Determination of biogenic amines in wines by ion-pair liquid chromatography and post-column derivatization with 1,2-naphthoquinone-4-sulphonate

A liquid chromatographic method with post-column derivatization for the determination of biogenic amines in wines is proposed. The method is based on the separation of amines by ion-pair chromatography using sodium heptanesulfonate (SHS) and on-line labeling of analytes with 1,2-naphthoquinone-4-sulfonate. The principal factors influencing the separation (acetonitrile and SHS concentration) have been considered for the optimization of the elution gradient through factorial design and multicriteria decision-making. Figures of merit have been established using red wine samples. Detection limits range from 0.2 to 3 mg L(-1), the peak area run-to-run repeatability from 1.6 to 4.6% and the retention time repeatability lower than 1.2%. Recoveries ranging from 92 and 108% prove the accuracy of the method for determining ethanolamine, ethylamine, histamine and tyramine in commercial red wines. The proposed method has been applied to the analysis of wines from different Spanish regions.     

Amperometric detection of amines using cobalt electrodes after separation by ion-moderated partition chromatography

A simple and low-cost amperometric sensor for amines has been developed using a cobalt wire electrode working in alkaline solution. The sensor may be used as a detector for high-performance liquid chromatography (HPLC) that avoids the need for derivatization or post-column reaction. Experimental conditions for flow injection analysis (FIA) and HPLC separation, including the applied potential, pH and concentrations of organic modifier and carrier solution, were optimized. A cobalt wire electrode, in the constant potential amperometric mode, gives an excellent response toward amines in ion-exclusion chromatography in unbuffered solution. The sensitivities of the detection and separation of amines on the column are affected by flow rate, the concentration of the mobile phase and the concentration of organic solvent in the mobile phase, whereas the applied potential only affects the sensitivity of the detector. A cobalt electrode is more sensitive than a copper electrode, and comparable in sensitivity to a UV detector for most amines tested. The detection sensitivity is comparable to that obtained with GC methods, but the procedures are far simpler. The detection limits of the order of nanomoles obtained under the chromatographic conditions used offer an alternative for the determination of amines in a variety of matrices, such as in environmental, biomedical and pharmaceutical samples.     

Determination of trace biogenic amines with 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene derivatization using high-performance liquid chromatography and fluorescence detection

A reversed-phase high-performance liquid chromatographic method based on chemical derivatization with fluorescence detection has been developed for analyzing biogenic amines in food and environmental samples. A BODIPY-based fluorescent reagent, 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene (TMBB-Su), was employed for the derivatization of these biogenic amines at 20 °C for 20 min in pH 7.20 borate buffer after careful investigation of the derivatization conditions including reagent concentration, buffer solution, reaction temperature and reaction time. Separation of biogenic amines with gradient elution was conducted on a C8 column with methanol-tetrahydrofuran-water as mobile phase. The detection limits were obtained in the range from 0.1 to 0.2 nM (signal-to-noise=3). This procedure has been validated using practical samples. The study results demonstrated a potential of employing high-performance liquid chromatography (HPLC) with 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene labeling as a tool for quantitative analysis of biogenic amines involved in various matrices.     

新型荧光衍生试剂用于脂肪胺的反相高效液相色谱-荧光检测分析

利用新型荧光试剂4-(1H-菲并[9,10-d]咪唑-2-)苯甲酸(PIBA)进行柱前衍生并经荧光检测对脂肪胺进行了高效液相色谱(HPLC)分离和在线质谱定性。激发和发射波长分别为ex=261nm,em=443nm。80℃下在吡啶溶剂中用N-乙基-N’-[(3-二甲氨基)丙基]碳二亚胺盐酸盐(EDC)做催化剂,衍生反应10min后获得稳定的荧光产物。在EclipseXDB-C8色谱柱(4.6150mm,5mm)上,梯度洗脱对12种游离脂肪胺衍生物进行了优化分离。采用大气压化学电离源(APCI)正离子模式,实现了各种脂肪胺衍生物的测定。多数脂肪胺的线性回归系数大于0.9999,检测限为10.5～53.4fmol。     

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.     

Application of high-performance liquid chromatography to the study of biogenic amine-related enzymes

The application of high-performance liquid chromatography to the study of biogenic amine-related enzymes is reviewed. Biogenic amines include catecholamines (dopamine, norepinephrine and epinephrine), indoleamines (serotonin and melatonin), imidazoleamines (histamine), polyamines (putrescine, spermidine and spermine) and acetylcholine. Three particular aspects are covered. The first aspect is the assay of enzyme activities of biogenic amine-related enzymes, such as tyrosine hydroxylase, tryptophan hydroxylase, aromatic L-amino acid decarboxylase, dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase. The introduction of highly sensitive assays of biogenic amines with electrochemical detection or fluorescence detection have made possible the non-isotopic assay of these activities, replacing the previously used radioisotopic methods. The second aspect is the purification of these enzymes. Since biogenic amine-synthesizing enzymes are generally unstable, rapid and efficient purification of these enzymes is very useful. The third aspect is the assay of biogenic amines (for example, acetylcholine and polyamines) using post-column derivatization with biogenic amine oxidases and electrochemical detection.     

Determination of organic colorants in cosmetic products by high-performance liquid chromatography

Summary High-performance liquid chromatography (HPLC) with UV-vis detection was used for the determination of organic colorants in cosmetic products. 126 colorants were characterized by their retention times in an ion-pair reversed-phase HPLC system with gradient elution, and by their UV-vis spectra, recorded with a diode array detector (DAD). The method is rapid and efficient, as is demonstrated by the analysis of 45 cosmetic samples.     

高效液相色谱-激光诱导荧光法分析水样中的胺类物质

通过色谱条件和衍生条件的优化,建立了微量胺类物质的高效液相色谱-激光诱导荧光检测分析方法。该方法灵敏度高,在优化的条件下分析亚精胺、腐胺和组胺,检出限达到10^-10 mol/L数量级,且稳定性好。连续进样5次,3种生物胺保留时间的RSD(n=5)小于0.3%,峰面积的RSD(n=5)小于3%,平均加标回收率为94.99%~104.7%。将该方法应用于实际水样中3种生物胺的检测及7种茶叶茶水中胺类物质的分析,取得了良好的结果。该方法灵敏度高,稳定性好,可用于水样中微量胺类物质的分析。     

An automated analyzer for methylated arginines in rat plasma by high-performance liquid chromatography with post-column fluorescence reaction

A fully automated analyzer for methylated L-arginine metabolites [N,N-dimethyl-L-arginine (ADMA), N-methylarginine (NMMA) and N,N'-dimethyl-L-arginine (SDMA)] by high-performance liquid chromatography with post-column fluorescence derivatization was developed. This system consists of an on-line extraction, a separation on a reversed phase ion-pair chromatograph, a post-column derivatization by o-phthaladehyde (OPA) and thiol reaction, and fluorescence detection. NMMA, ADMA and SDMA were separated in 40 min with isocratic elution by a combination of octanoate and cyclohexane carboxylate as ion-pair reagents. The eluate was monitored at 450 nm with excitation at 337 nm. The calibration curves for NMMA, ADMA and SDMA showed linearity over the range from 0.05 micromol l(-1) (0.5 pmol on column) to 5.0 micromol l(-1) (50 pmol on column). This method does not require any time-consuming pre-treatment and requires only 10 microl of plasma sample for assay.     

Determination of H2O2 and organic peroxides by high-performance liquid chromatography with post-column UV irradiation, derivatization and fluorescence detection

A method for the determination of hydrogen peroxide and several organic peroxides by high-performance liquid chromatography with post-column UV irradiation, derivatization and fluorescence detection is described. By means of post-column UV irradiation in the presence of water organic peroxides are converted into hydrogen peroxide and organic hydroperoxides, which react rapidly with the post-column derivatization agent p-hydroxyphenylacetic acid (PHPAA) under catalysis of horseradish peroxidase to yield the fluorescent PHPAA dimer that is detected at excitation and emission wavelengths of 285 and 400 nm, respectively. The detection limit for hydrogen peroxide is 14 ng/mL, for organic peroxides between 34 ng/mL and 5 μg/mL. No interference by other compounds was observed when their concentrations were below 10 mg/mL except ethers and phenols. Received: 6 August 1997 / Revised: 11 December 1997 / Accepted: 15 December 1997     

Determination of H2O2 and organic peroxides by high-performance liquid chromatography with post-column UV irradiation, derivatization and fluorescence detection

A method for the determination of hydrogen peroxide and several organic peroxides by high-performance liquid chromatography with post-column UV irradiation, derivatization and fluorescence detection is described. By means of post-column UV irradiation in the presence of water organic peroxides are converted into hydrogen peroxide and organic hydroperoxides, which react rapidly with the post-column derivatization agent p-hydroxyphenylacetic acid (PHPAA) under catalysis of horseradish peroxidase to yield the fluorescent PHPAA dimer that is detected at excitation and emission wavelengths of 285 and 400 nm, respectively. The detection limit for hydrogen peroxide is 14 ng/mL, for organic peroxides between 34 ng/mL and 5 μg/mL. No interference by other compounds was observed when their concentrations were below 10 mg/mL except ethers and phenols. Received: 6 August 1997 / Revised: 11 December 1997 / Accepted: 15 December 1997     

HPLC determination of catabolic amines in silage using their dansyl derivatives and an electrochemical detector

Summary The use of the electrochemical detector (EC) in the high performance liquid chromatography (HPLC) of dansyl derivatives of biogenic amines is reported. Isocratic and gradient elution patterns of synthetic mixtures of putrescine, cadaverine, 1,6-diaminohexane, tryptamine, histamine, tyramine, spermine and spermidine on a reversed phase column are shown. Hydrodynamic voltammograms of standard compounds are presented. The detection limits of the EC are compared with those of ultraviolet and spectrofluorimetric detectors. A chromatographic profile of amines from the proteolytic catabolism of maize silage byClostridia is shown.Presented at the 17th International Symposium on Chromatography, September 25–30, 1988, Vienna, Austria.     

高效液相色谱-柱后衍生法检测养殖水体及沉积物中11种磺胺药物残留

建立了养殖水体及沉积物中11种磺胺化合物的高效液相色谱-柱后衍生分析方法。养殖水体过滤后采用HLB固相萃取柱进行净化、富集;沉积物采用甲醇/EDTA-Mcllvaine缓冲液(1:1, v/v)提取,HLB固相萃取柱净化富集。经高效液相色谱分离,用荧光胺衍生试剂进行柱后衍生,荧光检测器检测。对柱后衍生系统参数进行了优化,确定了荧光胺溶液的浓度、流速和反应温度分别为0.2 g/L、0.15 mL/min和50 ℃,磺胺化合物在0.01~1.0 mg/L范围内线性显著,其相关系数r²值大于0.99995。11种磺胺类药物在养殖水体和沉积物中的加标回收率分别为79.3%~100.7%和74.6%~95.3%,相对标准偏差为2.2%~11.0%和2.6%~10.3%,检出限(LOD, S/N=3)为0.9~5.5 ng/L和0.3~1.3 μg/kg,定量限(LOQ, S/N=10)为3.0~18.1 ng/L和1.0~4.4 μg/kg。该法可应用于养殖环境中磺胺类药物的定性定量检测,具有较好的实用性。     

High-performance liquid chromatographic determination of clenbuterol and cimaterol using post-column derivatization.

A general high-performance liquid chromatographic method for the simultaneous and rapid determination of cimaterol and clenbuterol is described. Solid samples, such as animal tissues, faeces and feeding-stuffs, are extracted with dilute acid saturated with ethyl acetate. The resulting extracts or liquid samples, such as urine, plasma, blood and bile, are purified via Chem Elut columns. Separation is achieved by ion-pair chromatography on a Nova-Pak C18 column, and highly specific detection is obtained with an adapted version of the post-column derivatization described previously for the determination of clenbuterol in urine and animal tissues. Detection limits for liquids and solids are 0.1 ng/ml and 0.2 ng/g, respectively. The results are in complete agreement with analysis by high-performance thin-layer chromatography and gas chromatography-mass spectrometry, applied for confirmation after the same sample pretreatment. With this simple method, complete analysis of a liquid sample needs about 30 min and, even without an automatic sampler, 40 samples can be completely analysed in one day. This method has been used on a routine scale for nearly two years.     

Analysis of Biogenic Amines Using Immunoassays,HPLC, and a Newly Developed IC-MS/MS Technique in Fish Products—A Comparative Study

In this study, comparative analyses were carried out with ion chromatography mass-spectrometry (IC-MS/MS) which has no derivatization step, high-performance liquid chromatography (HPLC) technique, as well as two quantitative and two semi-quantitative immunoassays. The results demonstrated that HPLC and quantitative immunoassay methods were well-correlated with IC-MS/MS in determining histamine in various types of fish products. The best correlation was observed with the HistaSure ELISA Fast Track kit (R² = 0.9903). More than half of the values (68%) obtained by two methods were also statistically similar. The results of semi-quantitative test kits also supported histamine values estimated by quantitative methods, with some exceptions. The best results were found for HistaSure Lateral Flow in supporting the quantitative techniques. Therefore, these methods are found suitable for monitoring histamine in fish products in terms of food safety. Good correlations were also observed HPLC and IC-MS/MS in determining cadaverine, putrescine, and tyramine with the highest value observed for tyramine as R² = 0.9785. However, no correlation was observed for other biogenic amines, and the majority of the results were significantly different from each other for these amines (p < 0.05). The differences may be caused by the drawbacks reported previously for HPLC. However, further studies are required to confirm the possible effects. This study provides a comparative evaluation of several methods in terms of their suitability in determining biogenic amines in fish products for both monitoring and regulatory purposes.     

Luminol chemiluminescence HPLC reaction detector for amino acids and other ligands

A detector for HPLC is based on suppression of chemiluminescence from the Cu(II)-luminol-peroxide reaction. Analytes which complex Cu(II) reduce the free Cu(II) concentration and thus the observed chemiluminescence intensity. The degree of chemiluminescence suppression is a measure of the analyte concentrations. Detection limits are in the range of 1 pmole-1 nmole for amino acids (both primary and secondary without derivatization), CN^–, amines, catecholamines, catechol, and aminoglycoside antibiotics. The detection approach is demonstrated with an ion-exchange separation of amino acids, a reverse phase separation of catecholamines, and an ion-pair separation of the three components of gentamicin C in commercial gentamicin sulfate.     

Bioanalysis of the neuropeptide des-enkephalin-gamma-endorphin by high-performance liquid chromatography with on-line sample pretreatment using gel permeation and solid-phase isolation.

A bioanalytical method is described that allows the determination of a number of beta-endorphin-related peptides. The method is based on the application of fluorescence detection after high-performance liquid chromatography followed by post-column derivatization with o-phthaldialdehyde. Concentrations exceeding 10-25 ng/ml could be determined by using conventional fluorescence detection, whereas lower concentrations demand the use of laser-induced fluorescence detection. The sample pretreatment includes the use of on-line gel permeation, on-line solid-phase isolation and heart cutting of a peak from reversed-phase gradient elution. The sample pretreatment procedure does not discriminate between the dodecapeptide des-enkaphalin-gamma-endorphin (DE gamma E) and its metabolites in order to obtain similar recoveries for all components. The final chromatographic phase system is based on ion-pair formation, which permits the separation of DE gamma E from its metabolites and degradation products. The optimized procedure allows the determination of these peptides in plasma at concentration levels down to about 1 ng/ml, demanding a sample volume of 1 ml.     

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

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

Determination of neomycin in plasma and urine by high-performance liquid chromatography. Application to a preliminary pharmacokinetic study.

A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the determination of neomycin in plasma and urine. The plasma was deproteinated with trichloroacetic acid and centrifuged. The supernatant was mixed with ion-pair concentrate and centrifuged again. The resultant supernatant was analyzed by HPLC. Urine was centrifuged to remove debris, if any, mixed with ion-pair concentrate and analyzed directly by HPLC. The HPLC conditions consisted of an ion-pairing mobile phase, a reversed-phase column, post-column derivatization with o-phthalaldehyde (OPA) reagent and fluorescence detection. The overall average recovery of neomycin was 97 and 113% from plasma spiked at 0.25-1.0 micrograms/ml, using standard curves prepared in plasma extract and in water, respectively, and 94% for urine spiked at 1-10 micrograms/ml using a standard curve prepared in water. The method was used to detect neomycin in plasma and urine obtained from animals injected intramuscularly with neomycin. Various pharmacokinetic parameters of neomycin were also determined from its profile of plasma concentration versus time.     

High-performance liquid chromatographic determination of histamine N-methyltransferase activity

A method for the determination of histamine N-methyltransferase (HMT) activity by high-performance liquid chromatography based on post-column derivatization with omicron-phthalaldehyde is described. The determination involves the separation of the substrate, histamine, from its product. N tau-methylhistamine, using a weak cation exchanger, followed by on-line derivatization of these imidazoleamines with omicron-phthalaldehyde and their detection and quantitation with a fluorimetric detector. This assay method is suitable for the measurement of HMT activity during enzyme purification.