On-line solid-phase extraction with surfactant accelerated on-column derivatization and micellar liquid chromatographic separation as a tool for the determination of biogenic amines in various food substrates

A sensitive method was developed for the determination of biogenic amines at very low levels by combining solid phase extraction (SPE) and derivatization on-line with HPLC. The on-line derivatization and SPE were performed simultaneously on a commercially available ODS guard column, which was installed instead of the filling loop on the HPLC apparatus. Resolution of the peaks and quantification was further enhanced with micellar liquid chromatography and sensitization of the benzene ring absorption at 254 nm. Detection limits of the benzoyl derivatives of biogenic amines were in the vicinity of 0.1 microg L(-)(1), which is even lower than those obtained by fluorescence detection and is unparallel to any other UV approach. The correlation coefficients of determinations were 0.9850-0.9998. The method was applied to the determination of Biogenic amines, that is, putrescine, cadaverine, agmatine, tyramine, tryptamine, phenylethylamine, spermine, spermidine and histamine in fish, chicken, and wine samples. Recovery of the proposed method ranged from 94 to 106%.     

On-line preconcentration prior to on-column derivatization monolith octadecasiloxane capillary electrochromatography for the determination of biogenic amines

To expand the applications of the on-line preconcentration technique with capillary electrochromatography (CEC) to biogenic amines that have no specific chromophore or fluorophore in their molecules, a method of on-line preconcentration prior to on-column derivatization CEC is presented. A monolithic ODS capillary column (20 cm effective length x 75 microm i.d.) for CEC was fabricated using a thermal sol-gel reaction of tetraethyl orthosilicate to capture ODS particles (5-microm particle diameter) in a capillary tube. A standard model biogenic amine solution consisting of histamine, methylhistamine, and serotonin was electrokinetically injected from the anodic site of the capillary column with 5 kV, and these amines were effectively concentrated at the inlet site of the capillary column by a field-amplified sample stacking, a gradient effect mode, or both. This preconcentration occurred whenever the several types of solvent for reconstitution of the amines, e.g., water (noneluting solvent or low-conductivity solvent), 0.9% sodium chloride (noneluting solvent or high-conductivity solvent), or 60% acetonitrile in 10 mM borate buffer (pH 10) (eluting solvent) were employed. After concentration, the amines were subsequently derivatized, separated, and detected during CEC with an optimum CEC run buffer solution containing 60% acetonitrile in 5 mM o-phthalaldehyde/2-mercaptoethanol-10 mM borate buffer (pH 10) when 5 kV was continuously applied. Using the present system, equipped with a fluorescence detector instead of a UV/visible detector, the detection sensitivity for amines reached a 0.1 microM level, which increased sensitivity by a factor of 10(3) times greater than that of normal on-column derivatization CEC.     

Selective detection of biogenic amines using capillary electrochromatography with an on-column derivatization technique

A selective detection method for biogenic amines present in highly complex matrixes was devised by employing both electrokinetic injection and on-column-derivatization capillary electrochromatographic methods. The on-column derivatization capillary electrochromatography system was evaluated by use of a capillary column (total length of 45 cm, effective length of 25 cm) fabricated using a 100-mcirom (i.d.) fused-silica capillary tube packed with 5-microm (i.d.) ODS particles that were tolerant of an alkaline environment. The column was filled with a run buffer consisting of a derivatization reagent, o-phthalaldehyde/2-mercaptoethanol, in a mixture of borate buffer (pH 10). After electrokinetic injection of a mixture of five biogenic amines (histamine, serotonin, tyramine, putrescine, cadaverine) as a test sample, the free amines entered into the anodic site of the capillary column and started to travel along the column, during which time the analytes reacted with the derivatization reagent, separated out, and were detected with an absorbance at 340 nm when high voltage was applied to the column. When this system was applied to a mixture containing 5 biogenic amines and 17 amino acids, the 5 biogenic amines plus arginine selectively entered into the capillary with the electrokinetic injection and were observed on the electrochromatogram, but none of the amino acids lacking arginine were detected. The designated method was also tested for its ability to determine the presence of biogenic amines in the crude extracts obtained from two types of aged fish.     

高效液相色谱法测定地沟油中5种生物胺

本文建立了高效液相色谱柱前衍生化法测定地沟油中5种生物胺含量的方法。样品经0.5mol/L盐酸提取后用丹磺酰氯衍生,采用ZORBAX 80A Extend-C1(84.6×150mm5μm),以0.1mol/L乙酸铵和乙腈为流动相进行梯度洗脱,流速1.0mL/min,柱温30℃,检测波长260nm。该方法检测限为腐胺1.2μg/g,尸胺1.4μg/g,组胺1.3μg/g,酪胺、精胺1.5μg/g。样品添加浓度为10μg/g、20μg/g时,回收率为82%～89%,相对标准偏差为1.8%～3.5%。本法为地沟油的质量鉴定提供了简便、灵敏、可靠的检测方法。     

Comparison of Potential-Time Waveforms for the Detection of Biogenic Amines in Complex Mixtures following Their Separation by Liquid Chromatography

Pulsed amperometric detection (PAD), integrated voltammetric detection (IVD), and integrated square-wave detection (ISWD) at gold electrodes are compared for the flow injection detection of 1,3-diaminopropane in a liquid chromatograph (LC). These detection methods are especially significant for alkylamines and amino acids because (i) the majority of these compounds do not naturally possess chromophoric or fluorophoric functional groups and (ii) their amperometric detection at constant potential at Au electrodes fails because the electrode activity is severely attenuated by the formation of an inert surface oxide (AuO). The anodic response mechanisms for detection of amines require concomitant formation of AuO; therefore, a large background signal is observed with conventional PAD. In comparison, the background is much smaller for IVD and ISWD because the anodic charge for oxide formation (positive scan/step) is compensated by the cathodic charge for oxide reduction (negative scan/step). The limits of detection (S/N = 3) for 1,3-diaminopropane by LC-PAD, LC-IVD and LC-ISWD are about 3 × 10(2) pg (4 pmol), 5 × 10(1) pg (0.7 pmol), and 3 × 10(1) pg (0.5 pmol), respectively, for 25-μL injections. Results are also shown to demonstrate ISWD for detection of nine biogenic amines following their chromatographic separation.     

Development of an automated method for simultaneous determination of low molecular weight aliphatic amines and ammonia in ambient air by diffusion scrubber coupled to ion chromatography

A method has been developed for the simultaneous determination of low molecular weight aliphatic amines and ammonia in the atmosphere. Analyte gases are collected quantitatively in high-purity deionized water of a planar diffusion scrubber, and the resultant solution is analyzed by ion chromatography. A 1-h cycle analysis could be continuously repeated. The calibrations for the amines are linear between 5 and 500 pptv. The detection limits are a few pptv with RSD of less than 3%. The calibration for ammonia shows severe curvature at high concentrations so that second-order fitting is required for accurate determination. This method was successfully applied to the environmental air analyses. The major amines in the atmosphere were trimethylamine, dimethylamine, and methylamine, and the temporal variations were closely correlated with that of ammonia, implying their identical emission sources.     

Development of a solid-phase microextraction GC-NPD procedure for the determination of free volatile amines in wastewater and sewage-polluted waters.

An analytical procedure for the determination of free volatile C1-C6 amines in aqueous matrixes has been developed and applied to their determination in waste-water, primary and secondary effluents, and sewage-polluted river samples. The developed analytical procedure involves headspace sampling using solid-phase microextraction with a poly(dimethylpolysiloxane) coating (100 microns) followed by GC-NPD determination and GC/MS confirmation using a tailor-made PoraPLOT amines capillary GC column for volatile amines. Procedural detection limits were compound dependent but ranged from 3 to 56 micrograms L-1, being close to or lower than the odor threshold concentration, and the reproducibility was ca. 15% (N = 5) in real water samples. The developed analytical procedure is solvent free, cost-effective (no cryogenic trap needed), and faster than existing methods because no derivatization step is involved in the determination. Linearity was compound dependent but ranged at least from 50 to 600 micrograms L-1.     

Propylene carbonate as a nonaqueous solvent for capillary electrophoresis: mobility and ionization constant of aliphatic amines.

The two properties of aliphatic amines were investigated in propylene carbonate as solvent that are decisive for capillary electrophoretic migration: the actual mobilities and the pKa* values. Solutes were eight primary, secondary, and tertiary amines. Roughly, the actual ionic mobilities of the ammonium ions are inversely proportional to the solvent viscosity, fairly obeying Walden's rule. The pKa* values of the cation acids, HB+ (the corresponding acids of the amines, B), were related to the conventional pH* scale of the buffers. Determined from the effective mobilities as a function of the pH*, they are increased by approximately 7 units compared to water. This increase was interpreted based on the concept of the standard free energy of transfer of the individual species in the acid-base equilibrium. The corresponding medium effect on the proton, log mgammaH+ (the logarithm of the transfer activity coefficient mgammaH+) is approximately +8. The medium effect on the free base, B, was obtained from solubility data; it is about -1 and smaller. Plausible values for the medium effect on the cation HB+ (-1 to -2) lead to a sum of the increments, which corresponds with the overall effect, expressed by the change in pKa*. Examination of the individual contributions shows that the drastically lower basicity of propylene carbonate compared to water is mainly responsible for the increase in pKa upon transfer of the acid-base equilibrium of aliphatic ammonium/amine from the aqueous to the organic solvent.     

Micellar electrokinetic capillary chromatography-electrochemical detection for analysis of biogenic amines in Drosophila melanogaster

Micellar electrokinetic chromatography coupled to amperometric electrochemical detection was used to investigate the chemical environment of the fruit fly, Drosophila melanogaster. Preliminary studies focused on the employment and optimization of the system to separate electroactive amine-containing molecules present in the head and body of male and female flies. Ultimately, biogenic amines significant to the fly including L-3,4-dihydroxyphenylalanine, dopamine, tyramine, and serotonin were identified and their relative abundance quantified. Transgenic Drosophila with functionally ablated dopamine and serotonin neurons were analyzed to demonstrate the sensitivity of the technique. The separation method developed in this study should offer an advantage in elucidating the critical role that electroactive biogenic amines play in complex physiological processes correlated with Drosophila behavior.     

Chromogenic sensing of biogenic amines using a chameleon probe and the red-green-blue readout of digital camera images

We report on sensing spots containing an amine reactive chromogenic probe and a green fluorescent (amine insensitive) reference dye incorporated in a hydrogel matrix on a solid support. Such spots enable rapid and direct determination of primary amines and, especially, biogenic amines (BA). A distinct color change from blue to red occurs on dipping the test spots into a pH 9.0 sample containing primary amines. BAs can be determined in the concentration range from 0.01 to 10 mM within 15 min, enabling rapid, qualitative, and semiquantitative evaluation. In the “photographic” approach, the typically 4-7.5-fold increase in fluorescence intensity of the probe at 620 nm along with the constant green fluorescence at 515 nm of a reference dye are used for quantitation of BAs. The sensing spots are photoexcited with high-power 505 nm light-emitting diodes (LEDs) in a black box. A digital picture is acquired with a commercially available digital camera, and the color information is extracted via red-green-blue (RGB) readout. The ratio of the intensities of the red (signal) channel and the green (reference) channel yields pseudocolor pictures and calibration plots.     

Heterobimetallic Ru(II)-Eu(III) complex as chemodosimeter for selective biogenic amine odorants detection in fish sample

Gaseous biogenic amines such as putrescine, spermidine, aniline, and trimethylamine are important biomolecules that play many crucial roles in metabolism and medical diagnostics. A chemodosimetric detection assay has been developed for those gaseous amines by Ru(II)-Eu(III) heterobimetallic complexes, K{[Ru(II)((t)Bubpy)(CN)(4)](2)Eu(III)(H(2)O)(4)} (where (t)Bubpy = 4,4'-di-tert-butyl-2,2'-bipyridine). Synthesis, X-ray crystal characterization, and spectroscopic properties of this Ru(II)-Eu(III) heterobimetallic complex were reported. Binding properties of the Ru(II)-Eu(III) complex with common gases revealed that this complex is very selective to gaseous amine molecules. Sensitivity of this complex toward the amines was found as ～log k() = 4.5-4.8. Real time monitoring of gaseous biogenic amines was applied to real fish samples (Atlantic mackerel) by studying the spectrofluorimetric responses of the Ru(II)-Eu(III) complex toward different biogenic amine concentration. GC/MS studies were also used as a reference for the studies. A linear spectrofluorimetric response was found toward biogenic amine concentration in real fish samples. This complex was found to respond specifically to those biogenic amines down to 10 ppb.     

Electrooxidation mechanism of biogenic amines at amine oxidase modified graphite electrode

Amine oxidase (AO, EC. 1.4.3.6) was previously shown to be a very efficient biological recognition element of amperometric biosensors for monitoring biogenic amines. The enzyme was effectively working in both mono- and bienzyme electrode designs, based on either a direct or a mediated electron-transfer pathway. This work focuses on the elucidation of the electron-transfer mechanism of the monoenzymatic unmediated AO-modified biosensor. The observed unmediated catalytic currents were assumed to be caused by (i) a direct electron-transfer process, (ii) the electrooxidation of the formed product, or (iii) their combination. Experiments supporting these assumptions are discussed in detail.     

Capillary electrophoretic determination of different classes of organic ions by potentiometric detection with coated-wire ion-selective electrodes

Singly charged amines and sulfonic acids as cationic and anionic aliphatic model substances, respectively, were detected in capillary electrophoresis with all-solid-state ion-selective electrodes. The sensitivity for these compounds is a function of their lipophilicity. The signal detected is generally greater for molecules with a larger organic part. The utility of the method is further demonstrated by the detection of a group of aromatic compounds in the form of the anionic analgesics (S)-(+)-2-(4-isobutylphenyl)propionic acid, 2-[(2,6-dichlorophenyl)amino]benzeneacetic acid, and o-acetylsalicylic acid. Further determined were the artificial sweeteners cyclamate, acesulfame-K, and saccharin. Detection limits for the different substances were between 2.5 × 10(-)(6) and 1 × 10(-)(5) M.     

In situ derivatization/solid-phase microextraction: determination of polar aromatic amines

A solid-phase microextraction GC/MS method for the trace determination of a wide variety of polar aromatic amines in aqueous samples was developed. Prior to extraction the analytes were derivatized directly in the aqueous solution by diazotation and subsequent iodination in a one-pot reaction. The derivatives were extracted by direct-SPME using a PDMS/DVB fiber and analyzed by GC/MS in the full-scan mode. By diazotation/iodination, the polarity of the analytes was significantly decreased and as a consequence extraction yields were dramatically improved. The derivatization proved to be suitable for strongly deactivated aromatic amines and even the very polar diamino compounds can efficiently be enriched after derivatization. We investigated 18 anilines comprising a wide range of functional groups, which could be determined simultaneously. The method was thoroughly validated, and the precision at a concentration of 0.5 microg/L was 3.8-11% relative standard deviation for nonnitrated analytes using aniline-d(5) as internal standard and 3.7-10% for nitroaromatic amines without internal standard. The in situ derivatization/SPME/GC/MS method was calibrated over the whole analytical procedure and was linear over 2 orders of magnitude. Using 10-mL samples, detection limits of 2-13 ng/L were achieved for 15 of the 18 analytes. For two aminodinitrotoluene isomers and a diaminonitrotoluene, detection limits ranged from 27 to 38 ng/L. By allowing quantification at the 0.1 microg/L level, analysis of all target compounds meets EU drinking water regulations. The method provides high sensitivity, robustness, and high sample throughput by automation. Finally, the method was applied to various real water samples and in wastewater from a former ammunition plant the contents of several aromatic amines were quantified.     

Analysis of biogenic amines in a single Drosophila larva brain by capillary electrophoresis with fast-scan cyclic voltammetry detection

Drosophila, the fruit fly, is a common model organism in biology; however, quantifying neurotransmitters in Drosophila is challenging because of the small size of the central nervous system (CNS). Here, we develop neurotransmitter quantification by capillary electrophoresis with fast-scan cyclic voltammetry (CE-FSCV) detection, which allows peak identification by both migration time and the cyclic voltammogram, in contrast to traditional amperometric detection which provides no chemical identification. Tissue content of biogenic amine neurotransmitters was determined in a single CNS dissected from a Drosophila larva. Low detection limits, 1 nM for dopamine and serotonin, 2.5 nM for tyramine, and 4 nM for octopamine, were achieved using field-amplified sample stacking by diluting the homogenized tissue with percholoric acid and acetonitrile. Two different strains of wild-type flies, Oregon R and Canton S, have similar dopamine and serotonin levels but different octopamine content. When flies are fed NSD-1015, which inhibits dopamine decarboxylase (Ddc) a synthesis enzyme in the dopamine and serotonin pathways, dopamine significantly decreases by 52%. A genetically altered driver line, Ddc-GAL4, had lower serotonin and dopamine content as did w(118) flies. When the Ddc-GAL4 line was used to produce flies overexpressing the serotonin synthesis enzyme tryptophan hydroxylase (Ddc-GAL4;UAS-Trh), the serotonin tissue content was greater than for Ddc-GAL4 but not significantly different than the wild-type. These results show that CE-FSCV is useful for monitoring the impact of genetic and pharmacological manipulations on the content of multiple neurotransmitters in a CNS from a Drosophila larva.     

3,3',5,5'-tetramethyl-N-(9-anthrylmethyl)benzidine: a dual-signaling fluorescent reagent for optical sensing of aliphatic aldehydes

A new optical chemical sensor for continuous monitoring of aliphatic aldehydes has been proposed based on the reversible chemical reaction between a new sensing reagent, 3,3',5,5'-tetramethyl-N-(9-anthrylmethyl)benzidine (TMAB), and the analytes. TMAB, containing two receptors and two fluorescent reporters, can perform dual fluorescence responses corresponding to the reactions of hydrogen ion and carbonyl compound. When immobilized in a plasticized poly(vinyl chloride) membrane, TMAB extracts aliphatic aldehydes from aqueous solution into the bulk membrane phase and reacts with the analyte by forming a Schiff base. Since the extraction equilibrium and chemical reaction are accompanied by fluorescence increase of the sensing membrane, the chemical recognition process could be directly translated into an optical signal. At pH 3.20, the sensor exhibits a dynamic detection range from 0.017 to 4.2 mM n-butyraldehyde with a limit of detection of 0.003 mM. The forward response time (t95) of the sensor is 3-5 min, and the reverse response time is 5-7 min. The responses of the sensor toward different kinds of aldehydes and ketones depend on the lipophilicity and the reactivity of the analytes. Since the fluorescence enhancement of the sensing membrane at 296 nm/410 nm is only related to the formation of Schiff base, the measurement of aldehydes is independent of pH.     

Gas chromatographic detection of volatile amines found in indoor air due to putrefactive degradation of casein-containing building materials

The use of non-sterile casein in building materials, especially in self-levelling floor-topping compound (putty), led to various defects in buildings with these materials. Bubbles in the mattings, dark patches unevenly spread over the floor and a very unpleasant odour developed. The defects were commonly induced or accelerated if the humidity in the floor was high. Our earlier studies have concluded that the cause of these problems is due to the growth of alkalitolerantClostridia. The presence of volatile mono-amines in air samples taken in the buildings also confirms our early theories. Due to the high pH of the putty, amines formed in the topping compound will easily be emitted into the air. Some of the amines are commonly found in spent growth media ofClostridia as well as in head-space samples from bacterial suspensions. By the use of gas chromatography (GC) we have been able to detect triethylamine, pyrrolidine, di-iso-butylamine, 2-ethyl-hexylamine and n-octylamine in the air samples. Air sampling was carried out by impinging in weak acidic solutions and adsorption on silica gel. Along with the mono-amines, ammonia has also been detected. The sampling was carried out using Draeger Tubes, which also non-specifically indicate several volatile amines in addition to ammonia. The amount of amines recovered was well below official threshold limits. The amount was in the parts per billion (ppb) region, whereas official threshold limits are usually in the 1–25 parts per million (ppm) region.     

食品生物胺安全控制关联的包装技术研究进展

目的 综述包装技术在食品生物胺安全控制领域的研究现状及应用进展,为新型高效的食品生物胺控制包装的研发提供借鉴与参考,助力食品安全国家战略的实施。方法 通过收集和整理相关文献,在概述食品中生物胺的形成机制和危害的基础上,探讨传统食品包装在控制生物胺形成方面的成效和局限性,重点总结近年来出现的新型生物胺控制包装技术(气调包装、抑脱羧酶活性包装及抑菌活性包装)及其研究进展,并对其未来发展方向进行展望。结论 食品包装可实现生物胺的安全控制,研发新型、高效的生物胺安全控制包装技术对于保障消费者舌尖上的安全具有重要的现实意义。     

Studies of interaction of amines with TOPO/TOP capped CdSe quantum dots: Role of crystallite size and oxidation potential

This work reports the interaction of aliphatic (triethyl amine, butyl amine) and aromatic amines (PPD, aniline) with CdSe quantum dots of varied sizes. The emission properties and lifetime values of CdSe quantum dots were found to be dependent on the oxidation potential of amines and crystallite sizes. Smaller CdSe quantum dots (size 5 nm) ensure better surface coverage of amines and hence higher quenching efficiency of amines could be realized as compared to larger CdSe quantum dots (size 14 nm). Heterogeneous quenching of amines due to the presence of accessible and inaccessible set of CdSe fluorophores is indicated. PPD owing to its lowest oxidation potential (0.26 V) has been found to have higher quenching efficiency as compared to other amines TEA and aniline having oxidation potentials 0.66 and >1.0 V, respectively. Butyl amine on the other hand, plays a dual role: its post-addition acts as a quencher for smaller and enhances emission for larger CdSe quantum dots, respectively. The beneficial effect of butyl amine in enhancing emission intensity could be attributed to enhance capping effect and better passivation of surface-traps.     

Simultaneous determination of biogenic monoamines in rat brain dialysates using capillary high-performance liquid chromatography with photoluminescence following electron transfer

Simultaneous determination of biogenic monoamines such as dopamine, serotonin, and 3-methoxytyramine in brain is important in understanding neurotransmitter activity. This study presents a sensitive determination of biogenic monoamines in rat brain striatum microdialysates using capillary high-performance liquid chromatography with the photoluminescence following electron-transfer detection technique. Separation conditions were optimized by changing the concentration of an ion-interaction agent and the percentage of an organic modifier. The high concentration of ion-interaction agent enabled the amines as a class to be separated from interfering acids, but also made the separation very long. To shorten the separation time, 10% (v/v) acetonitrile was used as the organic modifier. Eight chromatographic runs during a 3-h period were analyzed in terms of retention times, peak heights, and peak widths. Chromatograms are very reproducible, with less than 1% changes in peak height over 3 h. Typical concentration detection limits at the optimum separation conditions were less than 100 pM for metabolic acids and approximately 200 pM for monoamines. The injection volume of the sample was 500 nL. Thus, the mass detection limits were less than 50 amol for metabolic acids and approximately 100 amol for monoamines. Typical separation time was less than 10 min. To validate the technique, the separation method was applied to the observation of drug-induced changes of monoamine concentrations in rat brain microdialysis samples. Local perfusion of tetrodotoxin, a sodium channel blocker, into the striatum of an anesthetized rat decreased dopamine, 3-methoxytyramine, and serotonin concentrations in dialysates. Successive monitoring of striatal dialysates at a temporal resolution of 7.7 min showed that the injection of nomifensine transiently increased dopamine and 3-methoxytyramine concentrations in rat brain dialysate.