Activated alpha-alkyl-alpha-arylacetic acid enantiomers for stereoselective thin-layer chromatographic and high-performance liquid chromatographic determination of chiral amines

The separation of racemic benoxaprofen into the two benoxaprofen enantiomers by preparative high-performance liquid chromatography and the application of the activated enantiomers as derivatization reagents for the simultaneous stereoselective determination of chiral amines in biological material is described. Activated (+)- and (-)-benoxaprofen are both shown to be very sensitive and stable chiral fluorescence markers, applicable to thin-layer chromatography as well as to high-performance liquid chromatography.     

Room-temperature phosphorimetric determination of biogenic indole compounds adsorbed on a thin-layer chromatographic plate

A simple and sensitive method is described for the determination of biogenic indole compounds adsorbed on cellulose or alumina plates for thin-layer chromatography by room-temperature phosphorimetry. The optimum conditions were investigated for 5-hydroxyindole-3-acetic acid and indole-3-acetic acid. The compounds are spotted on the plates, which are then sprayed successively with sodium citrate or sodium acetate, and sodiu iodide solutions. The plates are dried completely under a stream of dry nitrogen, and immediately dipped in molten paraffin. The phosphorescence is stable for at least 3 h even in moist air. The limits of detection for nine biogenic indole compounds tested are between 2 and 300 pmol per sample spot.     

Improvement of extraction procedure for biogenic amines in foods and their high-performance liquid chromatographic determination.

A high-performance liquid chromatography method is described for the simultaneous determination of the biogenic amines tryptamine, 2-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine in cheese. The optimization of the procedure for the extraction of amines from the matrix is described. The separation of dansyl derivatives of the amines was achieved by reversed-phase liquid chromatography with gradient elution, followed by UV detection at 254 nm. The mobile phase was acetonitrile-0.01 M phosphate buffer (pH 7)-water. Under these conditions, rapid elution of the amines in less than 13 min was obtained. Validation of the method included calibration experiments, addition of standard amines for the determination of amine recoveries and repeatability tests.     

Disposable biosensors for determination of biogenic amines

This work reports monoamine oxidase (MAO)/horseradish peroxidase (HRP) and diamine oxidase (DAO)/horseradish peroxidase (HRP) based biosensors using screen-printed carbon electrodes for the determination of biogenic amines (BA). The enzymes have been covalently immobilized onto the carbon working electrode, previously modified by an aryl diazonium salt, using hydroxysuccinimide and carbodiimide. The detection has been performed by measuring the cathodic current due to the reduction of the mediator hydroxymethylferrocene at a low potential, 250 mV vs screen-printed Ag/AgCl reference electrode. The experimental conditions for the enzymes immobilization, as well as for the main variables that can influence the chronoamperometric current have been optimized by the experimental design methodology. Under these optimum conditions, the disposable biosensors have been characterized. A linear response range from 0.2 up to 1.6 μM and from 0.4 to 2.4 μM of histamine was obtained for DAO/HRP and MAO/HRP based biosensors, respectively. The biosensor construction was highly reproducible, yielding relative standard deviations of 10% and 11% in terms of sensitivity for DAO/HRP and MAO/HRP based biosensors, respectively. The capability of detection, 0.18 ± 0.01 μM in the case of DAO/HRP and 0.40 ± 0.04 μM (α = 0.05 and β = 0.005) for MAO/HRP based biosensors, and the biosensor sensitivity towards different BA has also been analyzed. Finally, the developed biosensors have been applied to the determination of the total amine content in fish samples.     

Hydrazone-based ligands for micro-solid phase extraction-high performance liquid chromatographic determination of biogenic amines in orange juice

Eight hydrazone-based ligands were synthesized, trapped in a silica sol-gel matrix, and were subsequently used in the micro-solid phase extraction (μ-SPE) of biogenic amines (BAs). The BAs investigated were tryptamine, phenylethylamine, putrescine, histamine, tyramine and spermidine. Prior to the extraction, dansyl chloride was added to the samples which were heated to 70°C for 10 min. The samples were extracted with μ-SPE, after which analytes were desorbed using acetonitrile via ultrasonication. The extracts were analysed by high performance liquid chromatography (HPLC) with ultraviolet detection. Of the eight ligands investigated as sorbents, benzophenone 2,4-dinitrophenylhydrazone was found to be the most promising. The enhanced π-π interaction between the analytes and the ligand facilitated the adsorption process. Under the most suitable extraction conditions, the method demonstrated good linearity with correlation coefficient of more than 0.985 over a concentration range of 1-50 μg L(-1). Satisfactory repeatability with relative standard deviations of 7.43-11.30% (n=3) were obtained. Detection limits ranged from 3.8 to 31.3 ng L(-1). The μ-SPE method exhibited lower recoveries (71.5-87.4%) when compared to the solid phase extraction technique (79.7-95.0%), but enrichment factors of 94-460 were obtained. The proposed μ-SPE-HPLC method was applied to the determination of BAs in orange juice purchased from local supermarkets, with satisfactory results. The orange juices were characterized by the presence of relatively high levels of putrescine (range, 550-2210 μg L(-1)) but tryptamine and phenylethylamine were not detected in any of the tested samples.     

Optimization of chromatographic parameters for the determination of biogenic amines in wines by reversed-phase high-performance liquid chromatography

A method suitable for the determination of eight biogenic amines (histamine, tyramine, phenylethylamine, tryptamine, cadaverine, putrescine, spermidine and spermine) in wines has been developed. The method involves derivatization of the amines by treatment with dabsyl chloride, after which the derivates were analysed by reversed-phase liquid chromatography with gradient elution and spectrophotometric detection at 446 nm. Different variables affecting separation were optimized. Validation of the method included calibration experiments, the addition of standards amines for the determination of recovery and repeatability tests. Good linearity of the responses was obtained up to 500 microg l(-1), except for putrescine (up to 2100 microg l(-1)). The detection limits ranged between 10 and 60 microg l(-1) for standard solutions. The method was successfully applied to the analysis of five Spanish wines.     

Liquid chromatographic determination of biogenic amines in fermented foods after derivatization with 3,5-dinitrobenzoyl chloride

The reagent 3,5-dinitrobenzoyl chloride (DNBZ-Cl) was tested for pre-column derivatization of biogenic amines (BAs). Samples were derivatized within 3 min in 1 M NaOH at ambient temperature by adding 2-propanole and 50 mM DNBZ-Cl in acetonitrile. The reaction was terminated by addition of 2 M HCl. For high-performance liquid chromatography an encapsulated stationary reversed-phase and gradient elution using a ternary gradient system were used. The DNBZ derivatives were quantified by their UV-absorption at 260 nm. The structures of the derivatives were elucidated using coupling of HPLC with electrospray ionization mass spectrometry. Detection limits of BAs were approximately 124-864 microg l(-1) (injected amounts 203-1410 pg) at a signal-to-noise ratio of 3:1. The coefficients of determination were 0.989-0.996, with the exceptions of cadaverine (0.976) and serotonin (0.965). The method was applied to the quantitative determination of agmatine, cadaverine, histamine, octopamine, 2-phenylethylamine, putrescine, serotonin, spermidine, spermine, tryptamine and tyramine, in fermented cabbage juices, soy sauces, Misos (soy pastes), fermented fish sauces, and anchovy paste.     

New procedure of selected biogenic amines determination in wine samples by HPLC

A new procedure for determination of biogenic amines (BA): histamine, phenethylamine, tyramine and tryptamine, based on the derivatization reaction with 2-chloro-1,3-dinitro-5-(trifluoromethyl)-benzene (CNBF), is proposed. The amines derivatives with CNBF were isolated and characterized by X-ray crystallography and ¹H, ¹³C, ¹⁹F NMR spectroscopy in solution. The novelty of the procedure is based on the pure and well-characterized products of the amines derivatization reaction. The method was applied for the simultaneous analysis of the above mentioned biogenic amines in wine samples by the reversed phase-high performance liquid chromatography. The procedure revealed correlation coefficients (R²) between 0.9997 and 0.9999, and linear range: 0.10–9.00 mg L⁻¹ (histamine); 0.10–9.36 mg L^-1 (tyramine); 0.09–8.64 mg L⁻¹ (tryptamine) and 0.10–8.64 mg L⁻¹ (phenethylamine), whereas accuracy was 97%–102% (recovery test). Detection limit of biogenic amines in wine samples was 0.02–0.03 mg L⁻¹, whereas quantification limit ranged 0.05–0.10 mg L⁻¹. The variation coefficients for the analyzed amines ranged between 0.49% and 3.92%. Obtained BA derivatives enhanced separation the analytes on chromatograms due to the inhibition of hydrolysis reaction and the reduction of by-products formation.     

Separation and determination of biogenic amines in fish using MEKC with novel multiphoton excitation fluorescence detection

Biogenic amines are a group of biological molecules derived from the enzymatic decarboxylation of natural amino acids. They can be found in a variety of foods and some of them are involved in essential cellular pathways regulating cellular functions. To address the issues raised by conventional detection methods for biogenic amines, such as laborious sample preparation and limited sensitivity, a new micellar electrokinetic chromatography scheme was developed based on multiphoton excitation fluorescence (MPEF) detection. Six FITC-labeled biogenic amine species were used for the evaluation of this MEKC-MPEF method in comparison to single photon excitation fluorescence detection. The results indicated that MEKC-MPEF had superior resolution with a detection volume as low as aL. Quantitative analysis of varying concentrations of biogenic amine species has also been achieved suggesting a ymole mass detection limit, a linear dynamic range of about two orders of magnitude, and 95-105% recoveries. Furthermore, the biogenic amine profile of decayed oriental crucian carps was successfully determined and quantified using this new method.     

Electrophoretic determination of biogenic amines in biological fluids

The capabilities of the electrophoretic separation of biogenic amines (adrenalin, noradrenalin, dopamine, serotonin, metanephrine, and normetanephrine) under conditions of capillary zone electrophoresis and micellar electrokinetic chromatography with the introduction of complex-forming agents (18-crown-6, 4,13-diaza-18-crown-6, and sodium dodecyl sulfate as an ion-pair reagent) and acetonitrile as the constituents of a working electrolyte were demonstrated. A technique for the sampling of biological fluids (urine, blood plasma, and serum) with the use of solid-phase extraction on aluminum oxide and a C18 reversed-phase sorbent was developed. The capabilities of various versions of the preconcentration of biogenic amines were determined, which allowed us to decrease the limits of detection by a factor of hundreds.     

Enzymatic determination of biogenic amines with transglutaminase

Tyramine, histamine, putrescine and cadaverine, the most common biogenic amines indicating the food quality, were studied in the transglutaminase-catalyzed reaction. Transglutaminase (protein-glutamine gamma-glutamyltransferase EC 2.3.2.13) catalyzes an acyl transfer reaction between a donor substrate and an acceptor substrate (e.g. biogenic amine) and forms a cross-linkage between substrates with a release of ammonia. The reaction can be monitored by measuring the ammonia produced in the reaction. The concentration of produced ammonia was found to be proportional to the concentration of biogenic amine and could hence be used to determination of biogenic amines in food matrixes.     

Stability-indicating thin-layer chromatographic method for quantitative determination of ribavirin

A simple and accurate stability-indicating thin-layer chromatographic (TLC) method is developed and validated for the quantitative determination of ribavirin (RBV) in its bulk and with used for development consists of chloroform-methanol-acetic acid (60:15:15, v/v/v). The separated spots are visualized as bluish green spots after being sprayed with anisaldehyde reagent. RBV is subjected to different accelerated stress conditions. The drug is found to undergo degradation under all stress conditions, and the degradation products are well resolved from the pure drug with significantly different Rf values. The optical densities of the separated spots are found to be linear with the amount of RBV in the range of 5-40 microg/spot with a good correlation coefficient (r=0.9980). The limit of detection and limit of quantitation values are 1.40 and 4.67 microg/spot, respectively. Statistical analysis proves that the method is repeatable and accurate for the determination of RBV in the presence of its degradation products. The method meets the International Conference on Harmonisation/Food and Drug Administration regulatory requirements. The proposed TLC method is successfully applied for the determination of RBV, pure and in capsules, with good accuracy and precision; the label claim percentages are 98.8%+/-1.5%. The results obtained by the proposed TLC method are comparable with those obtained by the official method.     

Effect of matrix on recovery of biogenic amines in fish

Biogenic amines, such as putrescine, cadaverine, and histamine, in fish can be indicators of spoilage and/or safety. Methanol extraction used for AOAC Official Methods 996.07 and 977.13 has resulted in low amine recoveries in fish. Extraction methodology was evaluated to improve recovery from fish tissue. Samples were evaluated for recovery of known quantities of biogenic amines added to tissue prior to extraction. Addition of 25% 0.4 N HCI to the 75% methanol-water extraction solvent (according to AOAC Official Methods 996.07 and 977.13) resulted in higher recoveries (P < 0.05) of the biogenic amines. Putrescine recovery increased from 44 +/- 2 to 100 +/- 14% in flounder and scup, and from 53 +/- 21 to 119 +/- 27% in mackerel and butterfish; cadaverine recovery increased from 47 +/- 4 to 106 +/- 15% (flounder/scup) and 58 +/- 24 to 113 +/- 9% (mackerel/butterfish); histamine recovery increased from 54 +/- 13 to 89 +/- 28% (mackerel). MeOH extraction resulted in comparable recovery from canned tuna, indicating that protein denaturation from processing may eliminate possible matrix interference. Acidification of the solvent resulted in a more complete extraction of the added amines to fresh/frozen fish tissue. This finding could indicate that current extraction procedures may cause biogenic amines to be underestimated.     

Development apparatus for thin-layer gel chromatography in organic solvent systems

An apparatus is devised for development of thin-layer gel chromatograms, which is easy to handle and gives reproducible results even with volatile organic solvent systems. The print technique with a silicone-treated paper can facilitate detection of the samples chromatographed.