeterminationofaminocompounds…     

Analysis of amino acids in human serum by isocratic reversed-phase high-performance liquid chromatography with electrochemical detection

A simple, sensitive and reproducible isocratic high-performance liquid chromatography (HPLC) method has been developed for the determination of amino acids in human serum. The method involves precipitation of the serum proteins with methanol followed by pre-column derivatization of amino acids with o-phthalaldehyde-2-mercaptoethanol or o-phthalaldehyde-sodium sulfite. HPLC separation of the derivatives was performed using an ODS column with an isocratic mobile phase system and electrochemical detection (+0.75 V). The response was linear over the range 5-300 microM for all amino acids. The method allows quantitative determination of glutamic acid, asparagine, serine, glutamine, histidine, taurine, alanine, arginine, methionine, isoleucine, ornithine, leucine, phenylalanine, lysine and tryptophan at concentrations as low as 0.5-5.0 pmol (signal-to-noise ratio=2). Using this method, the levels of amino acids in serum from healthy donors and patients with ischemic stroke were determined.     

Quantitative analysis of fluorimated ethylchloroformate derivatives of non-protein amino acids using positive and negative chemical ionization gas chromatography-mass spectometry

The GC-MS characterization of the ethylchloroformate derivatives of amino acids in an aqueous medium has been applied to non-protein amino acids. Derivatization of non-protein amino acids using ethylchloroformate, trifluoroethanol, and pyridine produced strong [M + 1]⁺ and [M - 1]⁻ ions in positive and negative chemical ionization (CI) modes, respectively. Twenty-one out of the twenty-three non-protein amino acids studied produced detectable ion chromatograms in both ionization modes when methane was used as the CI reagent gas. Mass spectra of these non-protein amino acid derivatives showed characteristic [M - 19]⁺, [M + 1]⁺, [M + 29]⁺, and [M + 41]⁺ peaks in the positive chemical ionization mode, and [M - 1]⁻, and [M + 35]⁻ peaks in the negative chemical ionization mode. The detection limits and the linear dynamic range of trifluorethanol ethylchloroformate derivatives of non-protein amino acids were studied using positive chemical ionization. The detection limits are mostly in the femtomole range.     

Elution behavior of diaminopimelic acid and related diamino acids using the advanced Marfey's method

The advanced Marfey's method consists of a chromatography technique for the separation of amino acids into each enantiomer by derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA), and a detection method using liquid chromatography/mass spectrometry (LC/MS) which can determine the non-empirically the absolute configuration of various amino acids including the non-protein ones. However, this method has not been applied to the determination of the absolute configuration of an amino acid with a "meso" configuration such as diaminopimelic acid (A2pm). In the present study, this method was successfully applied to determine the absolute configurations of diaminosuccinic acid (DAS), A2pm, cystine (Cys), selenocystine (SeCys) and homocystine (HomoCys) using a racemization procedure and the DL-FDLA method, and the resulting elution behavior was summarized as follows: (1) the LL- and meso-isomers were eluted prior to the DD-isomer except for one case; (2) the LL- and meso-isomers are closely eluted and the elution was occasionally reversed; (3) the retention time for both the L- and D-derivatives of the meso-isomer was not changed; (4) the complementary use of the two solvent systems using CH3CN and MeOH was effective to obtain a chromatogram with a high resolution; (5) the abnormality, such as the elution order and peak shape, was observed in the elution behavior of DAS.     

A capillary electrophoresis-tandem mass spectrometry methodology for the determination of non-protein amino acids in vegetable oils as novel markers for the detection of adulterations in olive oils

A new analytical methodology based on capillary electrophoresis-mass spectrometry (CE-MS(2)) is presented in this work, enabling the identification and determination of six non-protein amino acids (ornithine, β-alanine, GABA, alloisoleucine, citrulline and pyroglutamic acid) in vegetable oils. This methodology is based on a previous derivatization with butanol and subsequent separation using acidic conditions followed by on-line coupling to an ion trap analyzer for MS(2) detection established through an electrospray-coaxial sheath flow interface. The electrophoretic and interface parameters were optimized obtaining the separation of all compounds in less than 15 min and with resolutions higher than 5. The proposed method was validated by assessing its accuracy, precision (RSD<7% for corrected peak areas), LODs and LOQs (between 0.04-0.19 ng/g and 0.06-0.31 ng/g, respectively) and linearity range (R(2)>0.99), and it was used in order to identify the selected non-protein amino acids in soybean oils, sunflower oils, corn oils and extra virgin olive oils. MS(2) experiments performed the fingerprint fragmentation of these compounds allowing to corroborate ornithine and alloisoleucine in seed oils but not in olive oils. The method was applied to identify and quantify olive oil adulterations with soybean oil detecting in a single run the amino acids in mixtures up to 2% (w/w). The results showed a high potential in using these compounds as novel markers for the detection of adulterations of extra virgin olive oils with seed oils. Thus, the developed method could be considered a simple, rapid and reliable method for the quality evaluation of extra virgin olive oil permitting its authentication.     

Application of UHPLC for the determination of free amino acids in different cheese varieties

A rapid ultra-high performance liquid chromatography (UHPLC) protocol for the determination of amino acids as their respective 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives was successfully applied for assessing free amino acid levels in commercial cheese samples representing typical product groups (ripening protocols) in cheesemaking. Based on the Waters AccQ.Tag? method as a high performance liquid chromatography (HPLC) amino acid solution designed for hydrolyzate analyses, method adaptation onto UHPLC was performed, and detection of AQC derivatives was changed from former fluorescence (λ _Ex 250 nm/λ _Em 395 nm) to UV (254 nm). Compared to the original HPLC method, UHPLC proved to be superior by facilitating excellent separations of 18 amino acids within 12 min only, thus demonstrating significantly shortened runtimes (>35 min for HPLC) while retaining the original separation chemistry and amino acid elution pattern. Free amino acid levels of the analyzed cheese samples showed a high extent of variability depending on the cheese type, with highest total amounts found for original Italian extra-hard cheeses (up to 9,000 mg/100 g) and lowest for surface mold- or bacterial smear-ripened soft cheeses (200–600 mg/100 g). Despite the intrinsic variability in both total and specific concentrations, the established UHPLC method enabled reliable and interference-free amino acid profiling throughout all cheese types, thus demonstrating a valuable tool to generate high quality data for the characterization of cheese ripening.     

Accurate evaluation method of the polymer content in Monomethoxy(polyethylene glycol) modified proteins based on Amino acid analysis

To overcome the uncertainty of the colorimetric or fluorimetric method so far employed for the evaluation of monomethoxy(polyethylene glycol) (MPEG) covalently bound to protein, a direct method based on amino acid analysis is proposed. The method exploits the use of MPEG, which was bounded with the unnatural amino acid norleucine (MPEG-Nle). MPEG-Nle was activated at its carboxylic group to succinimidyl ester for the binding to the amino groups of protein. After acid hydrolysis, the amino acid content is evaluated by conventional amino acid analyzer or by reverse-phase HPLC as phenylthiocarbamyl derivative. The number of bound MPEG chains is calculated from the amino acid composition, since one norleucine residue is released from each bound polymer chain. The method was verified with several proteins in comparison with colorimetric ones, also in the case of proteins that contain chromophores in the visible range, such cytocrome C. It was observed that in most of the cases, the colorimetric methods give an overestimation of the degree of protein modification.     

Use of high performance liquid chromatography in defining the abnormalities in the free amino acid patterns in the cerebrospinal fluid of patients with aseptic meningitis.

Free amino acids were quantitatively determined in cerebrospinal fluid (CSF) and plasma samples from patients with aseptic meningitis by a newly developed high performance liquid chromatographic (HPLC) method. The method of analysis was based on precolumn derivatization of orthophthaladehyde in the presence of 2-mercaptoethanol and detection was made at Eex = 340 nm and Eem = 450 nm. The method was sensitive and the limit for detection was less than 1 pmol for most of the amino acids. It took 45 min to separate 26 amino acids with highly reproducible results, giving a coefficient of variance for retention times and integrated areas less than 0.4% and 2%, respectively, after five replicate runs. The results accumulated in 10 patients were compared statistically with 11 age-matched healthy controls. Among the amino acids almost all the neurotransmitter candidates, such as aspartic acid, glutamic acid, glutamine, glycine, tyrosine, phenylalanine and gamma-aminobutyric acid (GABA), were significantly increased in the patients' CSF, whereas arginine and threonine were low. No change was observed in plasma amino acids in patients as compared to healthy controls. The higher levels of most of the neurotransmitters, especially GABA, aspartic acid and glutamic acid, could be used diagnostically in assessing the progression and remission in aseptic meningitis.     

Identification and isolation of human insulin A and B chains by high-performance liquid chromatography

A method for the isolation, identification and quantification of human insulin A and B chains by high-performance liquid chromatography (HPLC) is described. These chains were isolated from a peptide mixture produced by E. coli with modified genes obtained by genetic engineering. The method is based on the use of hydrophilic reagents, forming ion pairs in a reversed-phase column. Because some undesirable effects resulting from the use of phosphoric acid were observed, especially with the B chain, a new HPLC method was developed for each of the two human insulin chains. The use of trifluoroacetic acid as a counter ion for the A chain and of formic acid for the B chain led to the rapid isolation and purification of each chain by HPLC. The advantage of this method is that it provides a highly pure product, which was identified by polyacrylamide gel electrophoresis and amino acid analysis.     

Use of capillary electrophoresis and laser-induced fluorescence for attomole detection of amino acids

A capillary electrophoresis and laser-induced fluorescence (CE-LIF) method was developed to identify and quantitate at amol (10(-18)) concentration. Amino acids were derivatized with 3-(4-carboxybenzoyl)-2-quinoline-carboxaldehyde prior to CE-LIF analysis. The assay was developed by varying the sodium borate concentration, buffer pH, operating voltage, and operating temperature. A run buffer system containing 6.25 mM borate, 150 mM sodium dodecyl sulfate, and 10 mM tetrahydrofuran (pH 9.66) at 25 degrees C, and 24 kV provided analysis conditions for a high-resolution, sensitive, and repeatable assay of amino acids. The rate of derivatization, stability of the labeled amino acids, and amino acid quantitation varied for each amino acid. Amino acids were detected with greater efficiency by this method than automated HPLC amino acid analysis. The repeatability of the assay ranged from 0.3 to 0.9% within a day and 0.7 to 1.5% between analysis days. Bacterial amino acid utilization in a chemically defined medium was successfully monitored using this method. This work defines a sensitive and repeatable method for the detection of amino acids during bacterial metabolism.     

Amino acid analysis using DABS-CL

Summary Pre-column derivatization followed by reversed-phase HPLC has become widely used as a sensitive and speedy method of amino acid analysis.The technique has been improved by the development of simple and reproducible strategies for derivatization of the amino acids with DABS and for the HPLC analysis. Results obtained indicate that the method has a high sensitivity is well and suited to generate precise amino acid composition information from peptides and proteins.     

氨基酸的分析方法及其应用进展

从衍生试剂角度,介绍了不同衍生化氨基酸的分析方法,包括离子交换色谱法、高效液相色谱法、气相谱法和毛细管电泳法,以及无需衍生化的直接分析法高效阴离子交换色谱-积分脉冲安培法,并总结了蛋白质、食品和生理体液样品中的氨基酸分析方法。     

手性衍生-高效液相色谱法拆分和定量测定茶氨酸对映体

建立了以1-氟-2,4-二硝基苯基-5-L-丙氨酰胺（FDAA）为手性衍生试剂、高效液相色谱拆分茶氨酸对映体的方法。采用的色谱条件为：Kromasil C18色谱柱;三乙胺-磷酸缓冲液和乙腈为流动相,梯度洗脱，流速1.0 mL/min;检测波长为340 nm;柱温为35 ℃。L-茶氨酸的进样量在1.732×10-3～2.077 μg范围内峰面积与进样量之间的线性关系良好，加标回收率为97.3%～102.0%,检测限为4.973×10-4 μg,定量限为1.223×10-3 μg。D-茶氨酸的进样量在1.696×10-3～2.044 μg范围内峰面积与进样量之间的线性关系良好,加标回收率为97.2%～103.2%,检测限为5.871×10-4 μg,定量限为1.236×10-3 μg。该方法灵敏度高,分析过程中不发生消旋化。     

Fast Analysis of Free Amino Acids in Tobacco by HPLC with Fluorescence Detection and Automated Derivatization

A fast, simple, and sensitive HPLC method for the determination of free amino acids in tobacco was described. A fully automated sample processor performed precolumn derivatization of both primary and secondary amino acids with o‐phthalaldehyde/3‐mercaptopropionic acid and 9‐fluorenylmethyl chloroformate (FMOC‐Cl), respectively. All reactions were fully automated by means of an injector programme and accomplished in 10 min. Sample preparation consisted of a single step of extraction with 0.1 mol/L HCl at ambient temperature (assisted by sonication) in 30 min, followed by filtration of an aliquot and derivatization. By optimization of sample preparation and HPLC conditions, separation of 20 amino acids in 30 min was achieved. Detection limits ranged from 0.50 to 1.40 μg/g; coefficients of variation ranged from 1.8% to 3.9%; recoveries ranged from 84.6% to 108.5%. The method was applied to the analysis of amino acids contents of tobacco leaves in different varieties and flue‐curing period.     

Enantiomeric Separation of N-Protected Non-Protein Amino Acid Esters by Chiral High-Performance Liquid Chromatography

Abstract

We have found that high-performance liquid chromatographic analysis of enantiomeric N-protected amino acid esters on a cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase column (Daicel Chiralcel OD) can be utilized as one of the procedures for determining the optical purities of non-protein amino acids. The methyl esters of the N-benzyloxycarbonyl (Z) derivatives of a number of non-protein amino acids showed excellent to good enantiomeric separations using hexane - 2-propanol as a mobile phase. There was a regularity in the elution order of enantiomers: the L-isomer had a shorter retention time than the D-isomer. We have also investigated the effect of the N-protecting groups and the ester groups on the enantiomeric separation. The Z, 4-methoxybenzyloxycarbonyl (Z(OMe)), and 9-fluorenylmethoxycarbonyl (Fmoc) derivatives gave exceptionally good resolutions. By contrast, the formyl and t-butoxycarbonyl (Boc) groups impaired the enantiomeric separation. Almost all the alkyl esters examined and the benzyl ester gave resolutions better than or of the same order as the methyl ester. The resolution of β-amino acids was worse than that of the corresponding α-amino acids.     

Simple and low-cost high-performance liquid chromatographic method for determination of D- and L-amino acids

In this article, a simple and low-cost method for the analysis of amino acid enantiomers by using high-performance liquid chromatography (HPLC) is described. In this method, the amino acids are modified to diastereomers in order to be separated into enantiomers on a usual C(18) reversed-phase column. Methanol instead of acetonitrile is used as an elution solvent; the results of HPLC with methanol elution are comparable with those of HPLC with acetonitrile elution. Sub-nanomolar sensitivity is attained by measuring the absorbance at 340 nm in analysis of 15 amino-acid enantiomers.