在线固相萃取-毛细管高效液相色谱联用测定奶酪中的生物胺

采用双二元泵毛细管液相色谱,通过六通阀实现了样品的在线净化与分离定量的自动切换,建立了同时测定奶酪中的15种生物胺的在线固相萃取-毛细管高效液相色谱联用方法。通过优化毛细管高效液相色谱的分离条件,考察在线固相萃取流动相的组成、上样溶液pH值以及六通阀的切换时间对生物胺回收率的影响,确定最佳分析条件为：5%乙腈-水作为固萃柱(Zorbax SB-C18)的流动相,上样溶液pH=11,上样3 min后切换六通阀。采用内标法定量,15种生物胺标准曲线的线性范围为0.25~50.0 mg/L,检出限( LOD)为0.05~0.25 mg/L,定量限(LOQ)为0.15~0.80 mg/L。除了甲胺、乙胺、3-甲基丁胺和5-羟基色胺外,其余生物胺的不同添加水平(1,20和40 mg/kg)下的加标回收率为79.6%~118.7%;除3-甲基丁胺和5-羟基色胺外,其余生物胺的RSD在0.3%~14.9%之间,可用于奶酪中多种生物胺的快速检测。     

Method development and validation for the determination of biogenic amines in soy sauce using supercritical fluid chromatography coupled with single quadrupole mass spectrometry

Biogenic amines have been reported in many foods such as fish, meat, and soy sauce. The consumption of foods containing high concentrations of biogenic amines has been associated with health hazards. In this study, a green and efficient method using supercritical fluid chromatography coupled with single quadrupole mass spectrometry was developed for determination of biogenic amines in soy sauce. The chromatographic and mass spectrometry conditions were systematically optimized in terms of selectivity and peak shape. Nine biogenic amines were well separated within 25 min on a Cosmosil 5HP column using 5% (v/v) water and 0.2% (v/v) ammonia solution in methanol as mobile phase additives at a backpressure of 120 bar and temperature of 40°C. The established method was fully validated regarding the linearity, sensitivity, precision, and accuracy. The limits of detection and limits of quantification ranged from 0.03 to 10.50 μg/mL and 0.10 to 23.1 μg/mL, respectively. The relative standard deviations for intra‐ and interday precisions were all lower than 9.36% and the recoveries ranged from 75.82 to 99.63% and 80.10 to 99.89% for two levels of standards spiked in soy sauce, respectively. Finally, the established method was successfully applied to the quantitative analysis of biogenic amines in soy sauce.     

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%     

毛细管电色谱-激光诱导荧光检测法分析食品中的生物胺

以4-氟-7-硝基-2,1,3-苯并氧杂恶二唑(NBD-F)为衍生化试剂,建立了食品中5种痕量生物胺(色胺、组胺、酪胺、亚精胺、精胺)的毛细管电色谱-激光诱导荧光检测(CEC-LIF)分析方法。采用50 mmol/L硼酸盐缓冲溶液(pH 8.0)作为衍生介质,在75℃条件下对生物胺进行衍生化反应25 min。生物胺衍生产物的最优色谱条件:固定相为C18毛细管电色谱柱,流动相为乙腈-乙酸铵(20 mmol/L,pH 8.0)(75∶25,v/v),辅助压力为6.9 MPa,分离电压为-8 kV,流速为0.03 mL/min。实验结果表明,生物胺的检出限(LOD,S/N=3)为0.1~1.0μg/L,加标回收率为78.3%~113.9%。该方法可成功用于加工和发酵食品中生物胺的测定,结果与传统HPLC法的检测结果无显著性差异,且检出限更低、分析速度更快,对于食品中痕量污染物的残留监测具有应用价值。     

Determination of Biogenic Amines in Digesta by High Performance Liquid Chromatography with Precolumn Dansylation

A rapid high-performance liquid chromatographic method for the determination of nine biogenic amines in digesta sample from pig cecum and colon was developed using a simple direct derivatization with dansyl chloride. After precipitation with trichloroacetic acid and extraction by n-hexane, the amines were separated on a C₁₈ column using a water–acetonitrile gradient elution program. The calibration curve for each amine was linear over the range of 50 to 250 µmol/L, and the relative standard deviation for intra-assay precision was below 0.5%. The recovery ranged from 86.79% to 117.62% while the limit of detection was 0.1 µmol/L for the amines except for spermine with a value of 0.03 µmol/L. This procedure was successfully applied to identify and quantify biogenic amines in the hindgut digesta of the pig.     

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.     

Quantification of biogenic amines in human plasma based on the derivatization with N-hydroxy-succinimidyl fluorescein-O-acetate by high-performance liquid chromatography

An HPLC method for the determination of biogenic amines based on the precolumn derivatization with N-hydroxysuccinimidyl fluorescein-O-acetate (SIFA) has been developed. The derivatization was performed at 45 degrees C for 30 min in borate buffer (pH 8.0). The derivatives were separated on a ZORBAX Eclipse XDB-C8 column (150 mm x 4.6 mm id; 5 mum) and monitored by fluorescence detection (excitation, 469 nm; emission, 512 nm). The LODs (S/N = 3) for spermine, spermidine, putrescine, cadaverine, and phenethylamine were 0.4, 0.2, 0.3, 0.5, and 0.4 nM, respectively. The developed method has been successfully applied to the determination of biogenic amines in human plasma of three healthy volunteers and four cancer patients. Average recoveries for plasma samples ranged from 94 to 106% and coefficients of variation ranged from 1.8 to 4.6%. Deproteinization of plasma was accomplished with ACN to precipitate interfering substances and the centrifuged supernatant was used directly for analysis.     

Monitoring of biogenic amines and drugs of various therapeutic groups in urine samples with use of HPLC

A high‐performance liquid chromatography method for simultaneous separation and determination of biogenic amines [dopamine, epinephrine, serotonin and its six metabolites (normetanephrine, metanephrine, 3,4‐dihydroxyphenylacetic acid, 4‐hydroxy‐3‐methoxyphenylglycol, homovanilic acid and 5‐hydroxyindoloacetic acid)] with drugs from different therapeutically groups [analgesics (paracetamol, metamizol), diuretics (furosemide) and antibiotics (cefazolin, fluconazole)] was developed. A chromatographic column with pre‐column with octadecylsilane phase (C_18e) and two detectors – diode array serial connected and fluorescence – was used. Gradient elution of mixture of acetate buffer (pH 4.66) and methanol as a mobile phase was applied. The limit of detection (LOD) of 8–10 ng/mL and limit of quantitation (LOQ) of 24–30 ng/mL for biogenic amines, as well as the LOD of 50–100 ng/mL and the LOQ of 150–300 ng/mL for drugs, were determined. The applied sample preparation method allowed recoveries of 93% for the biogenic amines and 92% for the drugs to be achieved. The developed procedure has been applied to simultaneous determination of the examined compounds in urine samples and could be used in clinical analysis. Copyright © 2015 John Wiley & Sons, Ltd.     

Fluorometric Determination of Histamine in Biological Fluids and Tissue by High-Performance Liquid Chromatography

Abstract

High-pressure liquid chromatography was used to separate the fluorescent adduct formed from the reaction of histamine with o-phthalaldehyde (OPT) from other biogenic amines in tissue, cerebrospinal fluid (CSF), sweat and urine samples. Using off-column derivatization and isocratic elution techniques fluorescent OPT adducts can be detected in the low picogram range. Perchloric acid extracts of tissue samples from Aplysia california and urine specimens collected from healthy adult males, including internal standard, were derivatized with OPT buffer, pH 9.5 and extracted with ethylacetate to increase sensitivity and stabilization of the fluorescent adduct prior to chromatography. Sweat and CSF samples were reacted with OPT buffer and aliquots of this mixture injected directly onto the chromatographic column (μBondapak CN) with methanol/0.08 mol/liter acetic acid (52/48 by volume) as the mobile phase. Assay of pooled urine containing added histamine (1 μg/ml) gave a with-in run coefficient of variation of 2.5%. The use of o-phthalaldehyde as an off-column HPLC derivatization agent for fluorometric determination of low-levels of biogenic amines is rapid, sensitive and easily adapted to routine use in a clinical or neurobiological laboratory.     

Study of the naphthalene-2,3-dicarboxaldehyde pre-column derivatization of biogenic mono- and diamines in mixture and fluorescence?HPLC determination

A new fluorescence−HPLC method was developed for the simultaneous determination of eight biogenic monoamines (histamine, methylamine, tyramine, ethylamine, propylamine, tryptamine, 2-phenylethylamine, isoamylamine) and two biogenic diamines (putrescine, cadaverine) in the presence of heptylamine as the internal standard. The amines were pre-column derivatized with naphthalene-2,3-dicarboxaldehyde in the presence of cyanide ion as the nucleophile. The effect of the derivatization reaction conditions on the reaction yield was investigated. The derivatives were separated on an Inertsil ODS-3 column (250 × 4mm i.d., 5 μm) using gradient elution and detected fluorimetrically at excitation and emission wavelengths of 424 and 494 nm, respectively. Limits of detection between 0.002 and 0.4 ng, injected on-column (10-μL loop), were achieved. The within- and between-day relative standard deviations ranged between 0.2−3.4% and 0.3−4.8%, respectively. The utility of the method in assaying biogenic mono- and diamine mixtures in Greek cheeses is demonstrated. Ultrasound-assisted liquid−liquid extraction was applied prior to derivatization.     

On-line determination of aliphatic amines in water using in-tube solid-phase microextraction-assisted derivatisation in in-valve mode for processing large sample volumes in LC

This paper describes a cost-effective procedure for the analysis of short-chain aliphatic amines in water samples using a solid-phase microextraction device. Analyte preconcentration and derivatisation were effected into a capillary column coated with 95% polydimethylsiloxane–5% polydiphenylsiloxane, which was used as the injection loop of a Rheodyne injection valve. The coating was previously loaded with the derivatisation reagent, 9-fluorenylmethyl chloroformate. A volume of 1 mL of samples was then drawn into the capillary column, and the extracted analytes were left to react on the capillary coating for 5 min. Next, the capillary column was cleaned by passing water. Finally, the injection valve was rotated, and the derivatives formed were dynamically desorbed and transferred to the analytical column into the mobile phase. Methylamine, ethylamine, propylamine, n-butylamine and n-pentylamine were selected as model compounds. Excellent sensitivity was achieved, being the limits of detection of 15–200 μg/L when using UV detection and of 0.1–0.4 μg/L by fluorescence.     

High-performance liquid chromatographic analysis of biogenic amines in biological materials as o-phthalaldehyde derivatives.

A remarkably sensitive, simple and selective reversed-phase high-performance liquid chromatographic (HPLC) method has been developed, allowing, for the first time, the direct measurement of histamine, norepinephrine, octopamine, normetanephrine, dopamine, serotonin and tyramine in a single sample of plasma (2 ml), tissue (0.2 g), or urine. The biogenic amines were modified by pre-column derivatization with o-phthalaldehyde which stabilizes the molecules, aids in extraction, and improves HPLC detection at the nanogram level. To minimize losses during the sampling procedure a careful collection procedure was designed. We developed a simple sample cleanup in which the samples were thawed, neutralized with KOH, immediately derivatized, extracted into ethyl acetate (EtOAc) and then chromatographed by HPLC. The derivatives were stable in EtOAc for more then 24 h. Interfering amino acids were removed from the EtOAc by partitioning twice with Na2HPO4 buffer (pH 10.0). Complete separation was achieved in ca. 60--90 min on a muBondapak phenyl column using a stepwise gradient of acetonitrile and/or methanol-phosphate buffer (pH 5.1). A variable wavelength fluorometer with a 5-microliter flow-cell was used (excitation 340 nm; emission 480 nm). Linearity ranged from 200 pg to 50 ng onto the column. Precision (R.S.D.) for retention times was 1% and for derivatization and injection 2.5%. Recoveries of the seven biogenic amines from plasma spiked with 25 ng/ml averaged 70%, with a relative standard deviation of 6%. Separation studies were also done using a muBondapak C18 column. The effects of various eluents are presented. Gas-liquid chromatography was also investigated but lacked the sensitivity achieved by HPLC. The HPLC method is used routinely for the determination of biogenic amines in plasma from pigs with malignant hyperthemia and thermally stressed bovine. Significant differences in levels of biogenic amines were noted between stressed and non-stressed animals. Data on rat brain tissue samples were compared with the trihydroxyindole method and canine heart tissue was analyzed for ventricular norepinephrine and dopamine. Application of the method to urine from normal persons and a patient with a brain tumor has been demonstrated.     

对甲氧基苯磺酰氯柱前衍生生物胺的高效液相色谱分离

采用对甲氧基苯磺酰氯作为柱前衍生试剂,RP-HPLC为分析模式,建立了一种新的生物胺衍生化方法,并对葡萄酒中生物胺含量进行检测。通过液质联用对产物进行定性,研究并确定了最适衍生化条件:衍生温度50℃,缓冲液pH 9.0,衍生时间15 min。实验建立了7种生物胺的HPLC分离方法:Beckman ODS柱;流动相A为10 mmol/L的NH4Ac溶液(pH 6.37),B相为乙腈;采用梯度洗脱;流速1 mL/min;检测波长240 nm,室温。     

Pre-Column Fluorescent Derivatization for High Pressure Liquid Chromatography with o-Phthalaldehyde: Separation of Urinary Catecholamines

Abstract

High-pressure liquid chromatography was used to separate the flourescent adducts formed from the reaction of o-phthalaldehyde with primary biogenic amines. Using precolumn derivatization and isocratic elution techniques fluorescent o-phthalaldehyde adducts can be detected and quantified with fluorometry in the low picogram range. The relative retention values of the o-phthalaldehyde adducts strongly depend on the pH of the eluent and percent organic solvent in the mobile phase. The method was applied to the analysis of free norepinephrine in pooled urine samples and in small-volume (2-h) urine collections obtained from thermally stressed subjects. Samples were treated with alumina, and the catecholamines, including internal standard 3,4-dihydroxy-benzylamine, eluted from it were reacted with o-phthalaldehyde prior to injection onto a reverse-phase column (octadecyl-silica stationary phase) with methanol/0.08 mol/liter acetic acid (50/50 by vol) as the mobile phase. Assay of pooled urine specimens (n = 10) for norepinephrine gave within-run and day-to-day coefficients of variation of 4.3 and 5.6% respectively. The use of o-phthalaldehyde as a pre-column derivatization agent for fluorometric determination of primary amines is rapid, sensitive and specific and applicable to many important biogenic amines.     

Simultaneous analysis of free amino acids and biogenic amines in honey and wine samples using in loop orthophthalaldeyde derivatization procedure

This work presents a RP-HPLC method for the simultaneous quantification of free amino acids and biogenic amines in liquid food matrices and the results of the application to honey and wine samples obtained from different production processes and geographic origins. The developed methodology is based on a pre-column derivatization with o-phthaldialdehyde carried out in the sample injection loop. The compounds were separated in a Nova-Pack RP-C(18) column (150 mm x 3.9 mm, 4 microm) at 35 degrees C. The mobile phase used was a mixture of phase A: 10 mM sodium phosphate buffer (pH 7.3), methanol and tetrahydrofuran (91:8:1); and phase B: methanol and phosphate buffer (80:20), with a flow rate of 1.0 ml/min. Fluorescence detection was used at an excitation wavelength of 335 nm and an emission wavelength of 440 nm. The separation and quantification of 19 amino acids and 6 amines was carried out in a single run as their OPA/MCE derivatives elute within 80 min, ensuring a reproducible quantification. The method showed to be adequate for the purpose, with an average RSD of 2% for the different amino acids; detection limits varying between 0.71 mg/l (Asn) and 8.26 mg/l (Lys) and recovery rates between 63.0% (Cad) and 98.0% (Asp). The amino acids present at the highest concentration in honey and wine samples were phenylalanine and arginine, respectively. Only residual levels of biogenic amines were detected in the analysed samples.     

Separation and determination of triadimefon and its metabolites triadimenol enantiomers in fruit puree by supercritical fluid chromatography

A method was established for the separation and determination of triadimefon and its metabolite triadimenol enantiomer residues in major complementary fruit puree for infants and young children (banana puree, pineapple puree, and grape puree) by supercritical fluid chromatography. After the samples were extracted with acetonitrile and purified with a solid phase extraction cartridge, Acquity Trefoil CEL2 chiral chromatographic column was adopted for separation, and gradient elution was conducted at the flow rate of 1.0 ml/min under the mobile phase of supercritical carbon dioxide - 0.5% ammonia methanol, the detection wavelength was 220 nm and quantification was conducted with the external standard method. The limits of quantitation of triadimefon and triadimenol enantiomers were both 0.05 mg/kg, the linear ranges were 0.5–50 mg/L, and the linear correlation coefficients were greater than 0.9993. The recoveries in the spiked samples at 0.05, 0.2, and 3.0 mg/kg were from 80.1 to 106%, and the relative standard deviation reached 3.3–7.6%. The method is efficient, rapid, reproducible, and environmentally friendly, enabling accurate analysis of pesticide enantiomers, which can detect the enantiomer residues of triadimefon and its metabolite triadimenol in major complementary fruit puree for infants and young children.     

Simultaneous determination of biogenic amines and morphine in discrete rat brain regions by high-performance liquid chromatography with electrochemical detection

A simple and sensitive method has been developed for the simultaneous determination of norepinephrine, epinephrine, dopamine, 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, and morphine in discrete rat brain regions by reversed-phase high-performance liquid chromatography with electrochemical detection. Perchloric acid extracts of the tissue were directly injected into the chromatographic system. Each of these compounds gave a linear response over the range of 20-160 ng/ml cerebellar homogenate (0.4-3.2 ng on column). Recoveries of these compounds, added to the homogenates, were complete when compared with standards dissolved in perchloric acid. The average between-run coefficients of variation for all these compounds were lower than 7.4% over the range of 20-160 ng/ml, and the within-run coefficients of variation at 20 ng/ml were lower than 8.7%. The present method has been applied to a study of the effects of intraperitoneal administration of morphine on biogenic amines in several discrete rat brain regions.     

Determination of biogenic amines in wines and beers by high performance liquid chromatography with pre-column dansylation and ultraviolet detection

Summary A sensitive high performance liquid chromatographic method for the simultaneous determination of eleven biogenic amines, using 1,7-diaminoheptane as internal standard, has been developed. The method involves pre-column derivatization of the amines with dansyl chloride and subsequent solid phase extraction of the derivatives through C18 cartridges. The derivatization and solid phase extraction procedures were optimized. The separation of dansylamides was achieved on an Inertsil ODS-3 column (250×4 mm I.D. 5 μm) using a 35-min gradient elution method with a binary system of acetonitrile-water, a flow rate of 1 mL.min¹ with UV detection at 254 nm. Linearity of derivatization was obtained for concentrations ranging from 0.025 to 3.0 mg.L¹. The within- and between-day relative standard deviations ranged from 0.4 to 5.7% and 0.6 to 7.3% respectively. The overall process was successfully applied to identify and quantify biogenic amines in white, red and Retsina Greek wines and Greek beers, after their treatment with polyvinylpyrrolidone.     

High-performance liquid chromatographic determination of prifinium quaternary ammonium ion in human serum and urine

A simple, sensitive method for the determination of the prifinium ion, a quaternary ammonium ion, in human serum and urine is described. The method is based on extraction of the test solution with chloroform in the presence of saturated potassium bromide solution and normal-phase high-performance liquid chromatography using aqueous methanol as the mobile phase at pH 10. To prevent the dissolution of silica from the analytical column, the mobile phase is pre-saturated with silica by using a silica saturation column. Quantitation is possible down to 0.5 ng/ml of prifinium ion using 2 ml of serum and down to 5 ng/ml using a 1 ml of urine. The coefficients of variation of the method are less than 1.3% in both serum and urine. Serum levels and urinary excretion data obtained with this method are given for three healthy volunteers who had received a 60-mg oral dose of prifinium bromide.     

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.