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.     

Liquid chromatographic determination of polyamines in human urine based on intramolecular excimer-forming fluorescence derivatization using 4-(1-pyrene)butanoyl chloride

A liquid chromatographic method for highly sensitive and selective fluorometric determination of polyamines (putrescine, cadaverine, spermidine and spermine) in human urine is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoyl chloride (PBC), followed by reversed-phase liquid chromatography. The method offers higher sensitivity for determination of spermidine and spermine than previously reported method utilizing 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester as a derivatization reagent. Samples containing free polyamines in diluted human urine were directly derivatized with PBC and separated on an octyl column. The derivatives were detected at excitation 345 and emission 475 nm wavelengths. For determination of total polyamine content, the conjugated polyamines were first hydrolyzed in 4 M HCl. The detection limits (signal-to-noise ratio = 3) for polyamines in urine were 1.1-3.4 pmol/mL. At optimized derivatization and chromatographic conditions, interferences such as biogenic monoamines gave no peaks or the peaks did not interfere with the peaks of polyamine derivatives. In conclusion, the present derivatization method allows direct determination of polyamines in human urine samples without the need for sample clean-up procedures.     

The determination and characterisation of 2-naphthyloxycarbonyl chloride derivatised biogenic amines in pet foods

The need to monitor biogenic amines levels is essential for many areas of the food industry for two main reasons: the caustic nature and potential toxicity of these amines, and the potential to use amine levels as markers for freshness and quality in foodstuffs. Optimised analysis conditions used for the determination of biogenic amines derivatised with 2-napthyloxycarbonyl chloride has been applied to different pet food samples to assess the effectiveness of this method for complex sample matrices. Further to this, the use of high-resolution mass spectrometry has enabled the previously unconfirmed derivatised form of seven biogenic amines to be established. The derivatised forms identified include as mono substituted (tryptamine and histamine), bisubstituted (putrescine, cadaverine and tyramine), trisubstituted (spermidine) and tetrasubstituted (spermine). The methodology of biogenic amine determination was performed successfully to a range of pet food products highlighting the applicability to a variety of complex sample matrices.     

胶束电动毛细管色谱检测鱼肉中的七种生物胺

建立了一种利用胶束电动毛细管色谱同时检测鱼肉中组胺、腐胺、2-苯乙基胺、尸胺、色胺、亚精胺及精胺7种生物胺的方法。样品经6%过氯酸萃取后,由苯甲酰氯衍生化,以含0.06 mol/L脱氧胆酸钠的0.02 mol/L硼酸(pH 9.2)-甲醇(体积比为95∶5)混合液为电泳介质,电泳电压25 kV,温度25 ℃,检测波长214 nm,在12 min内实现了7种生物胺的完全分离。7种生物胺的浓度与其峰面积在一定的范围呈良好的线性关系,检出限除组胺为15 μg/g外,其余均为5 μg/g。迁移时间和峰面积的日内、日间相对标准偏差均小于5%。该法用于海鱼中7种胺类物质含量的测定,结果令人满意。     

Urine polyamines determination using dansyl chloride derivatization in solid-phase extraction cartridges and HPLC

The derivatization of biogenic amines such as putrescine, cadaverine, spermidine and spermine with dansyl chloride in solid phase extraction cartridges is described. Different types of filling materials were tested in order to have the highest retention of the different analytes. The best results were obtained by using C18 cartridges. The optimal conditions were: amine solution buffered at pH 12, 2 mM dansyl chloride (acetone-bicarbonate solution 20 mM (pH 9-9.5), 2 + 3 v/v) as reagent concentration, room temperature and 30 min reaction time. The developed procedure was applied to the determination of these polyamines in urine samples from healthy controls and cancer patients using HPLC with 1,7-diaminoheptane as internal standard. The concentrations ranged from 0.5 to 5 micrograms mL-1 and the detection limits were 10 ng mL-1 for all polyamines. By concentrating the urine extracts, the detection limits were improved down to 2 ng mL-1. The accuracy and the precision of the method were tested. The proposed dansylation method is advantageous with respect to solution dansylation. It improves the total analysis time, avoids high temperatures that can affect the thermal stability of the derivatives and could make possible the automation of the procedure.     

High-performance liquid chromatographic determination of free and total polyamines in human serum as fluorescamine derivatives

A highly sensitive and simple fluorimetric method for the determination of free and total polyamines, spermidine, spermine, putrescine and cadaverine, in human serum by high-performance liquid chromatography is described. The polyamines, obtained after clean-up of deproteinized serum by Cellex P column chromatography, are converted to their fluorescamine derivatives in the presence of nickel ion which inhibits the reaction of interfering amines with fluorescamine, and the derivatives are separated simultaneously by reversed-phase chromatography (LiChrosorb RP-18) with a linear gradient elution. The lower limits of detection are 10 and 15 pmole for spermine and the others in 0.5 ml of serum, respectively.     

Determination of polyamines in human erythrocytes by capillary gas chromatography with nitrogen-phosphorus detection

A capillary gas chromatographic method with nitrogen-phosphorus detection is used to determine simultaneously 1,3-diaminopropane, putrescine, cadaverine, spermidine and spermine in human erythrocytes. The compounds are isolated by adsorption on silica and converted into their heptafluorobutyryl derivatives. We give quality-control data and (age-dependent) normal values for 48 apparently healthy controls.     

Analysis of amino acids and biogenic amines in biological tissues as their o-phthalaldehyde/ethanethiol/fluorenylmethyl chloroformate derivatives by high-performance liquid chromatography. A deproteinization study

The extraction of ornithine, lysine, putrescine, cadaverine, 1,7-diaminoheptane, spermidine and spermine from biological tissues was optimized for HPLC quantitation as their o-phthalaldehyde/ethanethiol/fluorenylmethyl chloroformate (OPA/ET/FMOC) derivatives. In applying perchloric acid deproteinization two approaches have been followed: (i) deproteinization with subsequent neutralization by potassium hydroxide and lyophilization, and (ii) deproteinization without neutralization and lyophilization. Neutralization and lyophilization resulted in the loss of free biogenic amines. HPLC analysis of ornithine (Orn), lysine (Lys), putrescine (Put), cadaverine (Cad), 1,7-diaminoheptane (Dah), spermidine (Spd) and spermine (Spm) content of biological tissues as their OPA/ET/FMOC derivatives was performed in the supernatant of perchloric acid-deproteinized samples (model solutions and tissues) with an average reproducibility of < or =2.6% relative standard deviation (RSD), including recovery of sample treatment and chromatography.     

Automated Determination of Urinary Polyamines

Abstract

A system for the determination of urinary polyamines by ion exchange chromatography with fluorescence detection is presented. It provides a sensitive and specific assay of putrescine, cadaverine, spermidine and spermine in urine hydrolyzates. An internal standard is used, which makes the automated determination of series of samples easy.     

Determination of polyamines in hydrolysates of uremic plasma by high-performance cation-exchange column chromatography

The levels of putrescine, cadaverine, spermidine and spermine in uremic plasma were determined with an automatic polyamine analyzer with a 7.5 X 0.2 cm I.D. cation-exchange column using a stepwise sodium chloride gradient. All four polyamines were higher in ten patients with chronic renal failure than in eight normal subjects. The total polyamine content was also measured in the patients' plasma before and after maintenance dialysis; putrescine and spermidine levels were significantly lowered by the procedure.     

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.     

Simultaneous determination of 10 kinds of biogenic amines in rat plasma using high‐performance liquid chromatography coupled with fluorescence detection

We describe a simple, rapid, selective and sensitive HPLC method coupled with fluorescence detection for simultaneous determination of 10 kinds of biogenic amines (BAs: tryptamine, 2‐phenethylamine, putrescine, cadaverine, histamine, 5‐hydroxytryptamine, tyramine, spermidine, dopamine and spermine). BAs and IS were derivated with dansyl chloride. Fluorescence detection (λ_ex/λ_em = 340/510 nm) was used. A satisfactory result for method validation was obtained. The assay was shown to be linear over the ranges 0.005–1.0 μg/mL for tryptamine, 2‐phenethylamine and spermidine, 0.025–1.0 μg/mL for putrescine, 0.001–1.0 μg/mL for cadaverine, 0.25–20 μg/mL for histamine, 0.25–10 μg/mL for 5–hydroxytryptamine and dopamine, and 0.01–1.0 μg/mL for tyramine and spermine. The limits of detection and the limits of quantification were 0.3–75.0 ng/mL and 1.0–250.0 ng/mL, respectively. Relative standard deviations were ≤5.14% for intra‐day and ≤6.58% for inter‐day precision. The recoveries of BAs ranged from 79.11 to 114.26% after spiking standard solutions of BAs into a sample at three levels. Seven kinds of BAs were found in rat plasma, and the mean values of tryptamine, 2‐phenethylamine, putrescine, cadaverine, histamine, spermidine and spermine determined were 52.72 ± 7.34, 11.45 ± 1.56, 162.56 ± 6.26, 312.75 ± 18.11, 1306.50 ± 116.16, 273.89 ± 26.41 and 41.51 ± 2.07 ng/mL, respectively.     

Reversed-phase liquid chromatographic separation and simultaneous fluorimetric detection of polyamines and their monoacetyl derivatives in human and animal urine, serum and tissue samples: an improved, rapid and sensitive method for routine application

A highly sensitive and precise method for the determination of the polyamines putrescine, cadaverine, spermidine and spermine and all their monoacetyl derivatives in a single analysis in human and animal urine, serum and tissue samples is described. For polyamine separation, an ion-pairing reversed-phase high-performance liquid chromatographic (HPLC) method is used, followed by post-column derivatization with o-phthalaldehyde and consecutive fluorescence detection. Urine and serum samples are purified with a Bond Elut silica cartridge. The detection limit for polyamines is 0.5-1.0 pmol and excellent linearity is achieved in the range from 3 pmol up to more than 10 nmol. The influence of some modifications of different analytical steps such as the temperature of the HPLC column and the derivatization reaction coil and the o-phthalaldehyde flow-rate is described. Quality control data and measurements of the reproducibility of the method are presented. In order to establish a rapid analytical method for easy routine use, all steps for preparation and quantitative analysis are minimized. This method was applied to the determination of total polyamines in human urine and serum hydrolysate and of free and acetylated polyamines in human urine and pancreatic tissue of the rat. Values for normal polyamine concentrations in the urine and serum of fifteen male and fifteen female healthy volunteers and in the pancreas of ten normal rats are presented.     

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.     

Determination of polyamines in human urine by precolumn derivatization with benzoyl chloride and high-performance liquid chromatography coupled with Q-time-of-flight mass spectrometry

A simple and sensitive HPLC/Q-TOF MS method for simultaneous determination of 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine and acetyl-spermine in human urine was developed in electrospray-ionization source by positive ion mode. The samples were firstly pretreated by 10% HClO₄ and then derivatized by benzoyl chloride with 1,6-diaminohexane as internal standard. The derived polyamines were separated on a C₁₈ column by a gradient elution with methanol-water, and then sensitively detected with Q-TOF MS. The limits of detection for polyamines ranged from 0.02 to 1.0 ng ml⁻¹ with excellent linearity within the range from 1 to 1000 ng ml⁻¹ except acetyl-spermine from 5 to 1000 ng ml⁻¹. The intra- and inter-day R.S.D. for all polyamines were 2.0-14.7% and 3.9-12.9%, respectively. The method was applied to determine the polyamines in human urine from 10 cancer patients and 15 healthy volunteers. Results showed that the mean levels of polyamines in urine of patients were all higher than those in healthy volunteers. The cluster analysis was used to establish the distinction mode between cancer sufferers and healthy individuals.     

对酞内酰胺苯甲酰氯柱前衍生反应HPLC分析痕量多胺

本文提出了一种快速、简便、灵敏检测痕量多胺的新方法。用对酞内酰胺苯甲酰氯同多胺进行柱前衍生反应,以反相高效液相色谱定量测定多胺含量。在4～500 pmol范围内有良好的定量线性关系,腐胺、精脒和精胺的检测限均为0.1 pmol。     

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.     

Polyamine distribution in the rat intestinal mucosa

As the first step in a study of mucosal polyamine metabolism during intestinal adaptation, we have measured mucosal polyamine concentrations at different sites along the normal rat intestine. Putrescine, spermidine, spermine and cadaverine were measured by spectrofluorometric analysis after thin-layer chromatography of their dansylated derivatives. Spermidine was present in the largest amounts at each of the sampling sites. The ratio of the concentration of spermidine to that of spermine paralleled the established pattern of cellular proliferation in the normal intestine as did the putrescine concentration (nmol per 10 cm) which decreased from duodenum to colon. These results provide the essential background to an assessment of the role of polyamines in the intestinal adaptive response.     

Selective fluorometric detection of polyamines using micellar electrokinetic chromatography with laser-induced fluorescence detection

The polyamines putrescine, cadaverine, spermine and spermidine were separated and quantified by micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection. The derivatization reagent, 1-pyrenebutanoic acid succinimidyl ester (PSE), allowed for the selective detection of the polyamines at 490 nm. Multiple labeling of the polyamines with PSE allows the formation of intramolecular excimers that emit at longer wavelengths (450-520 nm) than mono-labeled analytes (360-420 nm). Optimal separation of the labeled polyamines was achieved using a separation buffer consisting of 10 mM phosphate pH 7.2, 30 mM cholate, and 30% acetonitrile. Using these conditions, the four polyamines were separated in under 10 min. Limits of detection for putrescine, cadaverine, spermine and spermidine were 6, 5, 15 and 13 nM, respectively. These are superior or comparable to those previously reported in the literature using fluorescence detection.     

The components of the venom of a spider Scodra griseipes. 1. Analysis of low molecular weight products using gas chromatography/mass spectrometry.

Low molecular weight venom constituents of the spider Scodra griseipes have been derivatized using bis(trimethylsilyl)-trifluoroacetamide and pentafluoropropionic anhydride. Examination of these derivatives by coupled gas chromatography/mass spectrometry using electron ionization or ammonia- or isobutane-chemical ionization allows their structural identification. Comparisons have also been made with retention time and mass spectra of the pure compounds. Four acids: phosphoric, lactic, citric, gamma-aminobutyric (neurotransmitter) and four basic biogenic amines: putrescine, cadaverine, spermidine and spermine have been found.