Analysis of free amino acids in fermented shrimp waste by high-performance liquid chromatography

This work presents an HPLC method for the quantification of free amino acids in lyophilized protein fraction from shrimp waste hydrolysate which is obtained by acid lactic fermentation and analyzed using pre-column derivatization with 9-fluorenylmethyl-chloroformate. The amino acids were separated in a Hypersil ODS 5 microm column (250 mm x 4.6 mm) at 38 degrees C. The mobile phase was a mixture of phase A: 30 mM ammonium phosphate (pH 6.5) in 15:85 (v/v) methanol/water; phase B: 15:85 (v/v) methanol/water; and phase C: 90:10 (v/v) acetonitrile/water, with flow rate 1.2 ml/min. Fluorescence detection was used at an excitation wavelength of 270 nm and an emission wavelength of 316 nm. Method precisions for the different amino acids were between 4.4 and 7.1% (relative standard deviation, RSD); detection limits were between 23 and 72 ng/ml; and the recoveries were between 89.0 and 95.0%. The amino acid present at the highest concentration was tyrosine.     

毛细管电泳-间接紫外检测法测定蜂蜜中的氨基酸

采用毛细管电泳-间接紫外检测法同时分离测定蜂蜜中的赖氨酸、色氨酸、谷氨酸等9种氨基酸。考察了磷酸浓度、进样方式和缓冲液pH对分离效率和重现性的影响。在分离电压为-15 kV、检测波长为220 nm条件下,以含有0.5 mmol/L十六烷基三甲基溴化铵、20 mmol/L烟酸、10%甲醇的10 mmol/L磷酸二氢钠缓冲溶液(pH 10.2)为运行缓冲液,9种组分在11 min内达到基线分离;检出限最低可达到0.3 mg/L;线性范围为1.0~1000 mg/L;日间及日内精密度为0.64%~5.83%。实际样品中除甲硫氨酸外的8种氨基酸的加标回收率为60.00%~118.37%。将该方法应用于不同蜜源植物和产地的蜂蜜样品的测定,在市售的5种蜂蜜中均检测到脯氨酸、丝氨酸和天冬氨酸,而只在荔枝蜜中检测到苏氨酸。该方法可以为蜂蜜的蜜源鉴别及质量评估提供借鉴方法。     

Rapid automated high performance liquid chromatography method for simultaneous determination of amino acids and biogenic amines in wine, fruit and honey

This paper reports a new, simple, rapid and economical method for routine determination of 24 amino acids and biogenic amines in grapes and wine. No sample clean-up is required and total run time including column re-equilibration is less than 40min. Following automated in-loop automated pre-column derivatisation with an o-phthaldialdehyde, N-acetyl-l-cysteine reagent, compounds were separated on a 3mm×25cm C(18) column using a binary mobile phase. The method was validated in the range 0.25-10mg/l; repeatability was less than 3% RSD and the intermediate precision ranged from 2 to 7% RSD. The method was shown to be linear by the 'lack of fit' test and the accuracy was between 97 and 101%. The LLOQ varied between 10μg/l for aspartic and glutamic acids, ethanolamine and GABA, and 100μg/l for tyrosine, phenylalanine, putrescine and cadaverine. The method was applied to grapes, white wine, red wine, honey and three species of physalis fruit. Grapes and physalis fruit were crushed, sieved, centrifuged and diluted 1/20 and 1/100, respectively, for analysis; wines and honeys were simply diluted 10-fold. It was shown using this method that the amino acid content of grapes was strongly correlated with berry volume, moderately correlated with sugar concentration and inversely correlated with total acidity.     

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.     

Analytical potential of 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein for the determination of amino compounds by capillary electrophoresis with laser-induced fluorescence detection

The analytical potential of a fluorescein analogue, 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein (SAMF), for the first time synthesized in our laboratory, as a labeling reagent for the labeling and determination of amino compounds by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was investigated. Biogenic monoamines and amino acids were chosen as model analytes to evaluate the analytical possibilities of this approach. The derivatization conditions and separation parameters for the biogenic amines were optimized in detail. The derivatization was performed at 30 degrees C for 6 min in boric acid buffer (pH 8.0). The derivatives were baseline-separated in 15 min with 25 mM boric acid running buffer (pH 9.0), containing 24 mM SDS and 12.5% v/v acetonitrile. The concentration detection limit for biogenic amines reaches 8 x 10(-11) mol.L(-1) (signal-to-noise ratio = 3). The application of CE in the analysis of the SAMF-derivatized amino acids was also exploited. The optimal running buffer for amino acids suggested that weak acidic background electrolyte offered better separation than the basic one. The proposed method was applied to the determination of biogenic amines in three different beer samples with satisfying recoveries varying from 92.8% to 104.8%. Finally, comparison of several fluorescein-based probes for amino compounds was discussed. With good labeling reaction, excellent photostability, pH-independent fluorescence (pH 4-9), and the resultant widely suited running buffer pH, SAMF has a great prospect in the determination of amino compounds in CE.     

Determination of ochratoxin A in wine by means of immunoaffinity column clean-up and high-performance liquid chromatography

A new and accurate method to quantify ochratoxin A (OA) in table wine has been developed. The method uses commercial immunoaffinity columns for clean-up and high-performance liquid chromatography (HPLC) with fluorescence detection for quantification of the toxin. Wine was diluted with a solution containing 1% polyethylene glycol (PEG 8000) and 5% sodium hydrogencarbonate, filtered and applied to an OchraTest immunoaffinity column. The column was washed with a solution containing sodium chloride (2.5%) and sodium hydrogencarbonate (0.5%) followed by water. OA was eluted with methanol and quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 333 nm, emission wavelength 460 nm) using acetonitrile-water-acetic acid (99:99:2) as mobile phase. Average recoveries of OA from white, rosé and red wine samples spiked at levels from 0.04 to 10 ng/ml ranged from 88% to 103%, with relative standard deviations (RSDs) between 0.2 and 9.7%. Detection limit was 0.01 ng/ml based on a signal-to-noise ratio of 3:1. The method was applied successfully to 56 samples of red (38), rosé (8), white (9) and dessert (1) wine. The levels of OA ranged from <0.01 to 7.6 ng/ml with red wines more contaminated than rosé and white wines. A good correlation (r=0.987) was found by comparative analysis of 20 naturally contaminated samples using this method and the method of Zimmerli and Dick with better recoveries of OA and better performances for the new method. Several advantages of this method with respect to the actually available methods have been pointed out, with particular reference to red wine which appears to be the most difficult to analyze.     

有机酸类化合物的反相高效液相色谱法的分离条件研究

 研究了在用RP HPLC法测定有机酸时如何对流动相的酸度进行控制 ,给出了流动相最佳pH的选择通式 :色谱柱允许的最低 pH值≤pH≤pKa- 2 ;研究了流动相中甲醇、乙腈存在时对此通式的影响。用研究得出的结论选择了测定甲酸、乙酸、丙酸、丁酸和丙二酸、丁二酸、戊二酸、己二酸时的色谱条件 ,并且研究了每种酸在各自浓度范围内测定的线性关系、回归方程、相关系数以及相对标准偏差。实验显示 ,在选定的色谱条件下进行分析测试时 ,各种酸的线性相关系数r >0 9995 ,相对标准偏差RSD <0 90 %。     

镧掺杂二氧化钛/沸石微柱在线预富集-分光光度法测定溶菌酶含量

以镧掺杂二氧化钛/沸石吸附剂微柱为萃取装置,建立了溶菌酶的分光光度检测法.考察了实验条件对萃取效率的影响.以甲醇为洗脱液,流速2.0 mL/min;样品的pH=5.0,流速1.2 mL/min,体积1.2 mL;检测波长280 nm.对蛋清、蜂蜜及葡萄酒中溶菌酶的含量进行了测定,日内与日间精密度分别为2.1%和2.8%...     

邻苯二甲醛-尿素柱前衍生高效液相色谱法快速检测枸杞中牛磺酸

干枸杞经粉碎、匀浆、离心后,通过阳离子交换柱脱去样品中其它氨基酸,再通过Ｚｏｒｂａｘ－Ｃ８柱进行柱前衍生分离。衍生剂：Ａ．４％ＯＰＡ甲醇溶液;Ｂ．尿素∶磷酸钠盐缓冲液（ｐＨ６．８）＝１∶３（Ｗ／Ｖ）。流动相：甲醇∶０．０１ｍｏｌ／Ｌ乙酸钠溶液（ｐＨ６．８）＝３５∶６５（Ｖ／Ｖ）。紫外检测波长３３０ｎｍ。牛磺酸浓度在０．１～１．０ｍｍｏｌ／Ｌ范围内可被定量测定。回收率可达１００．３１％±１．９８％,变异系数（ＣＶ）为１．９４％。     

Validation of a method for the analysis of biogenic amines: Histamine instability during wine sample storage

This paper reports on the development of an optimized method for the simultaneous analysis of eight biogenic amines (histamine, methylamine, ethylamine, tyramine, putrescine, cadaverine, phenethylamine, and isoamylamine). The analytical method thus proposed has the following advantages: the easy derivatization of wine, the quantification of biogenic amines and a complete degradation of excess derivatization reagent during sample preparation in order to preserve the column. It consists in reversed phase separation by HPLC and UV–vis detection of the aminoenones formed by the reaction of amino compounds with the derivatization reagent diethyl ethoxymethylenemalonate (DEEMM). The usefulness of this technique was confirmed by an alternative oenological analytical method for the validation, quality control and uncertainty assessment (OIV Oeno 10/2005). The method was validated and proposed as a reference method to the International Organization of Vine and Wine (OIV). As a specific application of the proposed method, the biogenic amine content of Rhône valley wines was investigated.     

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。     

Single-Run Separation and Determination of Aliphatic and Aromatic Carboxylic Acids in Wine and Human Urine Samples by Ion-Exclusion Chromatography

This work is focused on developing a new chromatographic method with UV detection for the separation and determination of thirty-two carboxylic aliphatic and/or aromatic acids in a single run. Ion-exclusion chromatography with a silica based, modified with C18, analytical column Alltech Prevail? organic acid 5 µm (150 mm × 4.6 mm I.D) was used for the solving of this problem. The developed method was based on ion-exclusion and/or hydrophobic interaction chromatographic separation mechanism. The effect of the concentrations of phosphate buffer and its pH as well as the column temperatures on the retention of the test acids has been investigated. Gradient elution of the mobile phase composed of aqueous phosphate buffer and methanol was used to achieve a required separation of carboxylic acids within 45 min. All measurements were done at 220 nm. Column temperature was set at 25 ± 0.1 °C. The LOD values for organic acids range from 0.002–2.224 mg L^?1. The repeatability of the procedure developed is characterized by the RSD, which varied between 0.52 and 2.85 % for the peak area. The proposed method was successfully applied for the determination of aliphatic and aromatic acids in dry white wine and human urine samples.     

Novel approach for the rapid determination of water-soluble organic acids in wine by co-electroosmotic flow capillary zone electrophoresis

An innovative protocol for the fast analysis of some organic acids in red wine by co-electroosmotic capillary zone electrophoresis and indirect UV detection using hexadimethrine bromide (HDB) as coating agent was proposed. The adsorption of HDB onto the capillary wall provided a stable electroosmotic flow and separation of small anions was carried out using background electrolytes containing no polymer additive. Low RSD% values (<3.6%) in terms of migration times and effective mobilities were obtained from the analysis of a mixture of nitrate and nitrite and of a mixture of organic acids. An experimental design approach was used to investigate the effects of temperature, separation voltage, and percentage of methanol added to the running buffer solution on the separation of the analytes. A faster method allowing the separation of the organic acids involved in the malolactic fermentation of wine was developed. Using a running electrolyte consisting of 35% (v/v) methanol in a solution of 22 mM benzoic acid at pH 6.10 adjusted with 1.0 M TRIS-base buffer, the separation of tartaric, malic, succinic, acetic, and lactic acids was feasible in less than 210 s. Application of the method to the quantification of the above-mentioned organic acids in Italian red wine samples is reported.     

Determination of biogenic amines by capillary electrophoresis

A method for determining biogenic amines in food using micellar electrokinetic capillary chromatography has been developed. Derivatization of the amines was performed with AccQ (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; Waters, Milford, MA, USA) reagent. The influence of buffer composition on the separation (including pH, SDS concentration and various additives) was investigated. The separation of seven biogenic amines (histamine, tyramine, tryptamine, spermine, spermidine, cadaverine and putrescine) could be achieved within 25-30 min with good repeatability. The biogenic amine profiles in three different food samples (wine, salami and chive) were determined and quantitated.     

Determination of amino acid neurotransmitters in rat hippocampi by HPLC‐UV using NBD‐F as a derivative

A simple, rapid and accurate high‐performance liquid chromatography method with ultraviolet–visible detection was developed for the determination of five amino acid neurotransmitters – aspartate, glutamic acid, glycine, taurine and γ‐aminobutyric acid – in rat hippocampi with pre‐column derivatization with 4‐fluoro‐7‐nitrobenzofurazan. Several conditions which influenced derivatization and separation, such as pH, temperature, acetonitrile percentage mobile phase and flow rate, were optimized to obtain a suitable protocol for amino acids quantification in samples. The separation of the five neurotransmitter derivatives was performed on a C₁₈ column using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0)–acetonitrile (84:16, v/v) at a flow rate of 1.0 mL/min with the column temperature at 30°C. The detection wavelength was 472 nm. Without gradient elution, the five neurotransmitter derivatives were completely separated within 15 min. The linear relation was good in the range from 0.50 to 500 µmol/L, and the correlation coefficients were ≥0.999. Intra‐day precision was between 1.8 and 3.2%, and inter‐day precision was between 2.4 and 4.7%. The limits of detection (signal‐to‐noise ratio 3) were from 0.02 to 0.15 µmol/L. The established method was used to determine amino acid neurotransmitters in rat hippocampi with satisfactory recoveries varying from 94.9 to 105.2%. Copyright © 2013 John Wiley & Sons, Ltd.     

Improvement of extraction procedure for biogenic amines in foods and their high-performance liquid chromatographic determination.

A high-performance liquid chromatography method is described for the simultaneous determination of the biogenic amines tryptamine, 2-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine in cheese. The optimization of the procedure for the extraction of amines from the matrix is described. The separation of dansyl derivatives of the amines was achieved by reversed-phase liquid chromatography with gradient elution, followed by UV detection at 254 nm. The mobile phase was acetonitrile-0.01 M phosphate buffer (pH 7)-water. Under these conditions, rapid elution of the amines in less than 13 min was obtained. Validation of the method included calibration experiments, addition of standard amines for the determination of amine recoveries and repeatability tests.     

Development of a precolumn derivatization HPLC method with diode‐array detection for the determination of amino sugars in peat and soil humic acids

The work is focused on the development of a high‐performance liquid chromatography method with diode‐array detection for the separation and quantitation of the three most abundant amino sugars; d ‐glucosamine, d ‐galactosamine, and d ‐mannosamine. The high‐performance liquid chromatography separation was carried out by reversed‐phase chromatography on Chromolith Performance RP‐18e monolithic column after acid hydrolysis (5 M HCl) and precolumn derivatization of samples using diethyl ethoxymethylenemalonate. Gradient elution and a mobile phase composed of ammonium formate buffer solution (10 mmol/L, pH 3.60) and methanol with flow rate of 1.0 mL/min were used. The monitoring wavelength was set at 280 nm. The limits of detection and quantitation for analytes ranged from 0.017 to 0.122 mg/L and from 0.057 to 0.407 mg/L, respectively. The proposed method was successfully applied for the determination of amino sugars in samples of humic acids isolated from different soils and peat.     

8-Phenyl-(4-oxy-acetic acid N-hydroxysuccinimidyl ester)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene as a new highly fluorescent-derivatizing reagent for aliphatic amines in disease-related samples with high-performance liquid chromatography

A novel fluorescent-activated ester, 8-phenyl-(4-oxy-acetic acid N-hydroxysuccinimidyl ester)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (TMPAB-OSu) has been designed and synthesized for amine labeling in HPLC. Being used 11 aliphatic amines as the models, the derivatization conditions were optimized. In 0.2 mol/l borate buffer (pH 8.8), amines reacted with TMPAB-OSu at 30 °C to form the derivatives in 10 min. The fluorescent quantum yield of TMPAB-OSu and its amine derivatives are high even compared with fluorescein. The separation of these amine derivatives was achieved with a C₈ column and gradient elution by using 0.1 mol/l sodium acetate buffer (pH 5.0) and methanol. With fluorescence detection at an emission wavelength of 509 nm and an excitation wavelength of 497 nm, the detection limits of aliphatic amines were 2–18 fmol, at a signal-to-noise ratio of 3:1. The proposed TMPAB-OSu-based HPLC method has been applied to the analysis of urine samples of health, hepatic and renal patients and lake water. Recoveries from different matrices are from 96 to 104%, depending on the sample investigated.     

胶束电动毛细管色谱检测鱼肉中的七种生物胺

建立了一种利用胶束电动毛细管色谱同时检测鱼肉中组胺、腐胺、2-苯乙基胺、尸胺、色胺、亚精胺及精胺7种生物胺的方法。样品经6%过氯酸萃取后,由苯甲酰氯衍生化,以含0.06 mol/L脱氧胆酸钠的0.02 mol/L硼酸(pH 9.2)-甲醇(体积比为95∶5)混合液为电泳介质,电泳电压25 kV,温度25 ℃,检测波长214 nm,在12 min内实现了7种生物胺的完全分离。7种生物胺的浓度与其峰面积在一定的范围呈良好的线性关系,检出限除组胺为15 μg/g外,其余均为5 μg/g。迁移时间和峰面积的日内、日间相对标准偏差均小于5%。该法用于海鱼中7种胺类物质含量的测定,结果令人满意。     

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.