Determination of catecholamines as their N-hydroxy-succinimidyl-3-indolylacetate derivatives by pre-column derivatization HPLC separation and fluorescent detection

N-Hydroxysuccinimidyl-3-indolylacetate (SIIA) is a new fluorescent derivatizing reagent with an indole ring and ¶an N-hydroxysuccinimide ester functionality. It can react with catecholamines under mild conditions to form corresponding amides, which have strong fluorescence at λ_ex/λ_em = 301 nm/¶365 nm. This paper covers the RP-HPLC separation and fluorescent determination of derivatized catecholamines with SIIA. In a mobile phase of methanol-water (36/64, v/v) containing H₃cit-Na₂HPO₄ buffer (pH = 4.00, 10 mmol/L), the derivatives of norepinephrine (NE), epinephrine (E) and dopamine (DA) were eluted within 15 min on a C₁₈ column. The detection limits were 0.043, 0.13 and 0.18 pmol, respectively, when the ratio of signal to noise (S/N) was 3. The excessive reagent is rapidly hydrolyzed to 3-indolylacetic acid (IA) that can be easily separated from derivatives.     

A simple and mild derivatization method for hydroxyl compounds using condensation agent carbazole-9-N-(2-methyl)-acetyl-benzene-disulfonate and its application for the determination of volatile alcohols from plasma by HPLC with fluorescence detection

A simple and sensitive HPLC method for the determination of hydroxyl compounds including volatile alcohols from spiked plasma, using a fluorescent condensation agent, namely carbazole-9-N-(2-methyl)-acetyl-benzene-disulfonate (CMABS), has been developed. A mixture of alcohols and pyridine in dichloromethane was treated with CMABS to give a quantitative yield of esters. The maximum fluorescence emissions for the derivatized alcohols are at 365 nm (λ_ex 335 nm). Studies on derivatization conditions indicate that alcohols react very fast with CMABS in presence of pyridine in dichloromethane to give the corresponding fluorescent derivatives. The method, in conjunction with a multi-step gradient, offers a baseline resolution of the common alcohol derivatives on a reversed-phase C₁₈ column, which is more convenient and more efficient than previous methods which require the prior conversion of the carboxylic acids to the acyl chlorides. The application of this method to the analysis of volatile alcohols in plasma was also investigated. The LC separation shows good selectivity and reproducibility for alcohol derivatives. The relative standard deviations ?(n = 5) for 100 pmol of each alcohol are < 4%. The detection limits are at the fmol level for C₁–C₃ alcohols and pmol level for C₄–C₉ alcohols. Received: 9 November 1998 / Revised: 3 May 1999 / Accepted: 6 May 1999     

4:3-β-naphthapyrone-4-acetic acidN-hydroxysuccinimidyl ester as a fluorescent labeling reagent for amino acids and oligopeptides in high-performance liquid chromatography

Summary 4:3-β-Naphthapyrone-4-acetic acidN-hydroxysuccinimidyl ester (NPA-OSu) is a highly sensitive and moderately reactive derivatizing reagent with a naphthapyrone moiety as fluorophore and anN-hydroxysuccinimidyl active ester as reactive group toward amino compounds. It is readily prepared in two steps. The fluorescence properties of NPA-OSu and its hydrolysis product have been studied in detail, and the conditions for derivatization and separation of the NPA-OSu derivatives of some amino acids and oligopeptides have been investigated. Atλ _ex = 352 nm andλ _em = 422 nm the detection limits (signal-to-noise ratio=3) for amino acids and oligopeptides reached fmol levels, for injection of 20 μL; this sensitivity was comparable with that obtained by use of 7-(diethylamino)coumarin-3-carboxylic acid succinimidyl ester as derivatizing reagent in the analysis of amino acids by capillary electrophoresis with laser-induced-fluorescence detection.     

Application of derivative UV spectrophotometry to the simultaneous determination of nitrate and nitrite ions in bath solutions for alkaline black-oxidation of steel

Second order derivative spectrophotometry was applied to the determination of nitrite and nitrate ions in bath solutions for alkaline black-oxidation of steel. The measurements were directly taken after dilution of the samples at λ = 336.4 nm for NO₃ ^– and λ = 390 nm for NO₂ ^–. The method was checked on artificial mixtures and applied to real samples containing approximately 5.5% NaNO₂ and 6% NaNO₃. The results agree well with those obtained by the standard manganometric method. Received: 8 July 1996 / Revised: 24 September 1996 / Accepted: 28 September 1996     

Determination of biogenic amines by RPHPLC with fluorescent detection after derivatization with 2-(9-carbazole)ethyl chloroformate (CEOC)

Summary The use of 2-(9-carbazole)ethyl chloroformate (CEOC) for pre-column derivatization of biogenic amines (BA) has been tested for the first time. The reagent reacts completely with BA within 3 min at ambient temperature in acetonitrile solution to form stable derivatives that are readily analyzed by reversed-phase HPLC. Study of the derivatization conditions revealed derivatization yields to be excellent in borate buffer over the pH range 9.0–10.0. Maximum yields were obtained by use of a three- to fourfold molar excess of reagent. The reaction is extremely tolerant of common buffer salts, no decrease in reaction yield is discernible in well-buffered samples. The emission maximum for the CEOC-derivatives is 360 nm (λ _ex = 293 nm). All the derivatives fluoresced strongly and direct injection of the reaction mixture was possible, with no significant disturbance from the major fluorescent reagent degradation by-products, 2-(9-carbazole)ethanol (CEOC-OH) and bis-(2-(9-carbazole)ethyl) carbonate (CEOC)₂. Separation of the derivatized BA by high-performance liquid chromatography with gradient elution was tested on a Hypersil BDS C18 column. Excellent response linearity was observed over the concentration range from 0.25 to 94.6 μmol L⁻¹ for the labeled BA. Detection limits were 117–840 fmol at a signal-to-noise ratio of 3∶1. Analysis of BA in a shrimp sauce extract was conducted to demonstrate the applicability of the technique to real sample matrixes; results were satisfactory.     

Sensitive and selective determination of metabolically formed trans-dihydrodiols and phenols of benzo[a]pyrene in water and urine samples by HPLC with amperometric detection

A method has been developed to separate hydroxylated metabolites of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene, i. e. trans-4,5-, 7,8-, 9,10-dihydrodiol and 1-, 3-, 7-, and 9-phenol, by HPLC with amperometric detection employing an isocratic methanol/water eluent (70:30, v/v) containing 0.5 g/L sulfuric acid and 1 g/L lithium perchlorate. Compared with the usually applied fluorescence (λ_ex = 265 nm, λ_em = 460 nm) and ultraviolet (λ = 265 nm) detection, the amperometric technique is about 2–12 times more sensitive for the determination of all metabolites investigated. The method was applied to the determination of the seven metabolites of benzo[a]pyrene in different water samples and in urine after solid-phase extraction (SPE). The results obtained by HPLC with amperometric detection after SPE enrichment from an aqueous extract of a soil sample and from the urine of a rat intragastrically treated with benzo[a]pyrene agreed well with the values determined with fluorescence and/or UV detection. Received: 20 December 1996 / Revised: 10 March 1997 / Accepted: 30 April 1997     

Methoxyl-induced fluorescence quenching in N,N′-bridged 9H-dipyrrinones and an X-ray crystal structure

Strongly fluorescent dipyrrinones can be prepared by bridging the pyrrole and lactam nitrogens with a carbonyl group, from reaction with N,N′-carbonyldiimidazole in the presence of a strong, non-nucleophilic base. The yellow, N,N′-carbonyl-bridged dipyrrinones typically have fluorescent quantum yields (φ_F) approaching 1.0. Thus, in chloroform, N,N′-bridged 9H-dipyrrinones with β-alkyl substituents: 2,3-diethyl-7,8-dimethyl has φ_F = 0.90 (λ_em = 465 nm) and 2,3-dimethyl-7,8-dimethoxy has φ_F = 0.84 (λ_em = 482 nm). In contrast, 2,3-dimethoxy-7,8-dimethyl and 2,3,7,8-tetramethoxy show red-shifted λ_em but with strongly reduced φ_F: φ_F = 0.10 (λ_em = 511 nm) and 0.08 (λ_em = 511 nm), respectively. Methoxy substituents on the lactam, but not the pyrrole ring act to quench the fluorescence and shift the emission and excitation wavelengths bathochromically. The first X-ray crystal structure of an N,N′-carbonyl-bridged dipyrrinone was obtained from 7,8-dimethoxy-2,3-dimethyl-10H-dipyrrin-1-one. Correspondence: David A. Lightner, Department of Chemistry, University of Nevada, Reno, Nevada 89557-0020, USA.     

Photostimulated changes in WO3 nanosized films

During the irradiation of WO₃ films d = 7–160 nm thick by light at λ = 320 nm (I = (1.5–7) × 10¹⁵ quantum cm⁻² s⁻¹), absorption band at λ = 850 nm appeared along with absorption band edge shift to shorter waves. The subsequent irradiation of samples at λ = 850 nm caused the disappearance of the longwave absorption band. The intrinsic absorption edge of WO₃ films was determined (λ = 320 nm). The degree of transformations of WO₃ films increased under atmospheric conditions as the intensity of incident light and the time of irradiation (1–140 min) grew and as film thickness decreased. A mechanism of photochemical transformations of WO₃ films was suggested. This mechanism included the generation of electron-hole pairs, the recombination of part of nonequilibrium charge carriers, the formation of [eV_a²⁺e] centers, and the isolation of photolysis products.     

Rapid and sensitive spectrofluorimetric determination of trace amounts of Hg(II) with o-vanillin-8-aminoquinoline

The fluorimetric determination of mercury ions with o-vanillin-8-aminoquinoline (OVAQ) in aqueous solutions was investigated. Hg(II) could react with the fluorescent reagent OVAQ (λ_ex/em = 278/314 nm) to form a nonfluorescent complex in an ethanol-water medium of pH 6.00. The linear range of the proposed method was from 2.5 to 80 μg/L, and the detection limit was 0.80 μg/L. The interferences of 24 foreign ions were also studied. The method was successfully applied to the determination of Hg(II) in sludge. The text was submitted by the authors in English.     

Sensitive determination of nucleotides and polynucleotides based on the fluorescence quenching of the Tb3+-Tiron complex

 A sensitive method using fluorescence quenching for the determination of nucleotides (ATP, ADP, AMP, CTP, UTP) and polynucleotides[poly(A), poly(I), poly(U)] is proposed. It is based on the ability of nucleotides and polynucleotides to inhibit the formation of a strongly fluorescent complex of Tb³⁺ ion with Tiron. The possibilities of spectrofluorimetric measurements of these systems were studied under optimal conditions (pH 6.9 in hexamethylene tetramine-HCl buffer, 1.2×10^-6 mol/L of Tb³⁺, 4.0×10^-6 mol/L of Tiron, λ_ex=317 nm, λ_em=546 nm). The results showed that the Tb³⁺-Tiron complex could be used as a fluorescence test for the phosphate moieties of nucleotides and polynucleotides. The detection limits are 0.3, 1.2, 3.7, 0.2, 0.3, 1.1, 0.6 and 0.9 ng/mL for ATP, ADP, AMP, CTP, UTP, poly(A), poly(I), and poly(U), respectively. The relative standard deviations (6 replicates) are within 4.0% in the middle of the linear range. The fluorescence quenching mechanism of these systems is also discussed. Received: 16 July 1996 / Revised: 13 November 1996 / Accepted: 13 November 1996     

Direct potentiometric titration of chloride and fluoride ions in reversed micelles of an ethoxylated surfactant

The possibility of the potentiometric titration of Cl^– and F^– ions directly in reversed micelles of the ethoxylated surfactant (Neonol APh₉-4) in n-decane is shown. The potential change of the indicator electrode (silver chloride and lanthanum fluoride) only depends on the ion concentration in the aqueous pseudophase of the reversed micelles and is independent of the aqueous phase concentration in n-decane in the region of concentrations from 4 to 0.2 vol %. The determination of the micelle size of Neonol APh₉-4 in the precipitation titration of Cl^– and F^– ions by photon correlation spectroscopy showed the formation of nanoparticles of AgCl and LaF₃ of dimensions limited by original micelle size (r_hd = 6 to 11 nm). The growth of AgCl and LaF₃ nanoparticles was studied at a shortage and excess of the titrant and in the point of equivalence. Received: 23 April 1999 / Revised: 15 July 1999 / Accepted: 19 July 1999     

Reagent peak-free liquid chromatography–fluorescence analysis of carboxylic acids using a fluorous scavenging–derivatization method

We developed a fluorous scavenging–derivatization method for reagent peak-free liquid chromatography (LC)–fluorescence analysis of carboxylic acids. In this method, carboxylic acids were fluorescently derivatized with 1-pyrenemethylamine in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-hydroxy-1H-benzotriazole. Residual excess unreacted reagent was tagged with 2-(perfluorooctyl)ethyl isocyanate and could be selectively removed by microfluorous solid-phase extraction before LC analysis. With use of this method, eight fluorescent derivatives of linear aliphatic carboxylic acids (C₁–C₈) can be separated within 30 min by reversed-phase LC with gradient elution. In the chromatogram obtained, the fluorous-tagged unreacted reagent peak is greatly decreased after microfluorous solid-phase extraction and does not interfere with the quantification of each acid. With use of microfluorous solid-phase extraction with 80% (v/v) aqueous methanol elution, over 99.9% of the unreacted fluorescent reagent was removed. The detection limits (signal-to-noise ratio of 3) for the carboxylic acids examined are 2.3–8.0 fmol per 10-μL injection. We also applied this method successfully to the analysis of highly polar carboxylic acids such as α-keto acids and tricarboxylic acid cycle metabolites.     

Kinetic Spectrofluorimetric Determination of Nitrite in Water Samples and Nitrogen Dioxide in the Atmosphere Sampled by the Liquid Droplet Method

 A simple, sensitive and selective kinetic spectrofluorimetric determination of NO₂ sampled by the droplet method in the atmosphere was proposed on the basis of the reaction of safranin O with nitrite. By this reaction, a diazonium salt is formed, which causes a fluorescent reddish-orange dye color of the solution to change into a non fluorescent blue color. The reaction was monitored fluorimetrically by measuring the decrease in fluorescence intensity of safranin O at (λ_ex/λ_em = 536 nm/579 nm) by a fixed-time method. The experimental conditions were optimized. Under the optimum conditions in the concentration range of 7.5–400 ng/ml, a linear calibration curve (r² = 0.9978) was obtained with a detection limit of 7.5 ng/ml. The method was applied successful ly to the determination of nitrite in spiked water and NO₂ in the atmosphere, as sampled by a liquid droplet method. Received August 23, 1999. Revision February 29, 2000.     

Extraction equilibria and spectrophotometric determination of vanadium(V) with 4-nitrocatechol and the ion-pair reagent thiazolyl blue tetrazolium

The formation and liquid-liquid extraction of a yellow ternary complex of vanadium(V) with 4-nitrocatechol (NC) and the ion-pair reagent Thiazolyl Blue Tetrazolium ⨑ub;3-(4,5-dimethyl-2-thiazol)-2,5diphenyltetrazolium bromide, MTT⫂ub; with 1: 2: 3 stoichiometry (V: NC: MTT) was studied. The optimum extraction conditions (pH, concentration of the reagents, extraction time), spectrophotometric parameters of the extract and key constants (extraction constant, association constant, distribution constant) were determined. Beer’s law was obeyed for concentrations ranging from 0.12 to 1.2 μg/mL of vanadium(V) with a molar absorptivity of ɛ = 3.13 × 10⁴ L/mol cm at λ_max = 400 nm. The effect of diverse ions was studied and extraction-spectrophotometric procedures for determination of vanadium in catalysts and steels were proposed.     

Spectrofluorometric determination of hesperidin by manual and flow-injection methods

A reliable and highly sensitive method for the determination of hesperidin is described. It involves the formation of a highly fluorescent complex between hesperidin and aluminium (III) in a micellar medium. There is a linear relationship between fluorescence intensity (λ_em = 496 nm, λ_ex = 391 nm) and hesperidin concentration over the range 5 × 10^–7– 2 × 10^–5 mol L^–1. The detection limit is 79 μg L^–1. The method can easily be adapted to a flow system using a three-channel manifold, the peak height being proportional to the hesperidin concentration over the range 1 × 10^–6– 1 × 10^–4 mol L^–1. Manual and flow-injection procedures have been successfully applied to the determination of hesperidin in orange peel and orange juice. Received: 21 October 1998 / Revised: 16 December 1998 / Accepted: 25 December 1998     

Determination of energizers in energy drinks

The method of UV/VIS derivative spectrophotometry for the determination of caffeine and B vitamins in energy drinks after solid phase extraction has been developed. Caffeine has been determined in the mixture with B₂ vitamin with zero-crossing technique from the I derivative spectra (λ = 266.8 nm), and B₃ in mixture with B₆ vitamin from the II derivative spectra (λ = 280.1 nm). B₁₂ vitamin has also been determined in a three-component mixture with vitamins B₃ and B₆. Taurine in drinks has been determined from the basic spectra after derivatization with ninhydrin (λ = 570 nm).     

Determination of Amoxycillin and Clavulanic Acid in Some Pharmaceutical Preparations by Derivative Spectrophotometry

 Derivative spectrophotometry was applied for the simultaneous determination of amoxycillin and clavulanic acid in pharmaceutical preparations: “Augmentin” inj. and tablets and “Amoksiklav” drops and tablets, in solutions after hydrolysis with sodium hydroxide. As the absorption spectra overlap strongly (amoxycillin λ_max = 247 nm and 290 nm, clavulanic acid λ_max = 258 nm) the first and the second derivative spectrophotometric procedure was elaborated for their determination. Amoxycillin was determined at λ = 257.9 nm (1-st derivative spectra) or λ = 273 nm (2-nd derivative) while clavulanic acid at λ = 280.3 nm (1-st derivative) or λ = 285 nm (2-nd derivative spectra). The Beer’s law is obeyed in the range of 0.004–0.04 mg/ml for amoxycillin and 0.002–0.02 mg/ml for clavulanic acid. Received December 6, 1999. Revision August 1, 2000.     

Neutral thallium clusters in aqueous solutions. Pulse radiolysis study

The formation of neutral Tl₂ (λ_max = 390 nm) and Tl₄ (λ_max = 360 nm) clusters in dilute aqueous solutions of Tl₂SO₄ containing formate ions was found by pulse radiolysis. The rate constants for the recombination of Tl⁰ atoms and Tl₂ clusters are equal to 1.5·10¹⁰ L mol⁻¹ s⁻¹ and 1.0·10¹⁰Lmol⁻¹ s⁻¹ (±30%), respectively, and the extinction coefficient of Tl₂ at 390 nm is −6.0·10³ L mol⁻¹ cm⁻¹ Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2367–2369, December, 1999.     

A fluorimetric liquid chromatography for highly sensitive analysis of very long chain fatty acids as naphthoxyethyl derivatives

Summary A simple and sensitive liquid chromatographic method is described for the simultaneous determination of biologically important very long chain fatty acids (docosanoic, tetracosanoic and hexacosanoic acids) as fluorogenic derivatives. The method is based on the derivatization of the fatty acids with 2-(2-naphtoxy)ethyl 2-(piperidino)ethanesulfonate (NOEPES) in toluene in the presence of potassium carbonate and 18-crown-6. Several parameters affecting the derivatization were studied, including reaction temperature, reaction time, reaction solvent, base catalyst and the amount of the reagent. The resulting derivatives were analyzed by HPLC with fluorimetric detection (λex=235 nm; λem=366 nm). The linear range for the determination of docosanoic, tetracosanoic and hexacosanoic acids was 0.028–1.4 μM with a detection limit of about 5.6 nM (S/N=3) (56 fmol per 10 μL injection). Application of the method to the analysis the non-esterified (free) very long chain fatty acids spiked in plasma proved feasible.