Impact of quality parameters on the recovery of putrescine and cadaverine in fish using methanol-hydrochloric acid solvent extraction

Methanol (MeOH) extraction by AOAC Official Method 996.07 has resulted in low amine recoveries in fresh fish tissue. Addition of 25% 0.4 M HCl to the 75% methanol-water extraction solvent resulted in higher recoveries of putrescine and cadaverine. Average putrescine recovery increased from 55 to 92% in flounder, scup, bluefish, and salmon; from 92 to 98% in mackerel; and from 83 to 107% in processed mackerel. Average cadaverine recovery increased from 57 to 95% in flounder, scup, bluefish, and salmon; from 91 to 97% in mackerel; and from 92 to 108% in processed mackerel. Fish stored on ice for 12 days also showed differences between background concentrations determined with the two solvents. However, the values decreased with storage time, indicating that degradation of the protein matrix may cause more comparable measurements between the two solvents. However, consistently higher putrescine and cadaverine measurements were determined using MeOH-HCl. Although significant differences in the extraction of amines from the high-fat fish tissue were not seen between MeOH and MeOH-HCl, it would be ideal to have one solvent for biogenic amine extraction. This study confirms that MeOH-HCl is a better solvent for complete extraction and recovery of putrescine and cadaverine in fresh and processed fish tissues.     

Aqueous sulfuric acid as the mobile phase in cation ion chromatography for determination of histamine, putrescine, and cadaverine in fish samples

Aqueous sulfuric acid can be used as the mobile phase in cation ion chromatography to separate the three biogenic amines, putrescine, cadaverine, and histamine, from fish. Various concentrations of aqueous sulfuric acid were investigated to optimize the separation of these three biogenic amines. Aqueous sulfuric acid (5.0 mM) was found to be optimum for the separation and was used to determine the three biogenic amines in fish. The LOQ, defined as the lowest level of the standard calibration curve, was 0.055 ppm (equivalent to 0.55 microg/g sample) for putrescine, 0.05 ppm (equivalent to 0.5 microg/g sample) for cadaverine, and 1.0 ppm (equivalent to 10 microg/g sample) for histamine. From statistical analysis of the LOQ, the method detection limit was 0.003 ppm for putrescine, 0.009 ppm for cadaverine, and 0.16 ppm for histamine. For sample preparation, the fish was composited, homogenized in methanol-water (75 + 25, v/v), incubated for 15 min at 60 degrees C, and centrifuged. The sample solution was micron-filtered before injection. The mobile phase flow rate was 0.8 mL/min under isocratic conditions at room temperature (15-25 degrees C). The three biogenic amines were separated in the order of increasing retention time, i.e., putrescine, cadaverine, and histamine, within 30 min. The chromatograms showed complete peak separation of the three amines regardless of the difference in fish matrixes.     

Rapid determination and confirmation of biogenic amines in tuna loin by gas chromatography/mass spectrometry using ethylchloroformate derivative

A gas chromatography-mass spectrometry (GC/MS) method is described for the easy rapid determination and simultaneous confirmation of the biogenic amines putrescine (PUT), cadaverine (CAD), histamine (HTA), and spermidine (SPD) in fresh frozen tuna loin. The method can also be used to monitor tyramine (TYR). The method involves homogenization of fish tissue, extraction of biogenic amines into trichloroacetic acid solution, centrifugation, alkalization, and derivatization of supernatant with ethylchloroformate. All seafood species were fortified to contain 2.5, 5.0, 10.0, 12.5, and 25.0 microg/g (ppm) PUT, CAD, and SPD; and 10.0, 20.0, 40.0, 50.0, and 100.0 microg/g (ppm) HTA. Determination was based on standard curves for each analyte using peak areas with matrix standards equivalent to a concentration range bracketing the spike level. A set of 5 matrix controls (unfortified tuna tissue) was also analyzed; only endogenous SPD was found in all samples. The interassay average recoveries ranged from 57 to 79% across analytes and spike levels.     

Determination of putrescine and cadaverine in seafood (finfish and shellfish) by liquid chromatography using pyrene excimer fluorescence

A liquid chromatography (LC) method is described for the easy determination of the biogenic diamines putrescine (PUT) and cadaverine (CAD) in canned tuna, frozen tuna loin, fresh mahimahi fillet, frozen raw shrimp, cooked lump crabmeat, and fresh and cold-smoked salmon. The method is also a useful screen for histamine (HTA). The method involves homogenization of fish tissue, extraction of biogenic amines into borate-trichloroacetic acid solution, centrifugation, and derivatization of supernatant with 1-pyrenebutanoic acid succinimidyl ester. The derivatized diamine species allow for the intramolecular excimer fluorescence of the pyrene moiety at a higher emission wavelength than is possible for the endogenous tissue monoamines, thus providing visual specificity of detection. All seafood species were fortified with 0.5, 1.0, 5.0, 10.0, and 15.0 microg/g (ppm) of PUT and CAD. Determination was based on standard graphs for PUT and CAD using peak areas with standard solutions equivalent to 0.375, 1.0, 5.0, 10.0, and 20.0 ppm in tissue. A set of five matrix controls (unfortified seafood tissue) were also analyzed; endogenous PUT was found in all samples except the canned tuna, and CAD found only in the shrimp, crab, and cold-smoked salmon. The background amines were thus subtracted prior to determining spike recovery. The intra-assay average recoveries ranged from 71 to 94% across species and spike levels.     

Validation study of a rapid ELISA for detection of histamine in tuna

Neogen Corp. has developed an improved Veratox histamine test kit for the detection of histamine in tuna tissue. The purpose of this study was to validate the method under the requirements of AOAC Research Institute (RI) Performance Tested Methods. Three AOAC Official Methods for histamine (954.04, 957.07, and 977.13) and one ELISA method have been performance tested by the AOAC RI. The most popular is AOAC Official Method 977.13, the fluorometric method, which is considered the reference method, but is complicated and time-consuming. The need for a rapid ELISA test kit to be validated by the AOAC RI exists.     

Determination of biogenic amines in fish tissues by ion-exchange chromatography with conductivity detection

Some biogenic amines, such as putrescine, cadaverine, spermidine and histamine, have been found to be useful as quality indices for the decomposition of fish, so research on the simultaneous analysis of various biogenic amines in food is of interest and importance. The intake of histamine may cause an allergic intoxication known as "scombroid poisoning" while secondary biogenic amines can potentiate the toxicity of histamine and in addition can react with nitrite to form carcinogenic nitrosamines. A new method for the simultaneous determination of underivatized biogenic amines based on ion-exchange chromatography with conductivity detector has been developed. The proposed method offers a number of advantages over previous pulsed amperometric detection and integrated pulsed amperometric detection methods such as simpler extraction procedure and sharp peaks. Separations were performed on a new low hydrophobic weak cation-exchange analytical column. This technique is simple, rapid and useful for routine checks in repetitive analyses.     

Vortex‐assisted surfactant‐enhanced‐emulsification liquid–liquid microextraction of biogenic amines in fermented foods before their simultaneous analysis by high‐performance liquid chromatography

A simple, rapid, sensitive, and environmentally friendly method, based on modified dispersive liquid–liquid microextraction coupled with high‐performance liquid chromatography was developed for the simultaneous determination of five biogenic amines in fermented food samples. Biogenic amines were derivatized with 9‐fluorenylmethyl chloroformate, extracted by vortex‐assisted surfactant‐enhanced emulsification liquid–liquid microextraction, and then analyzed by high‐performance liquid chromatography. Five biogenic amine compounds were separated within 30 min using a C₁₈ column and gradient elution with acetonitrile and 1% acetic acid. Factors influencing the derivatization and extraction efficiency such as type and volume of extraction solvent, type, and concentration of surfactant, pH, salt addition, and vortex time were optimized. Under the optimum conditions, the method provided the enrichment factors in the range of 161–553. Good linearity was obtained from 0.002–0.5 mg/L for cadaverine and tyramine, 0.003–1 mg/L for tryptamine and histamine, and 0.005–1 mg/L for spermidine with coefficient of determination (R²) > 0.992. The limits of detection ranged from 0.0010 to 0.0026 mg/L. The proposed method was successfully applied to analysis of biogenic amines in fermented foods such as fermented fish (plaa‐som), wine and beer where good recoveries were obtained in the range of 83.2–112.5%     

Determination of biogenic amines as their benzoyl derivatives after cloud point extraction with micellar liquid chromatographic separation

The advantages of micellar cloud point extraction combined with a surfactant-assisted separation in a HPLC system are presented as a method for the effective separation and determination of nine biogenic amines in fish substrates. Benzoyl derivatives of the amines are extracted inside the micelles of a non-ionic surfactant, Triton X-114, and separated with gradient elution micellar liquid chromatography. Quantification was performed by measuring the UV absorbance of the benzene ring at 254 nm. Detection limits of the nine biogenic amines were in the vicinity of 0.01 mg l(-1) which are approximately 10 times lower than those of the conventional method (HPLC-UV) and 100 times lower than those of micellar electrokinetic capillary chromatography. The correlation coefficients of determinations were 0.9911-0.9996. The method was applied for the determination of putrescine, cadaverine, agmatine, tyramine, tryptamine, phenylethylamine, spermine, spermidine and histamine in trout samples. Recovery of the proposed method ranged from 95 to 103.5%.     

New solvent systems for thin-layer chromatographic determination of nine biogenic amines in fish and squid

Different solvent systems were evaluated for their ability to separate biogenic amines by thin-layer chromatography (TLC). Dansyl derivatives of agmatine, putrescine, tryptamine, cadaverine, spermidine, histamine, spermine, tyramine and beta-phenylethylamine were separated using the solvent system chloroform-diethyl ether-triethylamine (6:4:1), followed by chloroform-triethylamine (6:1). After separation dansyl amines were quantified by fluorescence densitometry at 330 nm. Correlation coefficients of linear regressions were higher than 0.99 for all amines, except for agmatine (0.976). Detection limits were 10ng for tryptamine, tyramine, histamine and beta-phenylethylamine, and 5 ng for the other amines. The overall repeatability of the chromatography was 1.82% when including agmatine and barely 1.02% for the other amines. The accuracy ranged from 105.97% (agmatine) to 49.92% (tryptamine). This thin-layer chromatography method was found to be an effective and precise analytical procedure to separate and determine biogenic amines. Its main advantages compared to previous procedures are that it uses less harmful solvent (diethyl ether instead of benzene) and can separate a larger group of biogenic amines.     

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 biogenic amines in alcoholic beverages by ion chromatography with suppressed conductivity detection and integrated pulsed amperometric detection

The determination of biogenic amines in alcoholic beverages is important to assess the potential risks associated with the consumption of high concentrations of these compounds. In addition, product storage conditions and the length of storage can cause the formation of biogenic amines that reduce product quality. We report a new method using cation-exchange chromatography with either suppressed conductivity, integrated pulsed amperometry, UV, or a combination of these detection techniques to determine biogenic amines in alcoholic beverages. The main objective was to provide a direct comparison between IPAD and suppressed conductivity detection for determining biogenic amines in alcoholic beverages. Suppressed conductivity is the simplest detection approach for determining putrescine, cadaverine, histamine, agmatine, phenylethylamine, spermidine, and spermine with good sensitivity (0.004-0.08 mg/l) and was used to evaluate the influence of storage time and conditions on the evolution of biogenic amines in alcoholic beverages. Integrated pulsed amperometric detection (IPAD) detects more biogenic amines than suppressed conductivity detection, enabling the detection of dopamine, tyramine, and serotonin. Tyramine was simultaneously determined by UV detection and IPAD to provide confirmation and ensure the accuracy of the analytical results. The linearity of biogenic amine responses was within 0.1-20 mg/l and peak area precisions were 0.24-4.97% for IPAD, suppressed conductivity-IPAD, and UV detection. The sensitivity for the 10 biogenic amines using the 3 detection techniques varied considerably from 0.004-1.1 mg/l and recoveries were within 85-122%.     

Determination of biogenic amines by capillary zone electrophoresis with conductometric detection

A capillary zone electrophoresis (CZE) method with conductometric detection of biogenic amines (cadaverine, putrescine, agmatine, histamine, tryptamine and tyramine) is described. The optimised background electrolyte was the following: 15 mM histidine + 5 mM adipic acid + 1.5 mM sulphuric acid + 0.1 mM ethylenediaminotetraacetic acid + 0.1% hydroxyethylcellulose + 50% methanol. A clear separation of six biogenic amines from other components of acidic sample extract was achieved within 10 min. Method characteristics, i.e., linearity (0-100 micromol/ml), accuracy (recovery 86-107%), intra-assay repeatability (2-4%), and detection limit (2-5 micromol/l) were evaluated. Low laboriousness, sufficient sensitivity, speed of analysis, and low running cost are important attributes of this method. The developed method was successfully applied on the determination of biogenic amines in selected food samples.     

Determination of biogenic amines in fresh and processed meat by suppressed ion chromatography-mass spectrometry using a cation-exchange column

A new method for simultaneous determination of underivatized biogenic amines based on the separation by cation-exchange chromatography and suppressed conductivity coupled with mass spectrometry detection has been developed. The method has been applied to the analysis of cadaverine, putrescine, histamine, agmatine, phenethylamine and spermidine in processed meat products. The amines were extracted from muscle tissue with methanesulfonic acid without any additional derivative step or sample clean-up. Biogenic amines were separated by the IonPac CS17 column, a cation-exchange column used with gradient elution, and detection was done by suppressed conductivity and mass spectrometry. Tyramine was simultaneously analysed by using a spectrophotometer (275 nm) before the suppressed conductivity detection. Linearity of response was obtained in the range 0.25-25 microg mL(-1). The detection limits ranged from 23 microg L(-1) for putrescine to 155 microg L(-1) for spermidine (suppressed conductivity) and from 9 microg L(-1) for agmatine to 34 microg L(-1) for spermidine (MS). Average recoveries from meat samples ranged from 85 to 97% and coefficients of variation ranged from 4.5 to 9.7%. The analysis of biogenic amines in fresh and processed meats (dry-cured, cooked and fermented products) can be used as a quality marker of raw material and for studying the relationship between their changes and the fermentation process involved in dry sausage ripening.     

Condensation nucleation light scattering detection for biogenic amines separated by ion-exchange chromatography.

A sensitive method of analysis for biogenic amines, putrescine, cadaverine, histamine and an amino acid precursor, histidine is described herein using ion-exchange chromatography and condensation nucleation light scattering detection. The method was successfully used for the analysis of biogenic amines in fish samples. The method offers a number of advantages: fast elution of analytes with no need for mobile phase conductivity suppression, no derivatization and no electrochemical activity for the analyte's detection. The 3 sigma detection limits for these compounds were found to range from 8 to 20 ng/ml.     

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.     

Trace analysis of trichlorobenzenes in fish by microwave-assisted extraction and gas chromatography-electron-capture detection

An analytical method consisting of extraction, clean-up, and analysis by gas chromatography-electron-capture detection (GC-ECD) was developed for the determination of trichlorobenzenes (TCBs) in fish samples. Two extraction methods, saponification and liquid-liquid extraction (S-LLE), and microwave-assisted extraction (MAE), were evaluated. In both cases, n-pentane was used as the extraction solvent. For S-LLE, the recoveries ranged from 66.6+/-9.1% for 1-bromo-4-chlorobenzene (4-BCB) to 93.5+/-4.9% for 1,2,4-trichlorobenzene (1,2,4-TCB). The recoveries were significantly lower, between 31.0+/-3.9% for 1,2,3-trichlorobenzene (1,2,3-TCB) and 52.3+/-3.0% for 1,3,5-trichlorobenzene (1,3,5-TCB), in the absence of fish. Proteins and glycerides of the fish tissue seemed to compete with TCBs for the base, and hence decreased their decomposition rate. In the case of MAE, the recoveries were highly dependent on the pressure applied during extraction. At 5 bar, much higher recoveries were obtained, from 66.7+/-15.6% for 4-BCB to 79.9+/-13.6% for 1,2,4-TCB, than at 1 bar. Sulfur formation was, however, observed at 5 bar, and interfered with the GC-ECD analysis of TCBs. Sulfur was adequately removed by copper powder treatment, which was shown not to affect the recovery of analytes. The recoveries of target analytes by S-LLE and MAE did not differ statistically (t-test, alpha = 0.01). Both methods were appropriate for the detection of TCBs at concentration levels typically observed in marine biota, i.e. approximately 1 ng/g. S-LLE was, however, more time consuming, and required larger volumes of high-purity organic solvents than MAE.     

The Development of an Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry Method for Biogenic Amines in Fish Samples

Biogenic amines (BAs) are a group of substances that are formed from amino acids by decarboxylation or amination and transamination of aldehydes and ketones. They may have either an aliphatic, aromatic, or heterocyclic structure. Their quantity determines their effects and optimum amounts are essential for physiological functions, but excess BAs causes various toxic effects throughout the human body. In our study, to rapidly determine 14 BAs (histamine, tyramine, dopamine, tryptamine, serotonin, putrescine, spermine, spermidine, octopamine, benzylamine, 1-Phenylethanamine, cadaverine, 2-Phenethylamine, and agmatine) in real fish samples, an ultra-performance liquid chromatography–tandem mass spectrometry method was established. The fish sample was extracted by acetonitrile with 0.1% formic acid and stable biogenic amine derivatives could be obtained by benzoyl chloride derivatization with a shorter reaction time. The method showed good linearity with a linear range of 3–4 orders of magnitude and regression coefficients ranging from 0.9961 to 0.9999. The calculated LODs ranged from 0.1 to 20 nM and the LOQs ranged from 0.3 to 60 nM. Satisfactory recovery was obtained from 84.6% to 119.3%. The proposed method was employed to determine the concentration levels of biogenic amine derivatives in different fish. The results indicated that this method was suitable for the analysis of biogenic amines.     

Determination of biogenic amines in food samples using derivatization followed by liquid chromatography/mass spectrometry

A liquid chromatography (LC)/mass spectrometry method was developed for the determination of selected biogenic amines in various fish and other food samples. It is based on a precolumn derivatization of the amines with succinimidylferrocenyl propionate under formation of the respective amides and their reversed-phase liquid-chromatographic separation with subsequent electrospray ionization mass-spectrometric detection. Deuterated putescine, cadaverine, and histamine are added prior to the derivatization as internal standards that are coeluted, thus allowing excellent reproducibility of the analysis to be achieved. Depending on the analyte, the limits of detection were between 1.2 and 19.0 mg/kg, covering between 2 and 3 decades of linearity. The limit of detection and the linear range for histamine are suitable for the surveillance of the only defined European threshold for biogenic amines in fish samples. Compared with the established ortho-phthalaldehyde (OPA)/LC/fluorescence method, the newly developed method allows an unambiguous identification of the biogenic amines by their mass spectra in addition to only retention times, a fivefold acceleration of the separation, and independency from the sample matrix owing to the isotope-labeled internal standards. Various fish, calamari, and salami samples were successfully analyzed with the new method and validated with an independent OPA/LC/fluorescence method.     

Environmentally friendly solid‐phase microextraction coupled with gas chromatography and mass spectrometry for the determination of biogenic amines in fish samples

In this work, a facile and environmentally friendly solid‐phase microextraction assay based on on‐fiber derivatization coupled with gas chromatography and mass spectrometry was developed for determining four nonvolatile index biogenic amines (putrescine, cadaverine, histamine, and tyramine) in fish samples. In the assay, the fiber was firstly dipped into a solution with isobutyl chloroformate as derivatization reagent and isooctane as extraction solvent. Thus, a thin organic liquid membrane coating was developed. Then the modified fiber was immersed into sample solution to extract four important bioamines. Afterwards, the fiber was directly inserted into gas chromatography injection port for thermal desorption. 1,7‐Diaminoheptane was employed as internal standard reagent for quantification of the targets. The limits of detection of the method were 2.98–45.3 μg/kg. The proposed method was successfully applied to the detection of bioamines in several fish samples with recoveries ranging 78.9–110%. The organic reagent used for extraction was as few as microliter that can greatly reduce the harm to manipulator and environment. Moreover, the extraction procedures were very simple without concentration and elution procedures, which can greatly simplify the pretreatment process. The assay can be extended to the in situ screening of other pollutant in food safety by changing the derivatization reagent.     

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。