Laser-induced fluorescence detection in liquid chromatography after preliminary derivatization of carboxylic acid and primary amino groups

The development of selective derivatization for the determination of carboxylic acids, amino acids and peptides in aqueous solutions is described as a preliminary study for the determination of these compounds in biological materials. The derivatization reactions are completed before the liquid chromatographic separation and laser-induced fluorescence detection for which a continuous-wave argon-ion gas laser is used in the ultraviolet or visble mode. Carboxylic acid groups arre derivatized with 9-hydroxymethylathracene and primary amino groups are derivatized with fluorescein isothiocyanate. Detection limits, in aqueous solutions, for the carboxylic acid derivatives are ca. 190 fg (ultraviolet mode). In the visible mode, the detection limits are ca. 1 fg for the primary amino derivatives of amino acids and peptides. In al the chromatographic analyses, the derivatization mixtures are injected onto a standard reversed-phase or reversed- phase ion-pair system and conventional flow cells are used without expensive photon counting or optical systems.     

Rapid Determination of Free Amino Acids in Milk by Microchip Electrophoresis Coupled with Laser‐induced Fluorescence Detection

A simple and sensitive method for determination of free amino acids in milk by microchip electrophoresis (MCE) coupled with laser‐induced fluorescence (LIF) detection was developed. Seven kinds of standard amino acids were derivated with sulfoindocyanine succinimidyl ester (Cy5) and then perfectly measured by MCE‐LIF within 150 s. The parameters of MCE separation were carefully investigated to obtain the optimal conditions: 100 mmol·L^?1 sodium borate solution (pH 10.0) as running buffer solution, 0.8 kV as injection voltage, 2.2 kV as separation voltage etc. The linear range of the detection of amino acids was from 0.01 µmol·L^?1 to 1.0 µmol·L^?1 and the detection limit was as low as about 1.0 nmol·L^?1. This MCE‐LIF method was applied to the measurements of free amino acids in actual milk samples and satisfactory experimental results were achieved.     

Fluorimetric determination of amino acids and photochemical stability of their reaction products with ortho-phthalic aldehyde under irradiation by high-power pulsed laser

The opportunity of the highly sensitive determination of amino acids using the fluorescence of their derivatives that form in the reaction with aqueous solutions of ortho-phthalic aldehyde and nucleophilic agents, potassium cyanide, sodium sulfite, and N-acetyl-L-cysteine was studied as an example. The chemical stability of forming fluorescent compounds (substituted isoindoles) and their photochemical stability under the action of power pulsed laser radiation is higher, and the analytic characteristics of reagents are highly competive with a standard applied reagent for determining amino acids and a mixture ortho-phthalic aldehyde with 2-mercaptoethanol.     

Determination of Sulfophenyl Carboxylic Acids in Agricultural Groundwater Samples by Liquid Chromatography with Fluorescence Detection

Abstract

We have developed an analytical method to determine six sulfophenyl carboxylic acids (SPC) in agricultural groundwater samples. It involves a solid-phase extraction (SPE) procedure and subsequent separation and determination using liquid chromatography with fluorescence detection (LC–FD).The quantification limits ranged between 0.7 and 1.2 ng ml^?1. The proposed method was proved satisfactorily for the detection and determination of these compounds in groundwater samples during a study into the biodegradation of linear alkylbenzene sulfonates (LAS) in an agricultural soil sampled from the irrigated plain to the west of Granada (Spain).     

Homocyclic o-dicarboxaldehydes: Derivatization reagents for sensitive analysis of amino acids and related compounds by capillary and microchip electrophoresis

Amino acids are essential compounds for living organisms, and their determination in biological fluids is crucial for the clinical analysis and diagnosis of many diseases. However, the detection of most amino acids is hindered by the lack of a strong chromophore/fluorophore or electrochemically active group in their chemical structures. The highly sensitive determination of amino acids often requires derivatization. Capillary electrophoresis is a separation technique with excellent characteristics for the analysis of amino acids in biological fluids. Moreover, it offers the possibility of precapillary, on-capillary, or postcapillary derivatization. Each derivatization approach has specific demands in terms of the chemistry involved in the derivatization, which is discussed in this review. The family of homocyclic o-dicarboxaldehyde compounds, namely o-phthalaldehyde, naphthalene-2,3-dicarboxaldehyde, and anthracene-2,3-dicarboxaldehyde, are powerful derivatization reagents for the determination of amino acids and related compounds. In the presence of suitable nucleophiles they react with the primary amino group to form both fluorescent and electroactive derivatives. Moreover, the reaction rate enables all of the derivatization approaches mentioned above. This review focuses on articles that deal with using these reagents for the derivatization of amino acids and related compounds for ultraviolet-visible spectrometry, fluorescence, or electrochemical detection. Applications in capillary and microchip electrophoresis are summarized and discussed.     

Application of flow-injection potentiometric system for determination of total concentration of aliphatic carboxylic acids

In this work, flow-injection system with potentiometric detection was tested for determination of total carboxylic acid concentration. Detection part of the examined system consists of ion-selective electrodes (ISEs) with polymer membranes of different compositions. First electrode is based on Zr(IV)-tetraphenylporphyrin as ionophore selective towards carboxylic acid anions, the membrane of second one contains only liphophilic anion exchanger - tridodecylmethylammonium chloride. Final response of the system is a result of combination of EMF signals from both electrodes. Combination of two detectors enables significant decrease of differences between potentiometric signals induced by mixtures of studied anions of various concentrations as compared to results obtained only with metalloporphyrin-based ISE. The use of anion-exchanger based detector allows for elimination of the influence of aliphatic carboxylic acids lipophilicity.Proposed potentiometric flow-injection system was employed for determination of short-chain aliphatic carboxylic acids (so-called VFA - volatile fatty acids) in samples originating from an anaerobic digester. Results obtained for these relatively complicated samples are in good agreement with results obtained with the use of reference colorimetric method.Linear response towards carboxylic acids was observed in the concentration range of 10⁻⁴ to 10⁻² mol dm⁻³, with the slopes in the range of −110 to −150 mV dec⁻¹ (for acetate⁻ and butyrate⁻, respectively). System enables for determination of about 6 samples per hour. Life time of ISEs average about 2 months.     

Application of co-electroosmotic capillary electrophoresis for the determination of inorganic anions and carboxylic acids in soil and plant extract with direct UV detection

Summary Indirect UV detection in capillary zone electrophoresis (CZE) is frequently used for the determination of inorganic anions and carboxylic acids. However, there are few reports on direct UV detection of these solutes in real samples. This paper describes the use of direct UV detection of inorganic anions and organic acids in environmental samples using co-electroosmotic capillary zone electrophoresis (co-CZE) at 185 nm. The best separation and detection of the solutes was achieved using a fused silica capillary with an electrolyte containing 25 mM phosphate, 0.5 mM tetradecyltrimethylammonium bromide (TTAB) and 15% acetonitrile (v/v) at pH 6.0. Four common inorganic anions (Cl⁻, NO₂ ⁻, NO₃ ⁻ and SO₄ ²⁻) and 11 organic acids (oxalic, formic, fumaric, tartaric, malonic, malic, citric, succinic, maleic, acetic, and lactic acid), were determined simultaneously in 15 min. Linear calibration plots for the test solutes were obtained in the range 0.02–0.5 mM with detection limits ranging from 1–9 μM depending on the analyte. The proposed method was successfully used to determine inorganic anions and carboxylic acids in soil and plant tissue extracts with direct injection of the sample.     

Optically active polyamidoimides based on amino acids containing cyclohexane fragment

New amino carboxylic acids containing a cyclohexane fragment and an L-leucine or L-valine fragment were synthesized. The reactions of the synthesized amino acids with 4,4′-oxydiphthalic anhydride gave in quantitative yields the dicarboxylic acids, which were converted to the corresponding dichlorides. Low-temperature polycondensation of the synthesized dichlorides with 4,4′-diaminodiphenyl ether yielded optically active polyamidoimides, which are soluble in various organic solvents and exhibit enhanced heat resistance. The structures of the synthesized monomeric compounds and polymers were confirmed by elemental analysis, IR and ¹H NMR spectroscopy, and high-resolution mass spectrometry. Specific rotation angles of the synthesized amino carboxylic acids and polyamidoimides were determined.     

An expedient route for the reduction of carboxylic acids to alcohols employing 1-propanephosphonic acid cyclic anhydride as acid activator

A simple and efficient method for the synthesis of alcohols from the corresponding carboxylic acids is described. Activation of carboxylic acid with 1-propanephosphonic acid cyclic anhydride (T3P) and subsequent reduction using NaBH₄ yield the alcohol in excellent yields with good purity. Reduction of several alkyl/aryl carboxylic acids and N^α-protected amino acids/peptide acids as well as N^β-protected amino acids was successfully carried out to obtain corresponding alcohols in good yields. All the products were fully characterized by ¹H NMR and mass spectral analyses. The procedure is mild, simple and the isolation of the products is easy.     

Determination of highly polar compounds in water samples around former ammunition plants

Summary A method was developed for the determination of nitrobenzoic acids and nitrophenols as well as of diaminoaromatics and was applied to the analysis of water samples from the former ammunition plant at Elsnig (Saxony, Germany). The procedure is based on a preseparation into a neutral, an acidic and a basic fraction by multi-step extraction at different pH values followed by HPLC analysis with UV and electrochemical detectors, coupled in series. Applying optimized enrichment conditions, all investigated compounds were extracted from spiked distilled water with recoveries >80% and variation coefficients <7%. Similar results were obtained with spiked ground water samples. After enrichment, all compounds can be analysed by HPLC with UV detection at concentrations below 100 ngL⁻¹. The electrochemical detector (ELCD) allowed a selective and sensitive detection of the nitrophenols and especially of the diaminoaromatics and, therefore, provides, some advantages in the analysis of real samples. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Analysis of the compounds in the synthesis mixtures of hydroxy acids by a gradient programmed RP-HPLC with UV/vis and RI detectors

Summary A gradient programmed reversed phase high-performance liquid chromatographic (RP-HPLC) method is described for the quantitative determination of some aldehydes, hydroxy aldehydes, unsaturated aldehydes, hydroxy carboxylic acids, carboxylic acids, alcohols and polyols using an ultraviolet and a refractive index detector in series. The sample matrices are synthesis mixtures of hydroxy carboxylic acids. The structures of the hydroxy aldehyde intermediates are determined by¹³C NMR and the influence of sample preparation on analysis results is discussed. The limits of detection and the precision of the method are evaluated.     

Electrophoretic separation of tryptophan enantiomers in biological samples.

A method for the determination of D- and L-tryptophan (Trp) in biological samples is described. The amino acid enantiomers were precolumn-derivatized with a fluorescence tagging reagent, naphthalene-2,3-dialdehyde (NDA). In the presence of hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD) as the chiral selector, NDA-tagged Trp enantiomers were well resolved by micellar electrokinetic chromatography (MEKC). Using laser induced fluorescence (LIF) detection, a detection limit of 3.3 x 10(-8) M Trp was obtained. The method was applied to the determination of Trp enantiomers in biological samples including human urine and cerebrospinal fluid (CSF), rat brain tissue, and Aplysia ganglia. No interference from other amino acids or the endogenous compounds in the sample matrices was observed. D-Trp was found at the sub-microM level in human urine samples collected from several healthy subjects. Further, the determination of DL-Trp residues in small quantities (10 microg) of peptides after acid hydrolysis is demonstrated.     

Differential-pulse polarography of amino acids in acetaldehyde-borax medium

A differential-pulse polarographic method for the determination of amino acids is reported, based on the formation of Schiff's base compounds in borax buffer solution (pH 10.10) containing acetaldehyde. The compounds are reduced at a mercury electrode with peak potentials of about ?1.5 V (vs. SCE) and well defined polarographic waves are obtained which can be used to determine amino acids in borax medium. The differential-pulse polarographic method was found to be the most sensitive and suitable for the determination of amino acids in the concentration range 1 × 10^?6–8 × 10^?4 M (lysine) and 2.8 × 10^?6–1 × 10^?3 M (arginine). The polarographic characteristics of these waves were studied by differential-pulse polarographic and cyclic voltammetric methods. The waves are ascribed to the reduction of the imido group in the Schiff's base compounds. The procedure was applied to the determination of lysine and arginine in foodstuffs and the total proteins in serum samples.     

Development of new methods for the analysis of carboxylic acids and carbonyl compounds in size classified raindrops by CE for application in modelling atmospheric processes

Summary At the present time the formation processes of clouds and precipitation are not totally understood. Because cloud- and raindroplets are major sinks for chemical species in the atmosphere it is important to understand the physical and the chemical processes which occur during precipitation. The development of models is hindered by the scarcity of information about the scavenging of gases or aerosol particles by raindrops of different sizes. These processes can only be investigated by field experiments using microanalytical methods and analysing single raindrops as well as size-classified raindrop samples. Raindrops were collected according to their size by freezing them in liquid nitrogen (“Guttalgor” method). Sample volumes of the smallest raindrop sizes (radius <200μm) were usually smaller than 2 μL. The analysis of microvolumina in the size range of μL down to pL required the development of methods designed especially for this purpose. Analysis of rain samples was carried out by capillary electrophoresis. Organic acids were determined using a new electrolyte system for indirect detection. With this system it was possible to determine monocarboxylic acids (C₁−C₄) dicarboxylic acids (C₂−C₄, C₉) and inorganic anions (Cl⁻, NO₃ ⁻, SO₄ ²⁻) in the rain samples. Carbonyl compounds were analysed after derivatisation with dansylhydrazine using direct UV-detection. The system allows the identification of aliphatic carbonyl compounds (C₁−C₃, C₅) as well as benzaldehyde. It was found that carbonyl compounds and carboxylic acids showed concentration maxima at different raindrop radii. These concentration maxima are a consequence of particle scavenging. By using the results of a former experiment we concluded that the two species are located on different aerosol particle sizes. Reasons for the different particle sizes where these species are located are discussed. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Amino alcohol based chiral solvating agents: synthesis and applications in the NMR enantiodiscrimination of carboxylic acids

Four optically active amino alcohols were synthesized via the ring opening of (R)-N-(2,3-epoxypropyl)phthalimide with (R)-2-phenyl glycinol, (1R,2S)-cis-1-amino-2-indanol, (R)-2-amino-1-butanol and (S)-phenyl ethylamine in 73-93% yields. The enantioselective recognition of these receptors towards the enantiomers of racemic carboxylic acids was studied by ¹H NMR spectroscopy. The molar ratio and the association constants of the chiral compounds with each of the enantiomers of the guests were determined by using Job plots and a non-linear least-squares fitting method, respectively. Large non-equivalent chemical shifts (up to 30.0 Hz) can be achieved in the presence of chiral amino alcohols 2 and 5. Amongst the chiral receptors used, compound 5 was found to be the best chiral shift reagent, and was effective in the determination of the enantiomeric excess of chiral carboxylic acids.     

Determination of D- and L-amino acids in biological samples by two-dimensional column liquid chromatography

Summary A two-dimensional, column liquid chromatographic system is used for the determination of the D- and L-enantiomers of amino acids in biological samples. Separation of the amino acids is first on ion-exchange column by gradient elution with a sodium citratesodium chloride buffer. Enantioseparation is by subsequent injection of 3 l heart-cuts of the individual amino acids onto a second column with a chiral crown ether stationary phase. Finally, fluorescence detection is after post-column labelling of the amino acids using ano-phthalaldehyde-2-mercaptoethanol reagent solution. The high separation power and selectivity of the system allow processing of complex samples with hardly any additional treatment and the determination of small quantities of D-amino acids in the presence of excess L-form. Applicability of the system is illustrated by the determination of D- and L-aspartate, serine, glutamate and alanine in various complex biological samples, such as protein hydrolysates, urine and biotechnological and food samples. Data are given on detectability, repeatability and linearity.     

Determination of C1−C4 fatty acids as p-bromophenacyl esters using glass-capillary gas chromatography and electron-capture detection

Summary A method for the determination of low relative molecular mass carboxylic acids (C₁–C₄) in water is reported. The acids are converted to p-bromophenacyl esters prior to a glass-capillary gas chromatographic separation. By utilizing electron-caputre detection the detectability is substantially improved compared to flame-ionization detection. A comparison of three different ways to treat the water samples and to produce the derivatives is made. It is shown that the , p-dibromoacetophenone reagent decomposes to a small extent which limits the utility of the reagent. Nevertheless a detection limit for formic acid of approximately 2.5 mgl^–1 is obtained. The method is applied to the determination of formic and acetic acids in a paper kraft water sample.     

Pressure Capillary Spot-Test for the Detection of Pollutants in Crops,Vegetation and Environment

Abstract

A new, simple, sensitive and selective technique, pressure capillary spot test, has been developed for the detection and determination of pollutants containing amino, carbonyl, carboxylic or phenolic groups. The pressure has been reduced with the help of a suction pump and a capillary containing p-diemthylaminobenzaldehyde has been used as detector for the semi-quantitative determination of plant growth regulators, indoleacetic acids, in wheat shoots.     

ESI‐MS/MS analysis of protonated N‐methyl amino acids and their immonium ions

Methylation is one of the important posttranslational modifications of biological systems. At the metabolite level, the methylation process is expected to convert bioactive compounds such as amino acids, fatty acids, lipids, sugars, and other organic acids into their methylated forms. A few of the methylated amino acids are identified and have been proved as potential biomarkers for several metabolic disorders by using mass spectrometry–based metabolomics workstation. As it is possible to encounter all the N‐methyl forms of the proteinogenic amino acids in plant/biological systems, it is essential to have analytical data of all N‐methyl amino acids for their detection and identification. In earlier studies, we have reported the ESI‐MS/MS data of all methylated proteinogenic amino acids, except that of mono‐N‐methyl amino acids. In this study, the N‐methyl amino acids of all the amino acids ( 1 ‐ 21 ; including one isomeric pair) were synthesized and characterized by ESI‐MS/MS, LC/MS/MS, and HRMS. These data could be useful for detection and identification of N‐methyl amino acids in biological systems for future metabolomics studies. The MS/MS spectra of [M + H]⁺ ions of most N‐methyl amino acids showed respective immonium ions by the loss of (H₂O, CO). The other most common product ions detected were [MH‐(NH₂CH₃]⁺, [MH‐(RH)]⁺ (where R = side chain group) ions, and the selective structure indicative product ions due to side chain and N‐methyl group. The isomeric/isobaric N‐methyl amino acids could easily be differentiated by their distinct MS/MS spectra. Further, the MS/MS of immonium ions inferred side chain structure and methyl group on α‐nitrogen of the N‐methyl amino acids.     

Some observations on the automation by flow injection analysis of the spectrophotometric determination of amino acids and proteins witho-phthalaldehyde

Automation by flow injection analysis with Spectrophotometric detection of the determination of total amino acids and proteins witho-phthalaldehyde is not straightforward. The use of spectrophotometry, instead of spectrofluorimetry, and of N-acetyl-L-cysteine, instead of the conventional mercaptoethanol is advantageous because of the lower variability of absorptivities with respect to fluorescence yields, and the larger stability of the derivatives. Under adequate working conditions and with leucine as reference, the procedure can be used for the evaluation of total amino acids. A similar procedure is proposed for the analysis of proteins in a sample. Limits of detection are 1 × 10^–5 M for amino acids, and 1 × 10^–6 M for proteins, respectively.