Monitoring of biologically active amines in cereals and cereal based food products by HPLC

Summary Biologically active amines (putreanine sulphate, N-acetyl putrescine, putrescine, cadaverine, histamine, agmatine, N-acetyl spermidine, spermidine, spermine) were separated and quantified in cereal flour and cereal products by a liquid chromatographic method. The method consists of the separation of ion pairs formed between biologically active amines and octanesulphonic acid on a reversed-phase column, postcolumn derivatization with o-phtalaldehyde-2-mercapthoethanol and spectrofluorometric detection. Results of the reliability study were satisfactory. The method was linear for each amine at 1–10 mg L⁻¹. Putrescine and spermidine were the only amines always detected in cereal flour and cereal products, ranging from 2.45 to 47.83 mg kg⁻¹ for putrescine and 3.27 to 37.14 mg kg⁻¹ for spermidine. The most important differences among types of samples were found in polyamine derivatives. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997.     

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.     

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.     

Characterisation of Tokaj wines based on free amino acids and biogenic amines using ion-exchange chromatography

Summary Tokaj wines (Szamorodni and Aszu wines) of Hungarian origin were investigated on the basis of free amino acids and biogenic amines. The separation and determination of these compounds was performed by an amino acid analyser equipped with an ion-exchange resin column. The total amount of free amino acids and biogenic amines was higher in Aszu wines than in Szamorodni wines. The main amino acids were proline and arginine, while the major biogenic amines were tyramine and putrescine. The free amino acid and biogenic amine content of Aszu wines depended on the vineyards the wines originated from. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

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.     

Validation of an ultra high pressure liquid chromatographic method for the determination of biologically active amines in food

Biologically active amines include the so called biogenic amines, such as histamine, tyramine and cadaverine, and polyamines such as spermidine and spermine. Ultra high pressure liquid chromatography (UHPLC) is a new generation of separation techniques that takes full advantage of chromatographic principles to increase speed flow which drastically reduce analysis time. The aim of the present work was to validate a rapid method of UHPLC to detect the presence of biogenic amines and polyamines in food. Different food matrixes (wine, fish, cheese, and dry fermented sausage) were used in order to test the versatility of the method. The UHPLC method described in this article has been demonstrated as a reliable procedure to determine 12 biogenic amines and polyamines in less than 7 min of chromatographic elution. The method provides a satisfactory linearity and chromatographic sensitivity with a detection limit lower than 0.2 mg/L and a determination limit falling below 0.3 mg/L for all amines. The precision, in terms of relative standard deviation, was lower than 5% and the accuracy, as mean recovery, was between 93% and 98%, depending on the food matrix.     

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.     

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.     

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.     

Improved HPLC determination of aliphatic amines in air by diffusion and derivatization techniques

Summary Traces of C₁–C₄ aliphatic amines, sampled from ambient air on H₃PO₃-coated annular denuders, are derivatized with m-toluoyl chloride (MTC) in alkaline acetonitrile. The resulting derivatives are determined by reversed phase HPLC with ultraviolet detection at 230 nm. The detection limits of the individual amines are in the 1 to 5 picomole range corresponding to gasphase concentrations lower than 0.1 g m^–3 in air samples collected at 5 Lmin^–1 for one hour.     

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.     

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.     

HPLC determination of volatile phenols in wines

Summary An alternative to the traditional solvent extraction method used to extract and rapidly quantify ethyl-and vinylphenol and ethyl-and vinylgaiacol from wine is presented. The method is based on retention of volatile phenols on adsorbants. Among the tested resins, the most efficient, AG 2-X8 (anion exchange resin), worked as well with a synthetic solution as with wines. The percolation of clarified wine adjusted to pH 9 on this resin permits, in particular, the elimination of organic acids. Phenols are not eluted after rinsing the column with 1N HCl, but are eluted with methanol after this treatment. Good recovery (91 %) and good repeatability are observed. The eluate is directly analysed by HPLC on an RP18 column after two-fold dilution in water. The four volatile phenols were completely separated and detected by UV at 280 nm with high sensitivity (20–40 ppb). No interference with other compounds were noted in the different wines analysed.     

Determination of the triglyceride composition of grapes by HPLC

Summary The triglyceride composition of the Tempranillo grape (Vitis vinifera) has been examined by a combination of HPLC and GLC. To identify the triglycerides, equations were applied relating log k′ with the molecular variables: equivalent carbon number, chain length and number of double bonds in each of the fatty acids in the glycerides. Ten triglycerides were found, the principal ones being trilinolein (35.75%), dilinoleyl-olein (21.03%) and dilinoleyl-palmitin (17.02%).     

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.     

An improved analytical method for determination of heterocyclic amines in chicken legs

Summary Several extraction, separation and detection methods for heterocyclic amines (HAs) in chicken legs were evaluated by liquid chromatography. Results showed that the most appropriate extraction method includes the removal of macrosubstances by centrifugation and subsequent purification using a PRS (propylsulfonic acid silica gel) and a C₁₈ cartridge, and the recovery obtained ranged between 51 and 89 %. For HPLC separation, a binary solvent system consisting of acetonitrile and 0.05 M ammonium acetate solution (pH 3.6) with gradient elution with flow rate of 1.0 mL min⁻¹ and detection at 258 nm was used to resolve 16 HAs. With fluorescence nine HAs could be detected by employing a programmable wavelength, and the sensitivity was 100–400 times higher than that by UV detection. The detection limits for UV and fluorescence detection were 0.02≈0.5 ng and 0.05≈3 pg respectively, with a signal-to-noise ratio 3. The presence of HAs in fried chicken legs was also determined.     

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.     

Separation of aliphatic and aromatic amines by the ion interaction reagent RP-HPLC technique

Summary Ion interaction reagent RP-HPLC has been employed for the separation of some typical aliphatic and aromatic amines. The effects on retention of the alkyl chain length of the eluent, and of the eluent flow-rate have been studied. The use of solutions of hexylaminium-, octylaminium-and decylaminium-salicylate has been tested and compared, employing both conductometric and spectrophotometric detection. On the basis of the results obtained, the optimal experimental conditions can be chosen for each separation. Aliphatic and aromatic mono and diamines can be separated and detected, with an average sensitivity of the order of 40 ng without any pretreatment or derivatization. The retention data obtained for amines, compared with those obtained with the same ion interaction reagents for anions, help in the interpretation of the mechanism involved in the technique.     

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.     

HPLC coupled with fluorescence detection for the determination of procyanidins in white wines

Summary In this study a high-performance liquid chromatography method was developed for the determination of procyanidin dimers B1, B2, B3, B4 and procyanidin trimers C1 and T2 in white wine using fluorescence detection. This method allowed analysis of white wine with no prior treatment. Limits of quantification were form 12 μg.L⁻¹ to 16 μg.L⁻¹. Reproductibility data for replicate analysis was measured and the CV was less than 4.5%. This method was used for the determination of these compounds in six French white wines.