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.     

Liquid chromatographic fluorescence determination of amino acids in plasma and urine after derivatization with phanquinone

Phanquinone (4,7-phenanthroline-5,6-dione) has been investigated as a pre-column derivatization fluorogenic reagent for liquid chromatographic determination of primary amino acids in biological samples. The derivatization reaction was carried out at 68 degrees C both in the presence of aqueous phosphate buffer (pH 8) for 30 min and without buffer for 60 min to allow the determination of basic amino acids (Orn, Lys, Arg). The resulting derivatives were separated under reversed-phase HPLC and detected at lambda(em) = 460 nm with lambda(ex) = 400 nm. The proposed method was validated and applied to the determination of a variety of amino acids directly in urine and after deproteinization with 5-sulfosalicylic acid in plasma samples. The detection and quantitation limits were found in the range 10-450 and 35-1400 fmol, respectively.     

Amino acid analysis by micellar electrokinetic chromatography with laser-induced fluorescence detection: application to nanolitre-volume biological samples from Arabidopsis thaliana and Myzus persicae

Amino acids were derivatised with 4-fluoro-7-nitrobenzo-2,1,3-oxadiazol (NBD-F), separated by micellar electrokinetic chromatography (MEKC), and detected by argon-ion (488 nm) laser-induced fluorescence. The optimised MEKC background electrolyte conditions were: 40 mM sodium cholate, 5 mM beta-cyclodextrin in 20 mM aqueous borate buffer, pH 9.1, with 7% v/v acetonitrile. Using these conditions, 19 amino acids were separated within 17 min. The limits of detection were in the range of 7.6-42.2 pmol/mL and limits of quantitation from 0.05-0.14 nmol/mL. The method was systematically validated for injection volume error, migration time variation, calibration linearity, accuracy, precision, and recovery. Nanolitre volume samples of phloem sap of individual sieve element cells from the plant Arabidopsis thaliana and honeydew from the aphid Myzus persicae were directly analysed with this method. Quantitative amino acid concentrations in these two biological matrices were profiled for the first time. This method is particularly important because it allows the complete profile of the amino acids obtained from individual phloem elements, allowing cell to cell and plant to plant variation to be quantified, which to date has not been possible with Arabidopsis thaliana.     

Determination of methylated arginines by column-switching high-performance liquid chromatography-fluorescence detection

A column-switching high-performance liquid chromatography (HPLC)-fluorescence detection method for the determination of three methylated arginines, N(G)-monomethyl-L-arginine (L-NMMA), N(G),N(G)-dimethyl-L-arginine (asymmetric dimethyl-L-arginine, ADMA), and N(G),N(G)'-dimethyl-L-arginine (symmetric dimethyl-L-arginine, SDMA), which are endogenous nitric oxide synthase inhibitors, was developed. After fluorescence derivatization of plasma samples with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the samples were injected into the HPLC system. The NBD-derivatized methylated arginines were trapped on a cation exchange column with filter to remove proteins, separated within 42 min on a reversed-phase column, and detected at an emission wavelength of 530 nm with excitation at 470 nm. The detection limits were 10 fmol for L-NMMA and 20 fmol for ADMA and SDMA with a signal-to-noise ratio of 3. A good linearity for calibration curves for each methylated arginine was observed within the range of 50-5000 fmol using homoarginine as an internal standard. The proposed method was applied to the quantitative determination of L-NMMA, ADMA and SDMA in rat plasma. The concentrations of L-NMMA, ADMA and SDMA in rat plasma were 0.16 +/- 0.01, 0.73 +/- 0.02 and 0.41 +/- 0.05 micromol l(-1), respectively (n= 5).     

High-performance liquid chromatographic assay of N(G)-monomethyl-L-arginine, N(G),N(G)-dimethyl-L-arginine, and N(G),N(G)'-dimethyl-L-arginine using 4-fluoro-7-nitro-2, 1,3-benzoxadiazole as a fluorescent reagent

N(G)-Monomethyl-L-arginine (L-NMMA), N(G),N(G)-dimethyl-L-arginine (ADMA), and N(G),N(G)'-dimethyl-L-arginine (SDMA) are emerging cardiovascular risk factors. A high-performance liquid chromatographic method with fluorescence detection for the simultaneous determination of L-NMMA, ADMA and SDMA is described. The assay employed 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as a fluorescent derivatization reagent. After solid phase extraction with cation-exchange column, the methylated arginines were converted to fluorescent derivatives with NBD-F, and the derivatives were separated within 32 min on a reversed-phase column. Nomega-Propyl-L-arginine was Used as an internal standard. Extrapolated detection limits were 12 nM (12 fmol per injection) for L-NMMA and 20 nM (20 fmol per injection) for ADMA and SDMA, respectively, with a signal-to-noise ratio of 3. The calibration curves for L-NMMA, ADMA and SDMA were linear within the range of 50-5000 fmol. The method was applied to the quantitative determination of L-NMMA, ADMA and SDMA in 200 microl of rat plasma. The concentrations of L-NMMA, ADMA and SDMA in rat plasma were 0.16 +/- 0.03, 0.80 +/- 0.25 and 0.40 +/- 0.21 microM, respectively (n = 5).     

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…     

Amino acid analysis in five pooled single plant cell samples using capillary electrophoresis coupled to laser-induced fluorescence detection

In this study 21 amino acid standards, samples of pure phloem sap and samples of pooled mesophyll cells were derivatized with fluorescein isothiocyanate, separated by capillary electrophoresis and detected with laser-induced fluorescence at 488 nm. Two different background electrolytes, a sodium borate buffer containing sodium dodecyl sulfate and a sodium borate buffer containing alpha-cyclodextrin, were used for the separation. Using the sodium dodecyl sulfate buffer, 14 amino acid standards could be separated, spiking identified 12 amino acids in pure phloem sap and 13 amino acids in pooled mesophyll cells. With the alpha-cyclodextrin containing background electrolyte, a resolution of 20 amino acid standards could be attained, 17 amino acids in pure phloem sap and 10 amino acids in mesophyll cells could be assigned. Leucine and isoleucine comigrated in both buffer systems. All separations were performed with a voltage of +20 kV and completed within 30 min. The detection limits obtained were in the fmol range for the sodium dodecyl sulfate and in the pmol range for the alpha-cyclodextrin background electrolyte. Compared to the one published capillary electrophoresis-based method for the determination of amino acids from few plant cells, the procedure described here allows very high sensitivity due to the use of laser-induced fluorescence detection and opens the possibility to dilute and measure pl samples with an fully automated, commercially available CE system.     

Selective determination of cysteines through precolumn double-labeling and liquid chromatography followed by detection of intramolecular FRET

In this paper we introduce a novel approach for highly selective and sensitive analysis of cysteines (glutathione, cysteine, and homocysteine). This method is based on the detection of intramolecular fluorescence resonance energy transfer (FRET) in a liquid chromatography (LC) system after double-labeling of the amino and sulfhydryl groups of the cysteines. In this detection process, we monitored the FRET between the amine-derivatized and thiol-derivatized fluorophores. We screened 16 combinations of fluorescent reagents. As a result, FRET occurred most effectively when the sulfhydryl and amino groups of the cysteines were derivatized with 7-diethylamino-3-[{4'-(iodoacetyl)amino}phenyl]-4-methylcoumarin (DCIA, Ex/Em 390/480 nm) and 4-fluoro-7-nitrobenz-2-oxo-1,3-diazole (NBD-F, Ex/Em 480/540 nm), respectively, in this order. The double-labeled cysteines emitted NBD-F fluorescence (540 nm) through an intramolecular FRET process when they were excited at the wavelength of maximum excitation of DCIA (390 nm). The generation of FRET was confirmed by comparison with analysis of n-amylamine or tryptophan (amines without a sulfhydryl group) and 6-mercaptohexanol (thiol without an amino group) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the double-labeled cysteines (DCIA and NBD-F) when performing LC on an ODS column with isocratic elution. The limits of quantification (signal-to-noise ratio = 10) and detection (signal-to-noise ratio = 3) for the cysteines, for a 20-μL injection volume, were in the range 150-670 fmol and 46-200 fmol, respectively. The sensitivity of the intramolecular FRET-forming derivatization method is higher than that of a system which takes advantage of conventional detection of the derivatives. Furthermore, this method provides sufficient selectivity and sensitivity to determine the total cysteines present in the plasma of healthy humans.     

Determination of aromatic and branched-chain amino acids in plasma by HPLC with electrogenerated Ru(bpy)3(3+) chemiluminescence detection

An HPLC method is described for the electrochemiluminescence (ECL) detection of amino acids, following cycloaddition reaction of their amino groups with divinyl sulfone (DVS), using electrogenerated tris(bipyridine)ruthenium(III). The derivatization reaction conditions were examined, with the optimum conditions found to be 40 mM DVS (pH 8.0) at 50 degrees C for 15 min. Detection limits for the 15 amino acids examined varied greatly (0.04-8.0 pmol) using a standard solution by flow injection analysis (FIA). These optimized conditions were used for HPLC determination of the amino acids in human plasma. A linear relationship was obtained up to 100 pmol on a column for aromatic and branched-chain amino acids. Recoveries of Tyr, Met, Val, Leu, Ile, Phe and Trp when added to human plasma (1 micromol/10 ml plasma, n=5) were 101.5+/-1.1, 99.0+/-1.2, 98.0+/-1.4, 101.1+/-1.6, 95.1+/-1.6, 99.2+/-1.5 and 97.7+/-1.3 % (mean+/-S.D.) respectively. The concentrations of the amino acids in the plasma are in good agreement with other published data.     

Analysis of free amino acids in islets of Langerhans by high-performance liquid chromatography using pre-column derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole

A simple high-performance liquid chromatographic method with pre-column derivatization and fluorescence detection was developed and used for the analysis of free amino acids in islets of Langerhans; 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) served as pre-column derivatization reagent. Islets of Langerhans were separated from the pancreas of normal and obese rats, treated with pre-cooling methanol-water (80:20, v/v), and ultrasonicated to fragmentize the islets and effect deproteination. Several parameters influencing the derivatization reaction and chromatographic separation were optimized. Amino acid derivatives obtained under optimal conditions were separated on a C18 column with acetonitrile-acetate buffer as mobile phase and detected at 470 nm/540 nm (Ex/Em). Matrix effects were investigated and good linearities with correlation coefficients better than 0.9972 were obtained over a wide range of 0.42-42.11 microM for most of the amino acids. The detection limits (S/N = 3) were within the range of 6.1-51 nM. The precision of the method and recoveries were in the ranges of 1.43-10.76% (RSD%) and 85.07-108.82%, respectively. The analytical results showed that the serine content was markedly higher in normal rats than in obese rats, whereas methionine was of relatively lower content in both normal and obese rats.     

Coupling of sequential injection with liquid chromatography for the automated derivatization and on-line determination of amino acids

The principle of sequential injection (SI) was exploited to develop a fully automated pre-column derivatization procedure combined on-line to liquid chromatography (LC). Using SI-LC derivatization 14 amino acids were determined fluorimetrically in pharmaceuticals with o-phthaldialdehyde (OPA) as the derivatization reagent. The SI system was used for the handling of samples and reagents, on-line mixing and introduction to the LC injection system. Chemical (pH and reagents concentrations) and instrumental variables (sample and reagent volumes, reaction time and flow rate) were optimized to attain the highest reaction yield and detector signal. Reversed phase chromatographic resolution of 14 amino acids was achieved within 35 min using gradient elution. The automated operation of the coupled SI-LC system resulted in very satisfactory performance. The method was applied for the simultaneous determination of amino acids in pharmaceutical formulations.     

In-capillary derivatization and analysis of amino acids,amino phosphonic acid-herbicides and biogenic amines by capillary electrophoresis with laser-induced fluorescence detection

This paper describes a general approach for the in-capillary derivatization of amino compounds and the subsequent sensitive determination of the derivatives by micellar electrokinetic chromatography (MEKC) or capillary zone electrophoresis (CZE) with laser-induced fluorescence (LIF) detection. Amino acids, biogenic amines and amino phosphonic acid-herbicides were chosen as model analytes to evaluate the analytical potential of this approach. Fulfilment of the in-capillary reaction of the analytes using LIF detection hinged on the excellent labeling chemistry of 5-(4,6-dichloro-s-triazin-2-ylamino)fluorescein (DTAF) and the good resolution achieved in the separation of derivatized analytes. Careful optimization of the electrophoretic conditions in the mixing step of this protocol allowed the determination of amino acids, biogenic amines and phosphorus-containing amino acid-herbicides with concentration limits of detection at the nug/L level and relative standard deviations from 3.5 to 5.8%. The whole analysis is carried out within 20 min, resulting in a very simple, fast and practical approach for the fully automated analysis of amino acids and related compounds in low-volume and low-concentration samples.     

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.     

微波水解衍生高效液相色谱法测定饲料中的氨基酸

建立了一种微波水解衍生高效液相色谱同时测定饲料中17种氨基酸含量的方法。采用2,4-二硝基氯苯作为柱前衍生试剂,利用C_(18)色谱柱分离。二极管阵列检测器进行检测,检测波长为360 nm,并对微波水解时间及温度进行优化。17种氨基酸在2.5~50 mg/L范围内呈良好线性关系,相关系数为0.990 7~0.999 9;相对标准偏差为0.88%~4.2%;加标回收率为90.6%~107.2%;检出限为0.15~2.37 mg/L。研究结果表明,150℃下微波水解16 min的结果与传统加热水解(110℃,24 h)的效果基本相同。该方法分析时间较短,灵敏度较高,可用于饲料中氨基酸含量的检测。     

An automated analyzer for methylated arginines in rat plasma by high-performance liquid chromatography with post-column fluorescence reaction

A fully automated analyzer for methylated L-arginine metabolites [N,N-dimethyl-L-arginine (ADMA), N-methylarginine (NMMA) and N,N'-dimethyl-L-arginine (SDMA)] by high-performance liquid chromatography with post-column fluorescence derivatization was developed. This system consists of an on-line extraction, a separation on a reversed phase ion-pair chromatograph, a post-column derivatization by o-phthaladehyde (OPA) and thiol reaction, and fluorescence detection. NMMA, ADMA and SDMA were separated in 40 min with isocratic elution by a combination of octanoate and cyclohexane carboxylate as ion-pair reagents. The eluate was monitored at 450 nm with excitation at 337 nm. The calibration curves for NMMA, ADMA and SDMA showed linearity over the range from 0.05 micromol l(-1) (0.5 pmol on column) to 5.0 micromol l(-1) (50 pmol on column). This method does not require any time-consuming pre-treatment and requires only 10 microl of plasma sample for assay.     

Automated precolumn derivatization system for analyzing physiological amino acids by liquid chromatography/mass spectrometry

An automated method for high‐throughput amino acid analysis, using precolumn derivatization high‐performance liquid chromatography/electrospray mass spectrometry (HPLC/ESI‐MS), was developed and evaluated. The precolumn derivatization step was performed in the reaction port of a home‐built auto‐sampler system. Amino acids were derivatized with 3‐aminopyridyl‐N‐hydroxysuccinimidyl carbamate, and a 3 μm Wakosil‐II 3C8‐100HG column (100 × 2.1 mm i.d.) was used for separation. To achieve a 13 min cycle for each sample, the derivatization and separation steps were performed in parallel. The results of the method evaluation, including the linearity, and the intra‐ and inter‐precision, were sufficient to measure physiological amino acids in human plasma samples. The relative standard deviations of typical amino acids in actual human plasma samples were below 10%. Copyright © 2009 John Wiley & Sons, Ltd.     

Highly sensitive determination and validation of gabapentin in pharmaceutical preparations by HPLC with 4-fluoro-7-nitrobenzofurazan derivatization and fluorescence detection

A sensitive HPLC method with pre-column fluorescence derivatization using 4-Fluoro-7-Nitrobenzofurazan (NBD-F) has been developed for the determination of gabapentin in pharmaceutical preparations. The method is based on the derivatization of gabapentin with (NBD-F) in borate buffer of pH 9.5 to yield a yellow, fluorescent product. The HPLC separation was achieved on a Inertsil C(18) column (250 mm × 4.6 mm) using a mobile phase of methanol water (80:20, v/v) solvent system at 1.2 mL/min flow rate. Mexiletine was used as the internal standard. The fluorometric detector was operated at 458 nm (excitation) and 521 nm (emission). The assay was linear over the concentration range of 5 50 ng/mL. The method was validated for specificity, linearity, limit of detection, limit of quantification, precision, accuracy, robustness. Moreover, the method was found to be sensitive with a low limit of detection (0.85 ng/mL) and limit of quantitation (2.55 ng/mL). The results of the developed procedure for gabapentin content in capsules were compared with those by the official method (USP 32). Statistical analysis by t- and F-tests, showed no significant difference at 95 confidence level between the two proposed methods.     

Amino acid composition analysis of minute amounts of cysteine-containing proteins using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and 4-fluoro-7-nitro-2,1,3-benzoxadiazole in combination with HPLC

The simultaneous determination of amino acid composition including cysteine of egg albumin, a model protein containing a/s cysteine residue, is reported. All the thiol groups of the cysteine residue(s) of egg albumin were labelled with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole, a fluorogenic reagent for thiol groups. The labeled egg albumin was hydrolyzed in 6N HCl at 110 degrees C for 24 h. The hydrolysate was lyophilized, derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, a fluorogenic reagent for amines, and subjected to HPLC. 18 derivatized amino acids including double labelled cysteine were separated within 90 min on a Nucleosil ODS column (150 mm X 4.6 mm i.d.; 5 microns), and detected at 530 nm (ex. 470 nm) in a range from 90 fmol (aspartic acid) to 1.3 pmol (cysteine) (S/N = 3). Composition ratios of amino acids of egg albumin were similar to theoretical values except for methionine, which would be destroyed under the present acid hydrolysis condition. Analytical methods for cysteine residues are reviewed, and the availability of fluorogenic reagents having the benzofurazan structure is also discussed.     

Applications of automated amino acid analysis using 9-fluorenylmethyl chloroformate.

A rapid and sensitive fully automated method for the determination of primary and secondary amino acids in different matrices is described. Amino acids are derivatized with 9-fluorenylmethyl chloroformate using an automated precolumn derivatization technique. Data are presented to show that the technique is both reproducible and highly sensitive. Applications of the technique are presented, including the analysis of peptide and protein hydrolysates and the profiling of free amino acids in physiological fluids.     

High-performance liquid chromatographic determination of peptides in biological fluids by automated pre-column fluorescence derivatization with fluorescamine

Peptides containing a free alpha- or epsilon-amino group react with fluorescamine under mild alkaline conditions to generate a highly fluorescent but unstable reaction product and, consequently, practical high-performance liquid chromatographic (HPLC) approaches to analysis have typically involved the use of postcolumn derivatization. An automated precolumn approach is reported in which peptides are reacted with fluorescamine just prior to HPLC analysis by a commercially available autoinjector with derivatization capabilities. The autoinjector added base and fluorescamine reagent solutions to a sample vial containing peptide analytes, and the derivatization reaction was allowed to proceed for 5 min at room temperature prior to injection into the HPLC system. The derivatized peptides were analyzed by reversed-phase HPLC with fluorescence detection (excitation at 390 nm; emission 470-nm cut-off filter) on an octylsilica column. Optimization of the precolumn reaction conditions and the use of narrower HPLC columns (2 mm I.D.) resulted in a typical on-column detection limit of 30-50 fmol of peptide, which was substantially lower than that in previously reported post-column methods. This approach was applied to the HPLC of several naturally occurring and synthetic peptides containing alpha- and epsilon-amino groups. In combination with solid-phase extraction, prior to automated precolumn fluorescence derivatization and chromatographic analysis, the methodology was used for the determination of a synthetic growth hormone-releasing peptide in plasma samples.