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。     

新型衍生试剂柱前衍生氨基酸的高效液相色谱分析

以邻硝基苯磺酰氯为标记试剂,RP-HPLC为分析模式,建立了一种新的氨基酸衍生化方法.通过液质联用对产物进行定性,研究并确定了最佳衍生化条件:衍生温度25 ℃,缓冲液pH9.0,衍生时间10 min.实验建立了20种氨基酸的HPLC分离方法:选用Kromasil C18柱,流动相A为30 mmol/L的NH4Ac溶液(pH 7.5),B相为乙腈;采用梯度洗脱,检测波长275 nm,室温.20种氨基酸在0.025 ～6.4 mmol/L范围内线性关系良好,相关系数在0.998 0 ～0.999 8之间,检出限为0.01 ～0.113 nmol.     

对甲基苯磺酰氯柱前衍生法测定水中哌嗪

建立了一种高效液相柱前衍生化测定水中哌嗪含量的方法。选取对甲基苯磺酰氯(p-TSC)为衍生试剂对哌嗪进行衍生化,衍生产物利用液质联用(LC-MS)技术定性,高效液相色谱定量分析。方法采用Ultimate XB-C18色谱柱,以甲醇-水(体积比60:40)为流动相,流速为0.8 mL/min,检测波长为240 nm,室温下检测。实验结果表明,哌嗪的衍生化产物与过量衍生化试剂分离良好,在10～100 mg/L的范围内,哌嗪的浓度与其衍生产物峰面积呈现良好的线性关系,相关系数r>0.999。方法的检出限为0.09 mg/L,定量限为0.30 mg/L。回收率范围为88.2%～99.2%,相对标准偏差为2.5%～5.3%。通过正交反应试验确定了最佳衍生化条件:衍生温度为55℃,缓冲液pH为10.0,衍生时间10 min。     

邻硝基苯磺酰氯柱前衍生-高效液相色谱法分析DL-丝氨酸

以邻硝基苯磺酰氯(NBSC)为衍生化试剂,建立了柱前衍生DL-丝氨酸的反相高效液相色谱紫外检测的方法,对工业成品DL-丝氨酸及其杂质进行分离和测定,并对衍生化条件进行了优化。最佳反应条件为:DL-丝氨酸与NBSC的摩尔比为1:4,衍生温度为室温,反应时间为10 min,pH 9硼砂缓冲溶液。色谱采用Hedera-C18柱,流速0.7 mL/min,柱温为室温,检测波长为228 nm,流动相为体积比15:85的乙腈和磷酸盐缓冲液(pH 5.8)。实验结果表明,用0.1 mol/L的HCl溶液溶解的DL-丝氨酸浓度在5～100 mmol/L范围内线性关系良好,相关系数(r2)为0.9994,加标回收率94.0%～100.6%,DL-丝氨酸衍生产物峰面积的相对标准偏差为0.026%,检出限为0.3 mmol/L。该方法可以作为批量生产DL-丝氨酸产品质量控制的方法。     

草甘膦的邻硝基苯磺酰氯柱前衍生高效液相色谱分析

以邻硝基苯磺酰氯(NBSC)为衍生化试剂,建立了柱前衍生草甘膦的反相高效液相色谱紫外检测法,并对衍生化条件进行了优化.最佳衍生化条件为:衍生温度25℃,反应时间10 min,硼砂缓冲溶液浓度0.25 mol/L(pH 9.0),草甘膦与NBSC的摩尔比为1:5.HPLC分析条件为:采用Lichrospher C18柱,...     

高效液相色谱法鉴别及测定中药吴茱萸中的生物胺

建立了高效液相色谱法(HPLC)测定中药吴茱萸中生物胺的方法。以丹磺酰氯为柱前衍生试剂,采用岛津高效液相色谱仪(LC-10A),色谱柱SHIMADZU-C18(150 mm×4.6 mm×5μm)进行测定,确定了最佳色谱条件:乙腈和水为流动相,梯度洗脱,DAD检测器,检测波长为334 nm,流速为0.8 mL/min,柱温30℃,进样量20μL。10种常见生物胺在45 min内得到良好的分离,在给定的浓度范围内,各生物胺呈现良好的线性相关(R2>0.999),精密度RSD<2%,回收率在91.87%~101.35%之间,重现性RSD<8%。结果表明,吴茱萸叶中含有7种生物胺,枝中含有4种,果中含有5种。这是首次发现在中药吴茱萸中存在生物胺类成分。     

手性试剂柱前衍生化高效液相色谱法测定α-苯乙胺的光学纯度

建立了一种简便的手性试剂柱前衍生化反相高效液相色谱测定α-苯乙胺光学纯度的方法。采用2,3,4,6-四-O-β-D-吡喃葡萄糖异氰酸酯(GITC)对α-苯乙胺进行衍生化,优化了衍生化反应参数;使用Agilent Zorbax C18色谱柱分离衍生化产物,流动相为甲醇-磷酸盐缓冲溶液(pH 3.0)(体积比为58:42),流速1.0 mL/min,检测波长241 nm,柱温为30 ℃。实验结果表明,α-苯乙胺两个对映体的衍生化产物分离良好,在0.15～15.0 mg/L范围内呈现良好的线性关系。方法的检出限为0.05 mg/L,定量限为0.15 mg/L,日内和日间精密度考察中测定值的相对标准偏差(RSD)均小于0.5%。建立的方法适用于α-苯乙胺的质量控制。     

毛细管电泳分离-激光诱导荧光检测生物胺

采用新合成的6-氧-[(1-琥珀酰亚胺)氧酰甲基]-荧光素乙酯(SOFE)作为柱前衍生试剂,毛细管电泳分离-激光诱导荧光检测了乙醇胺(EOA)、组胺(His)、甲胺(MA)、乙胺(EA)、酪胺(TYR)及苯乙胺(PEA)6种生物胺。在硼酸-硼砂缓冲溶液(pH8.5)中,室温(25℃)下衍生10min。用pH8.5的含25mmol/LSDS15%(V/V)乙腈的40mmol/L硼酸盐溶液作为电泳缓冲溶液,6种衍生物在15min内完全分离,检出限在2.5×10-11～8×10-11mol/L之间。该方法应用于酱油中生物胺的测定,结果令人满意。     

柱前衍生法测定烟叶中的7种生物胺

建立了丹磺酰氯柱前衍生高效液相色谱法(HPLC)测定烟草基因编辑素材中的7种生物胺的方法。采用0.4 mol/L HClO_4进行样品超声提取,提取液经过pH调整后,利用丹磺酰氯(4 mg/mL)进行柱前衍生;衍生后的样品溶液利用优化的HPLC-DAD色谱方法进行检测;方法在1.0～50μg/mL范围内具有较好的线性(R~2 0.98),检出限在0.01～0.15μg/mL之间,定量限在0.05～0.25μg/mL之间,日间和日内精密度均小于5%,不同加标水平下,平均回收率在86.7%～124.0%之间。     

柱前衍生高效液相色谱法检测加巴喷丁衍生化条件的研究

采用紫外衍生剂对甲氧基苯磺酰氯(MOBS–Cl)对加巴喷丁进行衍生反应,建立了一种柱前衍生测定加巴喷丁的新方法。最佳衍生化条件:对甲氧基苯磺酰氯与加巴喷丁的质量比大于7.5∶1,于50℃水浴中反应30 min。以0.05 mol/L Na2HPO4缓冲液(pH 10.0)–甲醇混合液为流动相,分析柱为反相ODS柱(150 mm×4.6 mm,5μm),柱温为室温,流速为0.8 mL/min,检测波长为240 nm。加巴喷丁溶液质量浓度在1~100 mg/L范围内与色谱峰面积线性关系良好,线性方程为A=24.5667c+12.3025,相关系数r=0.9999,测定结果的相对标准偏差(RSD)为0.56%,加巴喷丁的检测下限为0.017μg/g。     

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.     

Determination of biogenic amines in HeLa cell lysate by 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein and micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

An MEKC-LIF method using 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxy-carbonyl) fluorescein (SAMF) newly synthesized in our lab as a labeling reagent for the separation and determination of eight typical biogenic amines was proposed. After careful study of the derivatization condition such as pH value, reagent concentration, temperature, and reaction time, derivatization reaction was accomplished as quickly as 10 min with stable yield. Optimal separation of SAMF-labeled amines was achieved with a running buffer (pH 9.3) containing 30 mM boric acid, 25 mM SDS, and 20% v/v ACN. The proposed method allowed biogenic amines to be determined with LODs as low as 0.25-2.5 nmol/L and RSD values from 0.4 to 4.5%. The present method has been successfully used to monitor biogenic amines in HeLa cells and fish samples. This study exploits the potential of MEKC-LIF with SAMF labeling as a tool for monitoring biogenic amines involved in complex physiological and behavioral processes in various matrices.     

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.     

Optimization of experimental variables in the dabsyl chloride derivatization of biogenic amines for their determination by RP-HPLC

Summary In the last few years special attention has been paid to the pre-column derivatization of biogenic amines with dabsyl chloride because proper experimental conditions for this reaction are very important. In this study, an experimental design (Doehlert design) was used to optimize the variables involved in the dabsylation of the following amines: histamine, tyramine, phenylethylamine, tryptamine, cadaverine, putrescine, spermidine, and spermine. The optimum experimental conditions for forming the dabsyl derivatives are: reagent concentration, 1.75.10⁻³ M; pH, 8.2; temperature, 70°C; heating time (t _h ), 21 min. Under these conditions good chromatographic repeatability is obtained.     

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.     

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.     

Sensitive determination of biogenic amines by capillary electrophoresis with a new fluorogenic reagent 3-(4-fluorobenzoyl)-2-quinolinecarboxaldehyde

An effective approach was proposed to the derivatization of seven biogenic amines using 3-(4-fluorobenzoyl)-2-quinolinecarboxaldehyde (FBQCA) as a fluorogenic reagent. The sensitive determinations of these derivatives were achieved by micellar electrokinetic capillary chromatography (MEKC) with laser-induced fluorescence (LIF) detection. The derivatization and electrophoretic conditions have been optimized. A running buffer was composed of mixtures of 25 mM pH 9.5 boric acid, 25 mM SDS, and 27% ACN. At 25 °C and 22.5 kV, the baseline separation of the derivatives was accomplished in 13 min. The detection limit (S/N = 3) was found as low as 0.4 nM. The proposed method was validated by the linearity of two orders magnitude and correlation coefficient in the range 0.9969–0.9998. Also, the procedure was successfully applied to the determination of biogenic amines in soy sauce, fish and wine samples.     

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.     

Poly(dimethylsiloxane) microchip for precolumn reaction and micellar electrokinetic chromatography of biogenic amines

We have demonstrated that precolumn derivatization and capillary electrophoresis separation on a poly(dimethylsiloxane) (PDMS) microchip can be realized as efficient as those on glass microchips. In an optimized condition of micellar electrokinetic chromatography (MEKC), using 25 mM sodium borate buffer (pH 10.0) with 25 mM sodium dodecyl sulfate (SDS) and 5% v/v methanol, the electroosmotic flow in an oxidized PDMS microchip is stabilized within 3% for days. By employing a fluorometric derivatization with o-phthaldialdehyde (OPA) in an optimally designed reaction chamber, four most important biogenic amines occurring in foods, histamine, tyramine, putrescine, and tryptamine, are quantitatively determined in less than 1 min at the levels applicable to real samples. The migration behaviors of anionic OPA-derivatized biogenic amines under the MEKC conditions are analyzed, and it has been found that under our separation conditions, the electrophoretic mobility of the SDS micelles is significantly greater than those of the anions in the aqueous phase. The channel manifold in a PDMS substrate is fabricated using replica molding against a thick photoresist, SU-8, pattern generated by photolithography. The plate with the microchannel pattern is strongly, irreversibly bonded to another PDMS plate by using a new bonding technique, which employs surface oxidation by corona discharge generated from a cheap, handy source, Tesla coil.