Determination of biogenic amines in cheese

A liquid chromatographic (LC) procedure for determining 10 biogenic amines in cheese is described. The method is based on ion-pair chromatography on a reversed-phase column with postcolumn derivatization with o-phthaldialdehyde and fluorometric detection. It allowed simultaneous determination of 10 amines in < 80 min: histamine, tyramine, tryptamine, 2-phenylethylamine, serotonin, agmatine, spermine, spermidine, putrescine, and cadaverine. Linearity for each amine was observed between 0.5 and 6.0 micrograms/mL. Detection limits ranged form 0.004 to 0.009 micrograms/20 microL, and determination limits ranged from 0.066 to 0.149 mg/100 g. Amino acids and other amines did not interfere with determination of biogenic amines. Three extractants--methanol, hydrochloric acid, and trichloroacetic acid--were compared in their efficiency to recover amines from spiked samples. Purification of the cheese extract was required prior to LC to avoid interference from compounds in the cheese matrix. Hydrochloric acid extraction followed by purification with diethyl ether gave best recoveries for all the amines (75.5-112.3%). The method is simple, fast, and reliable. It can be used to study the technological and toxicological implications of biogenic amines in cheeses.     

Structure of the gene encoding the ubiquitin-conjugating enzyme Ubcm4, characterization of its promoter, and chromosomal location

An improved method for the simultaneous determination of underivatized biogenic amines, cadaverine, putrescine, spermidine, histamine, tyramine and some amino acids precursors, histidine and tyrosine, in food products, based on ion-exchange chromatography (IC) with integrated pulsed amperometric detection (IPAD) has been developed. The method was successfully used for the analysis of biogenic amines and amino acids in food both of vegetable (kiwi, Actinidia chinensis) and animal origin, (fish, pilchard), as well as in fermented foods, such as cheese (Emmenthal) and dry sausages (salami). The method was also successfully used to study the changes in biogenic amines during the ripening of dry fermented sausages (salami). The analytes were extracted from foods with perchloric acid and the extracts were purified by liquid-liquid partition using n-hexane. Determination of biogenic amines was performed through cation-exchange chromatography with isocratic elution and IPAD. The detection limits for the analytes under investigation were found to range from 1.25 to 2.50 ng, at a signal-to-noise ratio of 3:1. Average recoveries ranged from 85.5 to 97.4% and R.S.D. values ranged from 3.4 to 8.8. The proposed method offers a number of advantages over our previous IPAD method, such as the application to a larger number of analytes and matrices, a simpler extraction procedure and clean-up, isocratic elution using low acid and base concentrations, an improved chromatographic separation and a lower detection limit.     

Polyamine metabolism in the thermotolerant mesophilic fungus Aspergillus fumigatus

Biomass production by Aspergillus fumigatus was greatest at 40-45 degrees C and was associated with an increase in concentration of the diamine putrescine and activity of its biosynthetic enzyme ornithine decarboxylase. Concentrations of the other amines, cadaverine, spermidine and spermine were considerably lower than putrescine concentration and did not change significantly over the temperature range 20-50 degrees C. This is surprising in view of the greatly increased flux of label from ornithine through to spermidine at 45 and 50 degrees C, indicating an increased formation of this triamine. It is suggested that there was increased formation of spermidine derivatives at these temperatures. Interestingly, there was greatly increased formation of the higher homologues of cadaverine, aminopropylcadaverine and N,N'-bis(3-aminopropyl)cadaverine, in A. fumigatus at 45 and 50 degrees C.     

Selective determination of secondary amines as their N-diethylthiophosphoryl derivatives by gas chromatography with flame photometric detection

A selective and sensitive method has been developed for the determination of secondary amines by gas chromatography (GC). After removal of primary amines by the reaction with o-phthaldialdehyde, secondary amines were converted into their N-diethylthiophosphoryl derivatives and then measured by GC with flame photometric detection using a DB-1701 capillary column. The derivatives were sufficiently volatile and stable to give single symmetrical peaks. The detection limits of secondary amines were ca. 0.05-0.2 pmol per injection. N-Methylcyclohexylamine was used as an internal standard. The calibration curves for secondary amines in the range 1-20 nmol were linear and sufficiently reproducible for quantitative determination. This method was successfully applied to small urine samples without prior clean-up. Overall recoveries of secondary amines added to urine samples were 91-105%. By using this method, secondary amines in urine samples could be analysed without any influence from primary amines and other coexisting substances. The analytical results of secondary amine content in urine samples of normal subjects are presented.     

Binding properties of (+/-)[3H]benidipine hydrochloride to rat heart membranes

A simple routine method for the gas chromatographic determination of methylamine, dimethylamine, ethylamine and methylethylamine in urine is presented. The method is based on a two-phase derivatization procedure with isobutyl chloroformate as reagent. The reaction is quantitative in 10 min. We found no artifact formation of either choline or trimethylamine (dietary amine compounds) or of dimethylethylamine or triethylamine (catalyst amines in the industrial setting). The chromatographic behaviour of the amine carbamates was excellent. The recoveries of methylamine, dimethylamine, ethylamine and methylethylamine in spiked urine samples were 82, 89, 100 and 96%, respectively, and the precision (the relative standard deviation) was 3.6, 1.8, 3.3 and 2.0%, respectively. The method was linear for the studied amine carbamates up to 250 mg/l. The endogenous amine concentrations in urine samples from ten normal subjects were: methylamine, 0.9 mg/l (mean; range 0.3-1.5); dimethylamine, 14.7 mg/l (mean; range 4.6-27.6); ethylamine, 0.8 mg/l (mean; range 0.2-2.3); methylethylamine, less than 0.02 mg/l.     

HPLC-based method for determination of absolute configuration of alpha-chiral amines

We introduce a novel, HPLC-based method for facile determination of the absolute configuration of alpha-chiral amines. Our method is easily applied to a variety of compounds, including amino acid derivatives. The method involves initial derivatization of the chiral amine analyte with the chiral derivatizing reagent, N-succinimidyl alpha-methoxyphenylacetate (SMPA), to produce the corresponding diastereomeric adducts. Inspection of a particular rotomer of the SMPA adduct and application of simple rules correlates absolute configuration and HPLC elution order. A key aspect of our method is that it can be used to determine absolute configuration without using enantiomeric standards of the amine analytes. Furthermore, it is of utmost significance that our method can also be used to determine absolute configuration even when only one analyte enantiomer of unknown absolute configuration is present, as is often the case for enzymatic products, naturally derived compounds, or enantiomerically enriched compounds prepared via chiral syntheses. We have observed strict adherence between predicted and observed absolute configuration for a wide variety of alpha-chiral amines. The chromatographic method we present in this paper is very practical and has several important advantages over NMR-based approaches which have been previously developed. For example, microgram quantities of an analyte in a complex enzymatic mixture can be directly analyzed by our HPLC-based method while the impurities often preclude definitive proton assignments in the NMR approach.     

Participation of cathepsins B and D in apoptosis of PC12 cells following serum deprivation

Biogenic amines in spoiled animal by-product feeds have been implicated in causing poor performance and intestinal lesions in broilers. This study was designed to determine if biogenic amines, at the concentrations found in animal by-product meals, would reduce performance in broilers or cause lesions. Twelve treatments were used in a 2 x 6 factorial arrangement with the main effects being either a corn-soybean meal diet or a corn-soybean meal diet with 10% animal by-products added and either no amines added or added levels of phenylethylamine (4.8 mg/kg), putrescine (49 mg/kg), cadaverine (107 mg/kg), histamine (131 mg/kg), or a combination of all these amines. Levels of biogenic amines used in this study simulated those found in areas with reported problems attributed to biogenic amines. Broilers were monitored for performance, gross lesions, and histologic evidence of lesions at 2, 4, and 6 wk. No consistent effects were observed on performance, and by the conclusion of the trial, no statistical differences were noted in the performance of any of the treatments. No gross lesions were observed on a consistent basis in any of the treatments. Histopathology was likewise unremarkable. On the basis of this study, it would appear that these four biogenic amines, at levels detected in the United States, do not pose a serious health concern for the broiler industry.     

Determination of total estrogen in urine with 3-methyl-2-benzothiazolinone hydrazone

The determination of total estrogens in urine with 3-methyl-2-benzothiazolinone hydrazone (MBTH) is reported. The method utilizes a colorimetric reaction consisting of the oxidative coupling of MBTH to the phenol portion of the steroid molecule. Colorimetric measurements were made at 530 nm manually or with an AutoAnalyzer at an analysis rate of 50 samples/hour. Comparison with a fluorometric method gave a correlation coefficient of .96 and with a gas chromatographic method, a coefficient of .94. Therefore this proposed method may be suitable as an alternative method for the determination of total estrogens in pregnancy urine samples.     

Simultaneous determination of the binary mixture of nifedipine and mefruside using derivate spectroscopy, capillary gas-liquid chromatography and high performance liquid chromatography

Accurate, precise first-derivative ultraviolet spectrophotometric, gas-liquid and high performance liquid chromatographic methods for the determination of nifedipine and mefruside in tablet dosage form were described. The first-derivative method depends on measurements of the derivative amplitudes by peak-to-base and zero-crossing techniques, at 390 and 282 nm, for the determination of nifedipine and mefruside, respectively. Calibration graphs were linear (r = 0.9999) ranging from 8-40 and 20-60 micrograms ml-1 for nifedipine and mefruside, respectively, in presence of one another. The gas-liquid chromatographic method (GLC) was based on using cross-linked methylsilicone gum capillary column with flame ionization detector for the determination of nifedipine and mefruside in the binary mixture. The high performance liquid chromatographic (HPLC) method was based on using a reverse-phase column with a mobile phase of methanol-water (55-45, pH = 4.5) with UV detection at 260 nm. The three methods showed good linearity, precision and reproducibility. The proposed methods were successfully applied to the determination of both drugs in pharmaceutical tablets.     

A case of Alstr?m syndrome associated with diabetes insipidus

The production of biogenic amines by 50 poultry-associated bacterial strains (25 Pseudomonas, 13 Salmonella and 12 Listeria) was investigated on amine agar plates containing lysine, histidine, ornithine, phenylalanine, tryptophan and tyrosine. Seventy-four per cent of all the strains produced cadaverine and putrescine, while phenylethylamine, histamine, tyramine and tryptamine were produced by 72, 56, 34 and 24% of strains, respectively. Different patterns of biogenic amine production amongst the three bacterial genera tested were apparent as well as amongst strains of the same genus. This study highlighted a high incidence of biogenic amine-producing bacterial strains associated with poultry.     

Determination of dimethindene in human tears by high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the determination of dimethindene in human tears. The tear samples were diluted in a 0.01 M hydrochloric acid-n-propanol mixture to prevent the irreversible adsorption of dimethindene. The diluted samples were directly injected into the chromatographic system to avoid sample pretreatment. The validation data demonstrate that the method is specific, precise and accurate within the calibration range of 12 to 1000 ng/ml dimethindene free base.     

Direct determination of biogenic amines in wine by integrating continuous flow clean-up and capillary electrophoresis with indirect UV detection

A flow-injection manifold for automating the determination of biogenic amines in wine using capillary electrophoresis (CE) with indirect UV detection was developed. The ensuing method involves clean-up and solid-phase extraction (SPE) of the target analytes in the sample. Various treatments involving different SPE minicolumns were tested and compared. The C18 minicolumn was chosen to concentrate the amines following addition of ammonium chloride and ammonium hydroxide as buffer to neutralize them. Additions of amine standards were used to determine recoveries. Biogenic amines can be separated and detected after SPE with limits of detection in the range 0.05-0.1 microgram ml-1 by using 4 mM copper(II) sulphate, formic acid and 18-crown-6 as running buffer. All the amines studied are eluted within 15 min under the optimum conditions established. The overall process was successfully used to identify biogenic amines in various types of wine from different Spanish regions.     

Improved column-switching liquid chromatographic method for the determination of the enantiomers of mefloquine

A liquid chromatographic method for the determination of the enantiomers of mefloquine has been improved. The chromatography involved two columns: an achiral cyanopropyl stationary phase for the quantification of (+/-)-mefloquine and a chiral naphthyl-urea stationary phase for the determination of the enantiomeric ratio. Compared with the previous method, which needed two detectors, this one used one detector-integrator to which the two columns are connected alternately by an automated column-switching system. The method is suitable for the quantification (0.05 microgram/ml) of mefloquine and the determination of enantiomeric ratios from 500-microliters plasma samples with ultraviolet detection.     

纸上色层分离-重量法测定铝铌合金中铌

铝铌合金中铌的含量对铝铌合金的性能和应用有直接影响。使用纸上色层分离-重量法测定铌时,铌与其他元素的分离效果受层析剂配比和环境的影响较大。实验通过考察称样量、层析剂配比、层析时间、共存元素的影响等,最终确定了最佳的试验条件,实现了铝铌合金中铌含量的准确测定。试样经氢氟酸和硝酸溶解,将浓缩后的溶液涂在色层纸上,利用铌与铝及其他共存元素在特定的层析剂中迁移速率的不同,实现铌与其他共存元素的分离;将含有铌的色层纸在900℃马弗炉中灼烧至恒量。对于铌质量分数为50%~80%的铝铌合金,称样量选择0.10g,层析剂中4-甲基-2-戊酮、丁酮、氢氟酸、硝酸体积比为125∶42∶32∶7,层析时间为6h时分离效果最佳,试样中共存的铁、钼、硅、镍、铬等微量元素对测定结果无影响,若样品中钽的质量分数大于0.01%,需从粗氧化铌沉淀中减去氧化钽的质量。实验方法用于3种铝铌合金样品中铌的测定,结果的相对标准偏差(RSD,n=11)为0.18%~0.29%;加标回收率为98%~102%。选择4家实验室协同验证,经科克伦检验测试结果无明显差异。     

纸上色层分离-重量法测定铝铌合金中铌

铝铌合金中铌的含量对铝铌合金的性能和应用有直接影响。使用纸上色层分离-重量法测定铌时,铌与其他元素的分离效果受层析剂配比和环境的影响较大。实验通过考察称样量、层析剂配比、层析时间、共存元素的影响等,最终确定了最佳的试验条件,实现了铝铌合金中铌含量的准确测定。试样经氢氟酸和硝酸溶解,将浓缩后的溶液涂在色层纸上,利用铌与铝及其他共存元素在特定的层析剂中迁移速率的不同,实现铌与其他共存元素的分离;将含有铌的色层纸在900℃马弗炉中灼烧至恒量。对于铌质量分数为50%~80%的铝铌合金,称样量选择0.10g,层析剂中4-甲基-2-戊酮、丁酮、氢氟酸、硝酸体积比为125∶42∶32∶7,层析时间为6h时分离效果最佳,试样中共存的铁、钼、硅、镍、铬等微量元素对测定结果无影响,若样品中钽的质量分数大于0.01%,需从粗氧化铌沉淀中减去氧化钽的质量。实验方法用于3种铝铌合金样品中铌的测定,结果的相对标准偏差(RSD,n=11)为0.18%~0.29%;加标回收率为98%~102%。选择4家实验室协同验证,经科克伦检验测试结果无明显差异。     

The effect of different amines added to eluents as silanol masking agents on the chromatographic behavior of some diuretics in reversed-phase high-performance liquid chromatography using C18 packings

A preliminary gradient separation in reversed-phase liquid chromatography of a mixture of 25 solutes (diuretics, probenecide, and atenolol) is carried out using several C18 columns and an aqueous phosphoric acid solution (pH 3.2)-acetonitrile mobile phase as a control. Using this separation, the chromatographic behavior of these solutes is studied using 11 water-soluble primary, secondary, and tertiary amine modifiers in the range of 0.7-7.5 mM and a Spherisorb C18 column. This study reveals the presence in the complex sample of two groups of solutes with positive (five typical solutes showed improvements in peak symmetry and retention) or negative responses using these amines as mobile phase modifiers. After experimentation in the presence of amines, these differences are related to solute structure. Hexylamine is found to be an effective masking agent of silanols because of its structure and small required concentration. On these bases, the silanophilic and hydrophobic character of typical solutes and several C18 packings are evaluated under isocratic elution and a relative effectiveness index for amines, and a method for their assessment is proposed. The role of the amine structure on solute retention and the importance of selecting amines of suitable hydrophobic character, molecular geometry, and concentration is discussed. A model of the formation and stabilization of the silanol-amine complex based on hydrophobic and ionic interactions is also proposed.     

Material of surface-ionization emitters for detecting amines

The material of a thermionic emitter with stable surface ionization parameters in air and a method for its activation are developed. A mock-up for the real-time detection of amines in air is created. The experience of using the mock-up for the determination of the freshness of protein products of an animal origin is described.     

Measurement of spin-lattice relaxation times and kinetic rate constants in rat muscle using progressive partial saturation and steady-state saturation transfer

The determination of neomycin sulfate by liquid chromatography using a column packed with a poly(styrenedivinylbenzene) co-polymer and pulsed electrochemical detection on a gold electrode is described. The mobile phase consisted of an aqueous solution containing 70 g/l of sodium sulfate, 1.4 g/l of sodium 1-octanesulfonate and 50 ml/l of a 0.2 M phosphate buffer (pH 3.0). Sodium hydroxide was added post-column. The influence of the different chromatographic parameters on the separation was investigated. The method shows good linearity and repeatability and is stability indicating. A number of commercial samples was analyzed using this method and the results were compared with results obtained with the European Pharmacopoeia method and a previously described thin-layer chromatographic method.     

High-pressure liquid chromatographic analysis of drugs in biological fluids. IV. Determination of clofibrinic acid

A rapid, sensitive and specific high-pressure liquid chromatographic method is described for the quantitative analysis of clofibrinic acid in plasma, saliva and urine. In contrast to previously reported gas-liquid chromatographic methods, which require derivatization of clofibrinic acid before chromatography, the present method involves a simple two-step extraction procedure and chromatographic determination of the underivatized clofibrinic acid. Concentrations between 1.0 and 25.0 microgram per sample can be measured with a coefficient of variation from 1 to 6%.     

Biogenic amines in silage, apparent postruminal passage, and the relationship between biogenic amines and digestive function and intake by steers

A 4 x 4 Latin square experiment was conducted to examine abomasal passage of biogenic amines in steers fed silage and their related effects on intake, digestibility, and digestive function. Thirty percent of the dry matter (DM) in the diets consisted of alfalfa forage, which was fed as either hay or silage. The DM from alfalfa silage DM was substituted at 0, 33, 67, and 100% for DM from alfalfa hay and was fed to four ruminally and abomasally cannulated steers. The roughage component of the diet constituted 50% of the DM and consisted of 60% alfalfa silage or hay and 40% tropical corn silage. The concentrate was composed mainly of ground corn. The concentrations of putrescine and cadaverine in abomasal digesta increased as alfalfa silage in the diet increased. Abomasal recovery of biogenic amines, a product of their concentration in abomasal digesta and the passage of DM through the abomasum, was negatively correlated with intake. Abomasal recovery of most amines was 5 to 20% of intake. Abomasal recovery of cadaverine was correlated with depressed intake. Total DM intake was reduced 8.3 to 25.8% as the proportion of alfalfa silage in the diet increased. Frequency of reticular contractions, intake, ruminal DM digestibility, ruminal outflow, volatile fatty acids, and total tract DM digestibility decreased in steers fed diets that contained more alfalfa silage. Ruminal fluid pH and NH3 concentration increased in steers fed more alfalfa silage; however, mass and the DM percentage of ruminal contents decreased linearly. Postprandial insulin concentrations were quadratically related to the proportion of alfalfa hay or silage in the diet. Intraruminal metabolism of biogenic amines is extensive based on the relatively low quantities recovered in abomasal digesta; however, the amounts recovered in abomasal digesta were related to intake depression and associated physiological effects.