Nitroaromatic Reagents for Determination of Amines and Amino Acids by Liquid Chromatography/Electrochemistry

Abstract

Trinitrobenzene sulfonic acid, 2,4-dinitrofluorobenzene and 2-chloro-3,5-dinitropyridine were compared in terms of properties relevant to use as precolumn derivatization labels for amino compounds. This included consideration of chemical characteristics as well as purely detector oriented behavior. Trinitrobenzene sulfonic acid appears to be superior in all respects, yielding the most detectable derivatives with minimum production of interfering by-products.     

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.     

Separation of Orthophthalaldehyde/Ethanethiol Derivatives of Taurine and Closely Eluting Amino Acids by High Performance Liquid Chromatography

Abstract

In studies of the reverse phase, HPLC analysis of amino acids employing precolumn derivatization with o-phthalaldehyde and ethanethiol, it was shown that α-amino-n-butyric acid, β-amino-isobutyric acid and taurine coeluted in the acetonitrile/aqueous phosphate solvent system. By using a ternary solvent system of acetonitrile/tetrahydrofuran/aqueous phosphate buffer and efficient 5- and 10-μm octadecylsilane packings, the co-elution problem has been resolved. This modified chromatographic system is now being used to quantitatively determine taurine and other closely eluting amino acids in a variety of physiological fluids in order to clarify the role of taurine in human development.     

Complete separation of ephedrines by graphite-layer open-tubular (GLOT) column

Summary A reversed phase high performance liquid chromatographic (RP-HPLC) method for the analysis of amino acids in kelp, using precolumn derivatization, is described. Phenylisothiocyanate (PITC) was used as the reagent for derivatization. The kelp samples were prepared by microwave hydrolysis in only 5 min; seventeen PTC amino acids were separated after hydrolysis and derivatization within 12 min. The coefficients of variation were >1.94 and the correlation coefficients for concentration versus response were >0.999 for all derivatives. The ratio of branched amino acid (BAA) to aromatic amino acid (AAA) was also studied. The method has the advantage of shorter hydrolysis and analysis times with optimum separation. In addition, it gives high repeatability of retention times and peak areas for all the amino acids present.     

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.     

Determination of Metoprolol Tartrate as Cu(II)-Dithiocarbamate Complex

Abstract

An HPLC method has been developed for the determination of the beta adrenergic blocking drug metoprolol tartrate. The method is based on the formation of a Cu (II) - dithiocarbamate complex by precolumn derivatization of secondary amino group of metoprolol with CS₂ and CuCl₂ in the presence of ammonia. The complex is extracted into chloroform and injected into the chromatograph. Chromatographic separation was performed by a RP (μ Bondapak C₁₈) column with methanol - pH 5.8 phosphate buffer (80 : 20) as the isocratic mobile phase. A variable wavelength absorbance detector (λ = 275 nm) was used. A linear relationship was obtained between peak areas and metoprolol tartrate concentrations over the range of 35 - 180 nmol ml^?1. The method was applied to the analysis of metoprolol tartrate tablets and the results were statistically compared with those obtained by official method using t - and F - tests.     

High-performance liquid chromatography with sequential injection for online precolumn derivatization of some heavy metals

HPLC was coupled with sequential injection (SI) for simultaneous analyses of some heavy metals, including Co(II), Ni(II), Cu(II), and Fe(II). 2-(5-Nitro-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (nitro-PAPS) was employed as a derivatizing reagent for sensitive spectrophotometric detection by online precolumn derivatization. The SI system offers an automated handling of sample and reagent, online precolumn derivatization, and propulsion of derivatives to the HPLC injection loop. The metal-nitro-PAPS complexes were separated on a C(18)-muBondapak column (3.9x300 mm(2)). Using the proposed SI-HPLC system, determination of four metal ions by means of nitro-PAPS complexes was achieved within 13 min in which the parallel of derivatization and separation were processed at the same time. Linear calibration graphs were obtained in the ranges of 0.005-0.250 mg/L for Cu(II), 0.007-1.000 mg/L for Co(II), 0.005-0.075 mg/L for Ni(II), and 0.005-0.100 mg/L for Fe(II). The system provides means for automation with good precision and minimizing error in solution handling with the RSD of less than 6%. The detection limits obtained were 2 microg/L for Cu(II) and Co(II), and 1 microg/L for Ni(II) and Fe(II). The method was successfully applied for the determination of metal ions in various samples, including milk powder for infant, mineral supplements, local wines, and drinking water.     

Chiral high-performance liquid chromatography analysis of alpha-amino acid mixtures using a novel SH reagent--N-R-mandelyl-L-cysteine and traditional enantiomeric thiols for precolumn derivatization

Several chiral thiols, i.e. traditionally used enantiomerically pure SH reagents and novel N-R-mandelyl-L-cysteine (R-NMC) containing additional chiral center, have been applied as co-reagents in precolumn derivatization with o-phthalaldehyde for enantiomeric HPLC analysis of individual alpha-amino acids and their mixtures. The R-NMC-derived isoindoles as well as adducts with other thiols have a characteristic absorption maximum at 340 nm, and are highly fluorescent allowing detection of 10 microg/l of an amino acid. Investigated 19 amino acids were analyzed separately and in a mixture by a gradient HPLC after precolumn derivatization. The chromatographic behavior of formed isoindoles substantially differs for each of the thiols used for modification. In contrast to traditional enantiomeric thiols application of diastereomeric R-NMC provides higher resolution for alpha-amino acid enantiomers, with L,D-elution order (except for Arg). Combined use of R-NMC and other thiol enlarges the possibilities of this method, allowing accurate chiral analysis of complex amino acid mixtures.     

A novel derivatization reagent possessing a bromoquinolinium structure for biological carboxylic acids in HPLC‐ESI‐MS/MS

A novel bromoquinolinium reagent, i.e. 1‐(3‐aminopropyl)‐3‐bromoquinolinium bromide (APBQ), was synthesized for the analysis of carboxylic acids. A simple and practical precolumn derivatization procedure using the APBQ in RP chromatography and MS (HPLC‐MS) has been developed using bile acids and free fatty acids, as the representative carboxylic acids in biological samples. The APBQ efficiently reacted with carboxylic acids at 60°C for 60 min in the presence of N,N‐dicyclohexylcarbodiimide and pyridine as the activation reagents. Because the APBQ possesses a bromine atom in the structure, the identification of a series of carboxylic acids was easily achieved due to the characteristic bromine isotope pattern in the mass spectra. The APBQ also has a quaternary amine structure, thus the positively charged derivatives are predominate for the highly sensitive detection of carboxylic acids. The APBQ was successfully applied to the selective determination of biological carboxylic acids in human plasma. The bile acids (chenodeoxycholic acid and deoxycholic acid) and several saturated (stearic acid and palmitic acid) and unsaturated free fatty acids (oleic acid and linoleic acid) were reasonably determined by HPLC‐MS under the proposed procedure. Based on the results of analyses of human plasma and saliva, the proposed procedure using APBQ seems to be applicable for the qualitative and quantitative analyses of a series of carboxylic acids in biological samples.     

Determination of captopril in human plasma with precolumn derivatization using solid phase extraction and HPLC

A rapid, accurate and sensitive method for the determination of captopril in human plasma was developed by solid phase extraction and high performance liquid chromatography (HPLC), using precolumn derivatization of captopril with chromophore label o-phethaldialdehyde (OPA). The extraction of captopril from human plasma was carried out by an amino propyl cartridge. A 0.01 M solution of HCl in methanol showed the best recovery and was chosen for elution of captopril in cartridge. This methanolic solution was applied to react with aqueous solution of OPA and glycine as a coderivatization reagent. The process of derivatization was completed within 2 min at room temperature. The derivatized captopril was injected into a reverse phase HPLC system. Mobile phase was consisted of water:acetonitrile:trifluoroacetic acid (85:15:0.1 v/v/v) with a flow rate of 1 ml min^?1 and detector was used at 345 nm. Linear dynamic range and limit of detection were found as 0.1–6 ppm and 0.1 ppm, respectively.     

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.     

Pre-Column Derivatization in HPLC of Amino Acids and Peptides

Abstract

The development of high preformance liquid chromatography with permanently bonded non-polar, so called reversed phase packings has been useful in the separation of a variety of peptides and amino acid derivatives. Limits to the detectability of underivatized substances are on the order of several nanomolar. Sensitivity enhancement became very important for the analysis and determination of peptide hormones, their metabolites and fragments, trace constituents and amino acids existing in very little quantity in biological and clinical samples. Therefore chemical derivatization techniques have been introduced into HPLC, too. By derivatization not only sensitivity enhancement, but selectivity can be achieved.     

High Performance Liquid Chromatographic Analysis for Free Valine in Plasma

Abstract

A precolumn derivatization method is presented with the use of a fluorescent derivative, 1-dimethylaminonaphhalene-5-sulfonyl-chloride, dansyl chloride, for the detection of free valine in plasma. Dansylated amino acids were determined in deproteinized samples by reverse-phase liquid chromatography. The level of detection is 100 femtoraoles (10^?15). Sample preparation required precipitation of proteins with trichoroacetic acid and removing the excess acid with water saturated ether. The deproteinized sample was adjusted to pH 9.0 and reacted with dansyl chloride. The dansylated products were detected by ultraviolet and fluorescence spectrometry. Elution time for valine subsequent to injection is 25 minutes, while the total assay requires less than 50 minutes.     

Analysis of Insulin by High Performance Liquid Chromatographic Method with Precolumn Derivatization with 4‐Chloro‐7‐Nitrobenzo‐2‐Oxa‐1,3‐Diazole

Abstract

A high performance liquid chromatographic method (HPLC) with precolumn derivatization and fluorescence detection for insulin was developed and applied for the quantification of insulin in spiked serum. To covalence couple with insulin, 4‐chloro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl) was selected as fluorescent reagent. The optimal derivatization conditions were as follows: temperature 50; time 2 h, in the dark; 0.1 M phosphate buffer (pH 9.0). Analytical separation was carried out on a C18 column and the mobile phase including acetonitrile‐water containing 0.1% trifluoroacetic acid (TFA) (v/v∶ 30/70). The excitation/emission wavelengths were 470/540 nm. Under the conditions, the retention time and capacity factor of the adduct of insulin‐NBD were 10.03 min (flow rate 1 mL/min) and 3, respectively. The recovery of insulin in serum was 95.06% and the detection limit was 90 nM. In the investigated concentration ranges (0.46 µM～16.10 µM), R² was 0.9934, which indicated the potential for the application of NBD‐Cl derivatization to the analysis of insulin in the biological matrices, although with the shortcoming of long analytical time.     

1,3,5,7-Tetramethyl-8-aminozide-difluoroboradiaza-s-indacene as a new fluorescent labeling reagent for the determination of aliphatic aldehydes in serum with high performance liquid chromatography

A BODIPY-based fluorescent derivatization reagent with a hydrazine moiety, 1,3,5,7-tetramethyl-8-aminozide-difluoroboradiaza-s-indacene (BODIPY-aminozide), has been designed for aldehyde labeling. An increased fluorescence quantum yield was observed from 0.38 to 0.94 in acetonitrile when it reacted with aldehydes. Twelve aliphatic aldehydes from formaldehyde to lauraldehyde were used to evaluate the analytical potential of this reagent by high performance liquid chromatography (HPLC) on C₁₈ column with fluorescence detection. The derivatization reaction of BODIPY-aminozide with aldehydes proceeded at 60 °C for 30 min to form stable corresponding BODIPY hydrazone derivatives in the presence of phosphoric acid as a catalyst. The maximum excitation (495 nm) and emission (505 nm) wavelengths were almost the same for all the aldehyde derivatives. A baseline separation of all the 12 aliphatic aldehydes (except formaldehyde and acetaldehyde) is achieved in 20 min with acetonitrile–tetrahydrofuran (THF)–water as mobile phase. The detection limits were obtained in the range from 0.43 to 0.69 nM (signal-to-noise = 3), which are better than or comparable with those obtained by the existing methods based on aldehyde labeling. This reagent has been applied to the precolumn derivatization followed with HPLC determination of trace aliphatic aldehydes in human serum samples without complex pretreatment or enrichment method.     

Derivatization of Ketosteroids for High-Performance Liquid Chromatography with Electrochemical Detection

Abstract

A highly sensitive and selective derivatization of ketosteroids for use in high-performance liquid chromatography with electrochemical detection (HPLC/EC) is described. When the detector response for three phenylhydrazone derivatives was compared with one another using dehydroepiandrosterone, the p-nitrophenylhydrazone showed the highest sensitivity with a detection limit of 200 pg. Dehydroepiandrosterone and other principal 17-ketosteroids in human blood were quantitatively derivatized into p-nitrophenylhydrazones and efficiently separated by HPLC/EC on a μBondapak C_{1 8} column using 0.5%. NH₄H₂PO₄/methanol (2:7, v/v).     

Analysis of Free Amino Acids and Protein Contents of Mature Human Milk from Turkish Mothers

Abstract

This study was designed to evaluate free amino acid (FAA) composition and total protein in mature human milk from Turkish mothers. Free amino acid concentrations in mature human milk were determined in all subjects using a high‐performance liquid chromatography (HPLC) with postcolumn derivatization system, with a fluorescence detector. Total protein content was determined by the classical biuret method. Total protein concentration was found to be 1.3±0.4 mg/dl. Glutamic asid plus glutamine is the most abundant amino acid (1275 µmol/L), followed by taurine (353 µmol/L) and alanine (261 µmol/L). Glutamic acid plus glutamine accounts for the most free amino acids in mature human milk and their sum represents 40% of total FAA. On the other hand, some amino acid derivatives such as citrulline, ethanolamine, ammonium, ornithine, ortophosphoserine, and phosphoethanolamine, not usually a part of protein, are determined and this fraction represented ～21% of the total FAA in mature human milk in the present study.     

Determination of urinary free cortisol by high performance liquid chromatography with sulphuric acid-ethanol derivatization and column switching.

A rapid and highly sensitive determination method for urinary free cortisol has been developed using reversed phase high performance liquid chromatography (HPLC) with a precolumn for sulphuric acid-ethanol fluorescence derivatization and column switching. Urinary cortisol, eluted from the octadecylsilane-bonded silica (ODS) minicolumn with 90% aqueous ethanol, was derivatized with the addition of sulphuric acid only at ambient temperature. Cortisol derivatives injected directly onto the ODS precolumn were purified on-line. After switching the columns, the cortisol derivative was separated on an ODS analytical column with a retention time of 15.3 min and monitored at an emission wavelength of 520 nm (exitation wavelength of 365 nm) to decrease the detection limit to 0.26 microgram/dL (signal-to-noise ratio = 3). The automated HPLC operation resulted in good reproducibility and recovery of the stable cortisol derivative at 5 degrees C.     

Laboratory investigation of annular denuders as sampling system for the determination of aliphatic primary and secondary amines in stack gas

A method for the sampling and analysis of aliphatic amines ranging from C₁ to C₅ and some diamines in exhaust gas is described. Two different annular denuder systems coated with phosphoric acid were used for the laboratory investigation of sampling gaseous amines. The denuder sampling systems were tested under different conditions for their readiness to work in stack gas.The reagent 2,4-dinitrofluorobenzene (DNFB) was used in pre-column derivatization of the amines investigated. The amino derivatives were separated using high performance liquid chromatography (HPLC) in conjunction with UV-detection. This method has been used for the examination of emissions from a landfill waste disposal and from animal husbandry with respect to traces of aliphatic amine emissions.     

Separation and Quantitation of O-Phthalaldehyde Derivatives of Taurine and Related Compounds in a High Performance Liquid Chromatography (HPLC) System

Abstract

A sensitive specific assay for taurine using high performance liquid chromatograpy and fluorescence measurement is described. The method employs precolumn derivatization with o-phthalaldehyde in the presence of ethanethiol. Taurine is clearly separated from other amino acids including its precursors hypotaurine and cysteine sulfinic acid. The fluorescence peak height is linear between 1 and 100 picomoles of taurine. There is clear separation of taurine from a contaminant of other taurine assays, α-glycerophosphoryl ethanolamine.