Gas chromatography-mass spectrometry assessment of amines in Port wine and grape juice after fast chloroformate extraction/derivatization

A simple, reliable, and sensitive gas chromatography-mass spectrometry method for the quantification of volatile and nonvolatile biogenic amines in Port wines and grape juices was developed and evaluated. The method was based on a previously reported two-phase derivatization procedure with isobutyl chloroformate in a toluene medium, which provides a quantitative reaction in 10 min. Following the derivatization step, the excess of reagent was eliminated by treatment with alkaline methanol. The derivatization procedure was performed directly on 1 mL of sample, avoiding any fastidious and time-consuming cleanup extraction steps. The method allows the simultaneous quantification of 22 amines, which can be found in wines: methylamine, dimethylamine, ethylamine, diethylamine, propylamine, isopropylamine, butylamine, isobutylamine, amylamine, isoamylamine, 2-methylbutylamine, hexylamine, pyrrolidine, piperidine, morpholine, 1,3-diaminopropane, putrescine, cadaverine, 1,6-diaminohexane, 2-phenylethylamine, histamine, and tyramine. Because of the fact that histamine and tyramine derivatives are degraded during the isobutyl chloroformate elimination step, the corresponding determination was made after removal of the excess of derivatizing reagent by evaporating an aliquot of the toluene layer obtained after the reaction. The presented method showed excellent analytical characteristics in what linearity, recovery, repeatability, and limit of detections were respected. It was used to assess the concentration of biogenic amines in juice grapes and Tawny and Vintage Port wines with different aging times. On the whole, the total content of amines in Port wines was low. Most of the amines found in wines have their origin in the raw material used for their elaboration, so the Port winemaking process is not prone to the production of this kind of compounds. Total biogenic amine contents have shown a decrease with the aging of both types of Port wines.     

Formation of amines and aldehydes from parent amino acids during thermal processing of cocoa and model systems: new insights into pathways of the strecker reaction

A method based on a derivatization with dansyl chloride and LC-MS-MS determination was developed for the quantitation of 2-methylbutyl-, 3-methylbutyl-, 2-phenylethyl-, 3-(methylthio)propyl-, and 2-methylpropylamine. Its application on unfermented, fermented, and roasted cocoas from Ghana and Sulawesi revealed an increase of all amines, except the 3-(methylthio)propylamine, during cocoa fermentation, suggesting an enzymic formation from the parent amino acids isoleucine, leucine, phenylalanine, and valine. However, a much more pronounced formation of most of the amines was measured after roasting of the cocoa, leading to concentrations in the milligrams per kilogram range. This result suggested a new "thermogenic" formation pathway of "biogenic amines". A comparison of the amounts of the amines and the aldehydes in roasted cocoa revealed similar concentrations, for example, for 2- and 3-methylbutanal and the respective amines, whereas the amounts of 2-phenylethylamine were much higher as compared to the amounts of phenylacetaldehyde. Strecker-type model systems, in which each parent amino acid was reacted with 2-oxopropanal, revealed the formation of both the amine and the aldehyde; however, in contrast to cocoa, the concentrations of the aldehydes were always much higher as compared to the amines. The results showed for the first time the thermally induced generation of "biogenic amines" from amino acids. Possible reasons for the different ratios of amines versus aldehydes formed during the roasting of cocoa or the model systems, respectively, are discussed.     

Principal component and linear discriminant analyses of free amino acids and biogenic amines in hungarian wines

Principal component analysis (PCA) and linear discriminant analysis (LDA) were used to classify 187 Hungarian white and red wines according to wine-making technology, geographic origin (wine-making region), grape variety, and year of vintage based on free amino acid and biogenic amine contents. Determination of free amino acids and biogenic amines was accomplished by ion-exchange chromatography. Six principal components accounted for >77% of the total variance in the data. The plots of component loadings showed significant groupings of free amino acids and biogenic amines. The component scores grouped according to wines made by different wine-making technologies. Using LDA the variables with a major discriminant capacity were determined. Almost complete classification (94.7%) was achieved concerning both white and red wines and wines made by different wine-making technologies. The results of differentiation between white wines according to geographic origin, grape variety, and year of vintage were 70.8, 62.4, and 73.5%, respectively. The same numbers for red wines according to geographic origin, grape variety, and year of vintage were 64.9, 71.6, and 82.4%, respectively.     

Quantitation of Amines in Cereal Products: Thermal Processes Are Able to Generate “Biogenic” Amines

On the basis of a derivatization with dansyl chloride prior to LC‐MS/MS measurement, a new quantitation method by means of stable isotope dilution analysis was developed for different amines, such as 2‐ and 3‐methylbutylamine, methylpropylamine, and 2‐phenylethylamine. The method was applied both to different cereal products and to a model system, revealing that the amines stemming from the parent amino acids l ‐isoleucine, l ‐leucine, l ‐valine, and l ‐phenylalanine can also be generated by a “thermogenic” pathway besides the well‐established enzymatic formation. A model system simulating the Strecker reaction by heating single amino acids with an α‐dicarbonyl compound (2‐oxopropanal) showed besides the formation of the well‐known desired aroma‐active Strecker aldehydes also the generation of the corresponding Strecker amines, which are undesired in food due to their negative physiological impacts on human health. Thus, a thermal formation of “biogenic” amines was shown, both in cereal‐based foods and in a model system. A contribution of these amines to the overall food aroma is also possible. With this knowledge at hand, mitigation strategies for the formation of amines undesired for health reasons in parallel with a favored generation of the corresponding aroma‐active aldehydes, depending on the processing parameters, can be suggested to food manufacturers.     

HPLC method for analysis of free amino acids in fish using o-phthaldialdehyde precolumn derivatization

Precolumn derivatization applying o-phthaldialdehyde (OPA) was used to analyze free lysine, histidine, and ornithine, precursors of the respective biogenic amines cadaverine, histamine, and putrescine, which are considered indicators of fish quality and safety. This method uses 75% methanol to eliminate the use of strong acids as the extraction solution. Each analysis took 35 min, was reproducible, and allowed separation of primary amino acids in fish samples. A binary solvent delivery system coupled with a fluorescence detector and an Ultrasphere ODS column were utilized for HPLC separation. Linearity of the calibration curves was very good (r(2) = 0.99) for the amino acids of interest. Minimum concentrations of detection were 40 pmol/mL for histidine and lysine and 70 pmol/mL for ornithine. Average recoveries were 72% for lysine, 93% for histidine, and 98% for ornithine. This method used solvent gradient elution to study the levels of these analytes in mahi-mahi, bigeye tuna, and flounder.     

Determination of biogenic amines in squid and white prawn by high-performance liquid chromatography with postcolumn derivatization

A simple method was developed for the determination of biogenic amines in aquatic food products using a reverse-phase high-performance liquid chromatography with postcolumn automatic o-phthalaldehyde derivatization and fluorescence detection. The linearity, repeatability, and recovery of the method for seven amines (tyramine, putrescine, cadaverine, histamine, agmatine, spermidine, and spermine) were evaluated. This optimized method was applied to detect biogenic amines in squid and white prawn during refrigerated storage. Sensory analysis, pH value, and total volatile base nitrogen value were combined to evaluate the freshness quality of these two raw materials. Agmatine and cadaverine in squid, cadaverine, and putrescine in white prawn were the most obviously changed amines during the storage at two different temperatures, and these biogenic amines could be the effective quality indicators for the freshness of the raw aquatic materials.     

Determination of free amino compounds in betalainic fruits and vegetables by gas chromatography with flame ionization and mass spectrometric detection

Amino acids and amines are the precursors of betalains. Therefore, the profiles of free amino compounds in juices obtained from cactus pears [Opuntia ficus-indica (L.) Mill. cv. Bianca, cv. Gialla, and cv. Rossa], pitaya fruits [Selenicereus megalanthus (K. Schumann ex Vaupel) Moran, Hylocereus polyrhizus (Weber) Britton & Rose, and Hylocereus undatus (Haworth) Britton & Rose], and in extracts from differently colored Swiss chard [Beta vulgaris L. ssp. cicla (L.) Alef. cv. Bright Lights] petioles and red and yellow beets (B. vulgaris L. ssp. vulgaris var. conditiva Alef. cv. Burpee's Golden) were investigated for the first time. Amino compounds were derivatized with propyl chloroformate. While gas chromatography (GC) with mass spectrometry was used for peak assignment, GC flame ionization detection was applied for quantification of individual compounds. Whereas proline was the major free amino compound of cactus pear and pitaya fruit juices, glutamine dominated in Swiss chard stems and beets, respectively. Interestingly, extremely high concentrations of dopamine were detected in Swiss chard stems and beets. Furthermore, the cleavage of betaxanthins caused by derivatization in alkaline reaction solutions is demonstrated for the first time. Amino acids and amines thus released might increase the actual free amino compound contents of the respective sample. To evaluate the contribution of betaxanthin cleavage to total amino acid and amine concentration, isolated betaxanthins were derivatized according to the "EZ:faast" method prior to quantification of the respective amino compounds released. On a molar basis, betaxanthin contribution to overall amino compound contents was always below 6.4%.     

High-performance liquid chromatographic method for determination of biogenic amines in feedstuffs, complete feeds, and animal tissues

Numerous methods to analyze biogenic amines in biological materials have been described. A versatile and rapid methodology to analyze these compounds in feedstuffs, complete feeds, and animal tissues, however, has not been reported. The current method was developed to address this need. Biogenic amines in feedstuffs, complete animal feeds, and animal tissues were extracted with 10% trichloroacetic acid, reacted with O-phthaladehyde using high-performance liquid chromatographic employing a cation exchange column. Detection limits were 50 pmol/mL for tyramine, histamine, putrescine, and spermine; 40 pmol/mL for cadaverine; and 25 pmol/mL for spermidine. Extraction efficiency of biogenic amines in feedstuffs, duodenum, liver, ileum + jejunum, and whole shrimp and shrimp hepatopancreas ranged between 99-105, 93-135, 80-85, 65-102, 88-98, and 88-97%, respectively. It can be concluded that the current method can be applied to individual feedstuffs, complete feeds, and animal tissues for the rapid and accurate determination of concentration of biogenic amines.     

以生物胺变化评价冷藏罗非鱼片腐败进程

为了探讨生物胺指标评价鱼肉腐败进程的可能性及其安全范围,应用反相高效液相色谱法测定冷藏罗非鱼片贮藏过程的生物胺变化,用以判断鱼片的腐败进程。结果表明:新鲜鱼片初始单胺和多胺总量较高,二胺总量很低。贮藏过程中单胺和多胺波动变化并呈下降趋势,尸胺、腐胺含量快速增加成为主要生物胺。含有尸胺+腐胺的二胺、生物胺指数、总生物胺等指标与其两者增长趋势相似。相关分析表明尸胺、腐胺、二胺、生物胺指数、总生物胺与贮藏时间、微生物数量和氨基态氮具有高度相关性。经回归分析表明假单胞菌、肠杆菌的增长与尸胺、腐胺具有重要的对应关系。尸胺、腐胺和二胺可简便有效评价鱼片腐败进程;但综合考虑组胺和酪胺的毒性,则生物胺指数(BAI)更适用,冷藏罗非鱼片生物胺指数的初步判别范围为:<20mg/kg,新鲜;20～40mg/kg,可接受;>40mg/kg,腐败。该研究为冷藏罗非鱼片生物胺的限量标准提供数据参考。     

Determination of ammonium,amides, and soluble carboxylates in plant tissues

Methods for extraction and determination of ammonium, amides and soluble carboxylates in plant tissues are compared and discussed. The procedure recommended involves extraction of finely ground plant tissues with 0.2 M formic acid, determination of ammonium and amides in the extract by overnight alkaline hydrolysis and distillation in Conway microdiffusion dishes, measurement of extracted ammonium using Carlson's rapid diffusion‐conductivity instrument, and estimation of soluble organic acids by titration after separation and purification with ion exchange resins. The method described is reasonably rapid, and gives complete recovery and highly reproducible results. The final solution of organic acids is suitable for direct chromatographic analysis.     

降解亚硝酸盐和生物胺乳杆菌筛选及其改善鱼肉香肠品质效果

为了筛选出优良亚硝酸盐和生物胺的降解植物乳杆菌,并研究在鱼肉发酵香肠中的应用。该文主要考察了19 株植物乳杆菌亚硝酸盐和生物胺的降解活性及相关基因的携带情况,从中筛选出优良菌株,并研究其对鱼肉香肠发酵过程中各种亚硝胺、生物胺、腐败微生物的抑制作用和对其他理化指标的影响。试验结果表明,L. plantarum CP3对亚硝酸盐和生物胺的降解能力最强,分别达到95%和77%,PCR结果显示此菌株中含有亚硝酸盐降解（假定蛋白（hypothetical protein,HP）、谷胱甘肽还原酶（glutathione reductase,GR）、一氧化还原酶（Oxidoreductase,ORD））和生物胺降解的相关基因（multicopper oxidases,suf I）,且不含生物胺生成相关基因（hdc,tdc,odc）。GC-MS测定结果显示L. plantarum CP3对发酵香肠中4种亚硝胺-二乙基亚硝胺（N-nitrosodiethylamine,NDEA）,二甲基亚硝胺（N-nitrosodimethylamine,NDMA）,亚硝基吗啉（N-nitrosomorpholine,NMOR）和甲基乙基亚硝胺（N-nitrosomethylamine,NMEA）的生成都具有显著的抑制作用,联合接种L.saki M4可以协同提高亚硝胺的降解能力。并且,与对照组相比,接种L. plantarum CP3+L.saki M4的鱼肉香肠,发酵48 h后腐胺、尸胺和酪胺的含量分别降低了76.83%,93.33%和88%。另外,接种乳酸菌处理组香肠中的肠杆菌、假单胞菌等腐败菌及pH值、硫代巴比妥酸（thiobarbituric acid,TBA）和挥发性盐基氮（total volatile basic nitrogen,TVB-N）的含量显著低于对照组。该研究结果体现了L. plantarum CP3作为香肠等肉制品发酵剂的良好开发应用前景。     

Chemical, microbiological, and AromaScan evaluation of mahi-mahi fillets under various storage conditions

The quality for mahi-mahi stored at 1.7, 7.2, and 12.8 degrees C for 0, 1, 3, and 5 days was determined using biogenic amine analysis, microbial counts, and sensory evaluation (by a sensory test panel and an AromaScan). Biogenic amines in methanol extracts from mahi-mahi samples were analyzed using capillary electrophoresis (CE) with ultraviolet detection at 210 nm and a gas chromatography (GC) method that can simultaneously determine the contents of putrescine, cadaverine, histamine, spermidine, and spermine within 20 min after pentafluoropropionic anhydride derivatization. A good correlation R2= 0.99) was found between CE and GC methods for detecting histamine in mahi-mahi. Fish quality deteriorated and correlated with increasing microbial numbers. Biogenic amines may be useful indicators for mahi-mahi quality and safety. AromaScan was able to correlate quality changes for mahi-mahi in microbiological and sensory analyses.     

Determination of theanine, GABA, and other amino acids in green, oolong, black, and Pu-erh teas with dabsylation and high-performance liquid chromatography

Dabsyl chloride (dimethylaminoazobenzene sulfonyl chloride), a useful chromophoric labeling reagent for amino acids and amines, was developed in this laboratory in 1975. Although several methods have been developed to determine various types of amino acids, a quick and easy method of determining theanine, GABA, and other amino acids has not been developed in one HPLC system. In this paper are analyzed the free amino acid contents of theanine and GABA in different teas (green tea, black tea, oolong tea, Pu-erh tea, and GABA tea) with a dabsylation and reverse phase high-performance liquid chromatography (HPLC) system coupled with a detector at 425 nm absorbance. Two reverse phase columns, Hypersil GOLD and Zorbax ODS, were used and gave different resolutions of dabsyl amino acids in the gradient elution program. The data suggest that the tea source or the steps of tea-making may contribute to the theanine contents variations. High theanine contents of high-mountain tea were observed in both green tea and oolong tea. Furthermore, the raw (natural fermented) Pu-erh tea contained more theanine than ripe (wet fermented) Pu-erh tea, and the GABA contents in normal teas were generally lower than that in GABA tea.     

Development of stable isotope dilution assays for the simultaneous quantitation of biogenic amines and polyamines in foods by LC-MS/MS

Microbial amino acid metabolism may lead to substantial amounts of biogenic amines in either spontaneously fermented or spoiled foods. For products manufactured with starter cultures, it has been suggested that certain strains may produce higher amounts of such amines than others; however, to support efforts of food manufacturers in mitigating amine formation, reliable methods for amine quantitation are needed. Using 10 isotopically labeled biogenic amines as the internal standards, stable isotope dilution assays were developed for the quantitation of 12 biogenic amines and of the 2 polyamines, spermine and spermidine, in one LC-MS/MS run. Application of the method to several foods revealed high concentrations of, for example, tyramine and putrescine in salami and fermented cabbage, whereas histamine was highest in Parmesan cheese and fermented cabbage. On the other hand, ethanolamine was highest in red wine and Parmesan cheese. The results suggest that different amino acid decarboxylases are active in the respective foods depending on the microorganisms present. The polyamine spermine was highest in salami and tuna.     

Fluorometric determination of nitrite in cured meats.

An indirect fluorometric method for determining sodium nitrite in meat products is presented. The extracted sodium nitrite is consumed in a diazotization reaction with a measured excess of sulfanilic acid. Fluorescamine, which acts selectively with primary amines such as sulfanilic acid, is a fluorogenic reagent for the excess amine. The amine consumed, calculated by difference from the total originally present, is directly related to the sodium nitrite content of the sample. Interferences from amino acids and soluble proteins in the meat extract are eliminated by judicious use of a secondary peak in the fluorescence spectra (436 nm excitation, 495 nm fluorescence) combined with measurement at low pH (3.30). The recoveries of sodium nitrite ranged from 83.2 to 99.6% with an average of 93.4 and a standard deviation of +/- 5.28% for 11 determinations.     

Free amino acid and cysteine sulfoxide composition of 11 garlic (Allium sativum L.) cultivars by gas chromatography with flame ionization and mass selective detection

Two garlic subspecies (n = 11), Allium sativum L. var. opioscorodon (hardneck) and Allium sativum L. var. sativum (softneck), were evaluated for their free amino acid composition. The free amino acid content of garlic samples analyzed ranged from 1121.7 to 3106.1 mg/100 g of fresh weight (mean = 2130.7 +/- 681.5 mg/100 g). Hardneck garlic had greater methiin, alliin, and total free amino acids contents compared to softneck garlic. The major free amino acid present in all but one subspecies was glutamine (cv. Mother of Pearl had aspartic acid as the major free amino acid). Cv. Music Pink garlic (a rocambole hardneck variety) contained the most methiin, alliin, and total free amino acids. The solid-phase extraction, alkylchloroformate derivatization, GC-FID, and GC-MS methods used in this study were simple and rapid, allowing 18 free amino acids in garlic to be separated within 10 min.     

Ion-exchange separation of amino acids with postcolumn orthophthalaldehyde detection

Moore's and Stein's classical ion-exchange separation of amino acids remains the standard by which all methods are judged. The adaptation of liquid chromatography (LC) equipment to amino acid analysis was inevitable because microprocessor control of gradients allowed almost infinite variation in gradient shape, producing superior resolution with only 2 buffers. The versatility of LC equipment allowed the instruments to be used for other assays. Adaptation of orthophthalaldehyde (OPA) to amino acid analysis increased detection sensitivity to the picomole range. A method for essential amino acids analysis in mechanically separated red meat and poultry products has been adapted to liquid chromatography using postcolumn hypochlorite oxidation, OPA derivatization, and fluorescence detection. Separation is achieved with 2 sequential concave exponential gradients combining ionic strength and pH increases with halide-containing buffers. Hydroxyproline and proline are detected with increasing sensitivity through the use of 3-mercaptopropionic acid in a stabilized OPA reagent. Sample preparation is a critical part of the method. A defatting procedure removes fat and other nonprotein nitrogenous substances. The hydrolysis procedure is designed to protect tryptophan which can be routinely assayed in hydrolysates with a modified flow program. Corrosion damage to the equipment by halide buffers has brought about a search for alternative methodology.     

Optimized procedures for analyzing primary alkylamines in wines by pentafluorobenzaldehyde derivatization and GC-MS

Biogenic primary alkylamines in wines are toxicologically significant and affect sensory properties. An optimized method for analysis in wines involving derivatization with pentafluorobenzaldehyde (PFB) to corresponding pentafluorobenzylimines, liquid-liquid extraction, and gas chromatography with mass selective detection is presented. Reaction parameters including pH, temperature, time, and derivatizing agent and amine concentration were varied in simulated wine solution (15% ethanol) to determine effect on reaction efficiency. Optimal reaction efficiency was characterized (pH 12, 24 degrees C, 30 min, and 10 mg/mL PFB), and parameters were used for the analysis of 10 biogenic alkylamines in 12 California wines. Alkylamine concentration in wines ranged from 0.048 to 91 mg/L. Amine recoveries from wines at five fortification levels (0.1-85 mg/L) were generally 81-100%.     

High-performance liquid chromatographic determination of biogenic amines in poultry carcasses

Biogenic amines, produced by bacterial decarboxylation of amino acids, have been associated with toxicological symptoms in broilers fed various poultry byproducts. A reversed-phase high-performance liquid chromatographic method is described for the quantitation of eight biogenic amines (tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine) in chicken carcasses. Amines were extracted with perchloric acid, derivatized with dansyl chloride, separated using gradient elution (methanol and water), and detected by fluorescence. Benzylamine was used as the internal standard. Linearity, repeatability, and recovery of the method were evaluated. The method was linear for all of the amines studied at concentrations ranging from 0.05 to 25 microg/mL. Average recoveries ranged from 92.6% to 96.8% for all amines except for histamine, which was 74.6%.     

Monitoring of the fermentation media of citric acid by the trimethylsilyl derivatives of the organic acids formed

In this approach, a derivatization method is described for monitoring of organic acids in fermentation media without any separation step. The aqueous phase of fermentation media was evaporated and heated in a silylation reagent to form trimethylsilyl (TMS) derivatives. The silylated compounds are analyzed by 29Si nuclear magnetic resonance (29Si NMR) and gas chromatography-mass spectrometry (GC-MS). 29Si NMR can qualitatively monitor the components produced in the Krebs cycle. Quantification of these compounds is investigated by using selected ion monitoring mode of mass spectrometry. In this mode, mass to charge (m/z) values of their [M - 15]+ ions, which are 465, 275, 247, 221, 335, 251, and 313 of TMS derivatives of citric, alpha-ketoglutaric, succinic, fumaric, l-malic, oxaloacetic, and palmitic (as an internal standard), acids, respectively, are used. The limit of detection and the linear working range for derivatized citric acid were found to be 0.1 mg L(-1) and 10-3 x 10(4) mg L(-1). The relative standard deviation of the method for five replicates was 2.1%. The average recovery efficiency for citric acid added to culture media was approximately 97.2%. Quantitative results of GC-MS are compared with those obtained by an ultraviolet-visible method.