Determination of biogenic amines in wines and beers by high performance liquid chromatography with pre-column dansylation and ultraviolet detection

Summary A sensitive high performance liquid chromatographic method for the simultaneous determination of eleven biogenic amines, using 1,7-diaminoheptane as internal standard, has been developed. The method involves pre-column derivatization of the amines with dansyl chloride and subsequent solid phase extraction of the derivatives through C18 cartridges. The derivatization and solid phase extraction procedures were optimized. The separation of dansylamides was achieved on an Inertsil ODS-3 column (250×4 mm I.D. 5 μm) using a 35-min gradient elution method with a binary system of acetonitrile-water, a flow rate of 1 mL.min¹ with UV detection at 254 nm. Linearity of derivatization was obtained for concentrations ranging from 0.025 to 3.0 mg.L¹. The within- and between-day relative standard deviations ranged from 0.4 to 5.7% and 0.6 to 7.3% respectively. The overall process was successfully applied to identify and quantify biogenic amines in white, red and Retsina Greek wines and Greek beers, after their treatment with polyvinylpyrrolidone.     

Separation of food-related biogenic amines by ion-interaction reversed-phase high performance liquid chromatography. Tyramine,histamine, 2-phenylethylamine,tryptamine and precursor aminoacids. Application to red wine

Summary Methods for the separation of food-related biogenic amines (histamine, tyramine, 2-phenylethylamine and tryptamine) have been developed based on ion-interaction reversed-phase liquid chromatography.Two different interaction reagents have been comparatively used, namely octylamine ortho-phosphate (at wave-lengths of 230, 254 and 280 nm) and octylamine salicylate (at a wavelength of 254 nm). The different elution sequence orders shown by the investigated amines for the two reagents are discussed and compared.The detection limits obtained were 20 ppb for tryptamine ( =280 nm), 500 ppb for 2-phenylethylamine (=254 nm), 400 ppb for tyramine (=230 or 280 nm) and 900 ppb for histamine (=230 nm).The method was applied to the analysis of a five years old Italian red wine, in which 2-phenylethylamine (at a concentration of 72±3 ppm) and tryptamine (at a concentration of 4.0±0.3 ppm) were found to be present.     

Study of biologically active amines in grapes and wines by HPLC

Summary The biologically active amines agmatine, cadaverine, histamine, phenethylamine, putrescine, spermidine, and tyramine have been determined in different varieties of grape, aszu grape, wine and aszu wine from the Tokaj region of Hungary. Ion pairs formed between the amines and octanesulphonic acid were separated by liquid chromatography on a μBondapak C₁₈ reversed-phase column, and spectrofluorimetric detection was performed after post-column derivatization witho-phthalaldehyde and 2-mercaptoethanol. The method was linear for the amines between 0.1 and 10 mg L⁻¹, and for spermidine between 1 and 30 mg L⁻¹. Comparison of the results revealed that the qualitative and quantitative content of biologically active amines was mostly determined by the vintage of the wine and the technology used for wine-making. The biogenic amine content of Tokaj wines is well below suggested limits for any of the amines, showing that the wine-making technology of the Tokaj region is of high quality. The levels of biologically active amines (identified and quantified by HPLC) in grapes, wines and aszu wines can provide useful information about the weather, growth ofBotrytis cinerea in Tokaj, and aspects of the methods used for wine-making. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Determination of biogenic amines as dansyl derivatives in intestinal digesta and feces by reversed phase HPLC

Summary A new method was developed for the determination of fifteen biogenic amines in the intestinal digesta and feces of animal or human origin. The method involves the addition of an internal standard (heptylamine), extraction of the amines, and precipitation of the proteins with perchloric acid. The amines are derivatised with dansyl chloride, separated on a C18 column using gradient elution with 0.2M ammonium acetate at pH 5, water and acetonitrile, and detected with a fluorescence detector. The separation was achieved in a 40 min run. Recoveries ranged from 67 to 110%, the relative standard deviation for intra-assay precision being <5% and the limit of determination 1–5 mg kg⁻¹. The method is specific for biogenic amines in intestinal and fecal samples.     

Determination of biogenic amines in wine after clean-up by solid-phase extraction

Summary A suitable method for the determination of 16 biogenic amines in wine has been developed. The method involves clean-up of wine samples using ion-exchange cartridges and a preconcentration step, under controlled vacuum, before derivatization of the amines by treatment with phthalaldehyde (PA) and reversedphase HPLC with gradient elution and fluorimetric detection. Linearity of response was obtained for all the biogenic amines from 100 g L^–1 to mg L^–1. Limits of detection for the amines were similar for all PA-derivatives (25–50 g L^–1) and the quantitation limits were about 0.1 mg L^–1. After clean-up and preconcentration, the concentration levels increased 10-fold for all amines except putrescine and cadaverine, which gave poor recovery by this method unlike the rest which gave recoveries of almost 90%. The overall process was successfully applied to identify and quantify biogenic amines in several red wines from the Tarragona region.     

A weak cation-exchange phase for the separation of biogenic amines by open tubular liquid chromatography

Summary The preparation and performance of a weak cation-exchange stationary phase for Open Tubular Liquid Chromatography (OT-LC) was investigated. The stationary phase was prepared in 5.4 μm I.D. fused silica capillaries byin situ photopolymerization of a mixture of silicon acrylate and acrylic acid. The influence of pH, counter ion concentration and organic modifier concentration of the mobile phase on the retention was studied with catecholamines as test solutes using LIF detection. Other biological amines like amino acids, small peptides and nucleic acid derivatives could be separated on this stationary phase as well. The kinetic performance of the stationary phase was studied with several cations and neutral solutes.     

Direct separation and detection of biogenic amines by ion-pair liquid chromatography with chemiluminescent nitrogen detector

Analysis of biogenic amines is critical to pharmaceutical and food industry due to their biological importance. For many years, the determination of biogenic amines has relied on high performance liquid chromatography (HPLC) coupling with pre-, on-, or post-column derivatization procedures to enable UV or fluorescent detections. In this study, 14 biogenic amines were separated on a Phenomenex Luna Phenyl-Hexyl column by an ion-pair liquid chromatography method using perfluorocarboxylic acids as ion-pair reagents and detected by a chemiluminescent nitrogen detector (CLND). This direct separation and detection HPLC method eliminated the time consuming and cumbersome derivatization procedures. Compared with HPLC-UV (post-column derivatization with ninhydrin) and HPLC-charged aerosol detector (CAD) methods, this HPLC-CLND technique provided narrower peaks, better baselines, and improved separations and detections. Excellent linearity was acquired by CLND for each of the 14 biogenic amines ranging from less than 1 ng to about 1000 ng (on-column weights). The relative response factors determined by this LC-CLND method were proportional to the numbers of nitrogen atoms in each compound, which has been the characteristic of the equimolar determinations by CLND. In addition, a number of samples including beer, dairy beverage, herb tea, and vinegar were analyzed by the LC-CLND method with satisfactory precision and accuracy.     

Determination of biogenic amines in wines by pre-column derivatization and high-performance liquid chromatography coupled to mass spectrometry

A new HPLC method for determining biogenic amines in wines is developed. This method is based on pre-column amine derivatization, further separation of derivatives and on-line hyphenation of HPLC to atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Biogenic amines have been derivatized with 1,2-naphthoquinone-4-sulfonate at 65 °C and pH 9.2 for 5 min. The separation of derivatives has been accomplished in a C₁₈ analytical column using an elution gradient based on increasing the percentage of methanol. Derivatives have been ionized in positive mode and detected by selected ion monitoring. The operating conditions of the APCI-MS system (voltages, temperatures and gases) have been thoroughly optimized to obtain the maximum sensitivity for all analytes. In the selected conditions, APCI-MS spectra display little fragmentation and good signal-to-noise ratio. Depending on the amine characteristics, the main spectral peaks are due to mono- and di-derivative products. Figures of merit of the method have been established under the selected conditions using red wine samples. Recoveries ranging from 94% to 106% have been obtained which prove excellent accuracy of the method in the determination of histamine, putrescine, cadaverine, tryptamine, phenylethylamine, tyramine and serotonin in red wines. The proposed method has been applied to the analysis of commercial wines from different Spanish regions.     

High-speed assay of amino acids using reversed-phase liquid chromatography

A procedure is described by which 20 commonly occurring amino acids may be separated by reversed-phase liquid chromatography at room temperature in a total time, including derivatisation with o-phthalaldehyde/2-mercaptoethanol reagent, of approximately 15 minutes. Further, an increase in column temperature enables increased resolution of certain amino acids to be obtained with small but acceptable losses in fluorescence intensity. The only major drawback to the method is that the derivatisation reagent does not react with proline or hydroxyproline.     

Optimization of experimental variables in the dabsyl chloride derivatization of biogenic amines for their determination by RP-HPLC

Summary In the last few years special attention has been paid to the pre-column derivatization of biogenic amines with dabsyl chloride because proper experimental conditions for this reaction are very important. In this study, an experimental design (Doehlert design) was used to optimize the variables involved in the dabsylation of the following amines: histamine, tyramine, phenylethylamine, tryptamine, cadaverine, putrescine, spermidine, and spermine. The optimum experimental conditions for forming the dabsyl derivatives are: reagent concentration, 1.75.10⁻³ M; pH, 8.2; temperature, 70°C; heating time (t _h ), 21 min. Under these conditions good chromatographic repeatability is obtained.     

Direct automatic determination of biogenic amines in wine by flow injection-capillary electrophoresis-mass spectrometry

A capillary electrophoresis-electrospray mass spectrometry (CE-ESI-MS) method for the separation and determination of nine biogenic amines is proposed. Operational variables, such as the voltage, temperature, sheath liquid composition, flow-rate, and MS parameters, were optimized. Samples are injected in the hydrodynamic mode into a 75 cm x 50 microm ID coated capillary and separated by using 25 mM citric acid at pH 2.0. Heptylamine is used as internal standard. The experimental setup includes a flow manifold coupled to the CE system for automatic insertion of samples into the CE vials. The proposed method allows amines to be determined with limits of detection from 0.018 to 0.09 microg x mL(-1) and relative standard deviation (RSD) values from 2.4% to 5.0% (except 6.8% for histamine). The method was successfully used to determine biogenic amines in red and white wines.     

Simultaneous determination of twelve biogenic amines in serum by high performance liquid chromatography

In this study, we established a method for simultaneously determining twelve biogenic amines in serum by using reversed phase high performance liquid chromatography (RP-HPLC). The biogenic amines were first extracted from human serum by perchloric acid solution and derivatized by dansyl chloride. An ODS column was selected as separation column at 40 °C. The mobile phase solutions were consisted of A, 0.1 mol/L ammonium acetate and B, acetonitrile. A gradient elution was carried out with a flow rate at 1.0 ml/ml. The results show that the detection limit for twelve biogenic amines ranged between 0.0621 and 0.628 μg/L. All the correlation coefficients were above 0.999. The linearity was over the range from 0.001 to 20 mg/L depending on individual biogenic amine. The intra-day and inter-day coefficients of variations were from 0.53% to 7.50%,and from 1.10% to 7.25% respectively. The average analytical recovery in serum was from 92.02% to 107.65%. Moreover, the serum concentrations of tryptamine, tyramine and histamine in healthy females were found lower than that in healthy males significantly. The method is sensitive, convenient, and reliable, and suitable for simultaneous analysis of multiple biogenic amines in the clinical diagnosis and drug discovery.     

Determination of biogenic amines in food by automated pre-column derivatization with 2-naphthyloxycarbonyl chloride (NOC-CI)

Summary The fluorogenic reagent 2-naphthyloxycarbonyl chloride (NOC-Cl) has been used for the automated precolumn derivatization of biogenic amines (BAs) at ambient followed by liquid-chromatographic separation of the derivatives formed. For optimized derivatization samples in 0.5 M borate buffer (pH 9.0) were derivatized with 5 mM NOC-Cl in acetonitrile (MeCN) for 3 minutes. Excess of reagent was scavenged by addition of 20 mM glycine in water. For HPLC a Superspher^? RP-18e column and gradient elution using 0.1 M sodium acetate buffer (pH 4.4) and MeCN were used. The NOC-derivatives were detected by fluorescence at an emission wavelength of 335 nm at an excitation wavelength of 274 nm. This method allows the detection of BAs (2-phenylethylamine, putrescine, histamine, cadaverine, tyramine, spermidine, spermine) found in food and beverages (fruit juices, wines, various vinegars, fermented cabbage juice, and salmon). Detection limit of BAs are approximately 49–113 μg kg⁻¹ with the exception of histamine (747 μg kg⁻¹) (injected amounts: 18–41 pg histamine 267 pg), at a signalto-noise ratio of 3:1. The limits of determination are approximately 82–189 μg kg⁻¹ (histamine 1245 μg kg⁻¹) at a signal-to-noise ratio of 5:1. The correlation coefficients of linearity are 0.9910–0.9976. Recoveries from different matrices range from 65 to 109%, depending on the sample investigated. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996.     

Determination of primary amino acids in wines by high performance liquid magneto-chromatography

Eight amino acids (ethanolamine, glycine, alanine, β-aminobutyric acid, leucine, methionine, histidine and asparagine) were identified and quantified in Spanish wines by high performance liquid magneto-chromatography (HPLMC) with UV-V spectrophotometry. For this method, the amino acids are first complexed with mono(1,10-phenanthroline)-Cu(II) to confer them paramagnetic properties, and then separated by application of a low magnetic field intensity (5.5 mT) to the stationary phase contained in the chromatographic column. Principal components analysis of the results obtained grouped together the wine samples according to their denomination of origin: “Ribera del Duero”, “Rueda” or “Rioja” (Spain). Through cluster analysis, a series of correlations was also observed among certain amino acids, and between these groupings and the type of wine. These clusters were found to reflect the role played by the amino acids as primary or secondary nutrients for the bacteria involved in alcoholic and malolactic fermentation.     

A new ultra-pressure liquid chromatography method for the determination of biogenic amines in cheese

A fast and reliable ultra-performance liquid chromatography (UPLC™) method for the determination of biogenic amines (ethanolamine, methylamine, agmatine, histamine, dimethylamine, ethylamine, octopamine, pyrrolidine, dopamine, isopropylamine, propylamine, tyramine, putrescine, butylamine, cadaverine, tryptamine, 2-phenylethylamine, 3-methylbutylamine, spermidine, spermine) in cheese was established. After pre-column derivatization with 6-aminoquinolyl-N-hydroxy-succinimidyl carbamate (AQC), 20 primary and secondary biogenic amines were separated on an Acquity™ UPLC™ column (BEH C₁₈, 1.7 μm; 2.1 mm × 50 mm) within 9 min. Limits of detection (mg/100 g cheese) ranged from 0.04 (ethanolamine) to 1.62 (spermine), and limits of quantification were between 0.16 (ethanolamine) and 6.09 (spermine). The UPLC™ method was applied to the analysis of 58 cheese samples as retailed in Austria. About 13.8% of samples had a histamine content above 10 mg/100 g, and 22.4% had a tyramine content above 10 mg/100 g. Moreover, 8.6% of samples had a putrescine or cadaverine content higher than 10 mg/100 g. The total concentration of biogenic amines in two cheese samples was about 194 mg/100 g. Thus, obligatory monitoring of biogenic amines should be considered to ensure quality of cheese in future.     

Application of column liquid chromatography (HPLC) to special problems in food chemistry a laboratory note

Summary Liquid chromatography has been used for the determination of amino acids, sugars and biogenic amines in food. Some special problems, for example, determination of patuline in apple juice, hyoscyamine and scopolamine in french beans preserves, were also solved by HPLC.     

Selectivity of dansyl amino acids in micellar liquid chromatography with an ion-exchange-induced stationary phase

Summary The retention behavior of dansyl amino acids in micellar liquid chromatography has been examined by using ionexchange-induced stationary phases. Several parameters affected the retention of the analytes, including the type and concentration of micellar agent and modifier ion and the concentration of acetonitrile in the mobile phase. The order of elution of dansyl amino acids obtained with the micellar mobile phase was very different from that observed in conventional reversed-phase liquid chromatography. Fluorescence intensities of some dansyl amino acids were enhanced by the micellar mobile phase.     

A systematic approach toward comparing electrospray ionization efficiencies of derivatized and non‐derivatized amino acids and biogenic amines

Ionization efficiency (IE) in mass spectrometry (MS) has been studied for many different compounds, and different IE scales have been constructed in order to quantitatively characterize IE. In the case of MS, derivatization has been used to increase the sensitivity of the method and to lower the limits of detection. However, the influence of derivatization on IE across different compounds and different derivatization reagents has not been thoroughly researched, so that practitioners do not have information on the IE‐enhancing abilities of different derivatization reagents. Moreover, measuring IE via direct infusion of compounds cannot be considered fully adequate. Since derivatized compounds are in complex mixtures, a chromatographic method is needed to separate these compounds to minimize potential matrix effects. In this work, an IE measurement system with a chromatographic column was developed for mainly amino acids and some biogenic amines. IE measurements with liquid chromatography electrospray ionization mass spectrometry (LC/ESI/MS) were carried out, and IE scales were constructed with a calibration curve for compounds with and without derivatization reagent diethyl ethoxymethylenemalonate. Additionally, eluent composition effects on ionization were investigated. Results showed that derivatization increases IE for most of the compounds (by average 0.9 and up to 2‐2.5 logIE units) and derivatized compounds have more similar logIE values than without derivatization. Mobile phase composition effects on ionization efficiencies were negligible. It was also noted that the use of chromatographic separation instead of flow injection mode slightly increases IE. In this work, for the first time, IE enhancement of derivatization reagents was quantified under real LC/ESI/MS conditions and obtained logIE values of derivatized compounds were linked with the existing scale.     

Separation of butyltin and phenyltin species by ion-exchange chromatography with complexing mobile phases

Summary Butyltin and phenyltin species have been separated by ion-exchange chromatography using silica-based and polymer-based columns. Mobile phases consisted of methanol-water mixtures containing polyfunctional carboxylic acids, which can act as complexing agents for organotin species. The best results were achieved with a system based on a methanol mobile phase containing malic and oxalic acids and a polymer-based column, which allowed the separation of tri- and diorganotin compounds and some resolution between monobutyltin and monophenyltin.