Fast separation and sensitive detection of carcinogenic aromatic amines by reversed-phase micro-liquid chromatography coupled with electrochemical detection

A micro-LC method was developed for the fast and sensitive analysis of aromatic amines by electrochemical detection. The chromatographic separation of nine carcinogenic aromatic amines was performed on an ABZ + PLUS column with detection limits up to pM L(-1) levels. Mobile phase comprised of methanol-acetate buffer of pH 5 (45:55, v/v) used at a flow rate of 0.2 ml min(-1). The detection was performed with a 6 mm glassy carbon electrode at an applied potential of 0.8 V versus Ag/AgCl. An intraday RSD for retention time and peak area were between 0.22% and 0.73% and 1.86% and 4.03%, respectively. The interdays RSD for retention time and peak area were between 0.47% and 1.35% and 2.04% and 4.42%, respectively. The applicability of the assay has been demonstrated by analyzing these aromatic amines in lake water and synthetic food colour additives. A comparison is given between electrochemical and UV detection.     

Ionic liquids as mobile phase additives in high-performance liquid chromatography with electrochemical detection: Application to the determination of heterocyclic aromatic amines in meat-based infant foods

The beneficial effects of several ionic liquids (ILs) as mobile phase additives in high-performance liquid chromatography with electrochemical detection for the determination of six heterocyclic aromatic amines (HAs) have been evaluated for first-time. The studied ionic liquids were 1-butyl-3-methylimidazolium tetrafluoroborate (BMIm-BF₄), 1-hexyl-3-methylimidazolium tetrafluoroborate (HMIm-BF₄) and 1-methyl-3-octylimidazolium tetrafluoroborate (MOIm-BF₄). Several chromatographic parameters have been evaluated in the presence or absence of ILs, or using ammonium acetate as the most common mobile phase additive, with three different C18 stationary phases. The effect of the acetonitrile content was also addressed. In general, best resolution, lower peak-widths (up to 72.1% lower) and lower retention factors are obtained when using ILs rather than ammonium acetate as mobile phase additives. The main improvement was obtained in the baseline noise, being 360% less noisy for BMIm-BF₄, 310% for HMIm-BF₄, and 227% for MOIm-BF₄, when compared to ammonium acetate at +1000 mV. Different chromatographic methods using the best conditions for each IL were also evaluated and compared. Finally, the best chromatographic conditions using 1 mM of BMIm-BF₄ as mobile phase additive, the Nova-Pak^® C18 column, 19% (v/v) of acetonitrile content in the mobile phase, and +1000 mV in the ECD, have been applied for the chromatographic analysis of six HAs contained in meat-based infant foods. The whole extraction method of meat-based infant foods using focused microwave-assisted extraction and solid-phase extraction has also been optimized. Extraction efficiencies up to 89% and detection limits ranged between 9.30 and 0.165 ng g⁻¹ have been obtained under optimized conditions.     

毛细管电泳-场强放大样品堆积法检测染发剂中的7种苯胺类物质

建立了毛细管电泳-场强放大样品堆积测定染发剂中4,4′-二氨基二苯甲烷、苯胺、邻甲氧基苯胺、对氨基苯甲醚、3,4-二甲基苯胺、间氨基苯酚、1-萘胺7种苯胺类物质的分析方法。在优化的缓冲溶液体系(0.15 mol/L NaH2PO4,0.015 mol/L 三乙醇胺, pH 2.3)下7种分析物在6.5 min内实现基线分离。考察了样品中添加的磷酸浓度和乙腈浓度、水柱长度、电动进样时间与电压对场强放大富集效率及重现性的影响。最佳的富集条件为: 水柱注入3.45 kPa(0.5 psi)×6 s,样品中添加40%(v/v)乙腈和0.6×10~3mol/L磷酸,进样电压与进样时间为10 kV×10 s。线性范围为3～1000 μg/L(R2＞0.996),检出限为0.26～2.75 μg/L,将已有方法的检测灵敏度提高了1～3个数量级。在2种市售黑色染发剂中均检测到间氨基苯酚,含量分别为7.32 mg/g和1.34 mg/g。平均加标回收率为74%～108%。该方法灵敏度高、快速、重现性好、成本低,可供多种样品基质中痕量苯胺类污染物及其他阳离子物质的测定借鉴使用。     

高效液相色谱法同时测定染发剂中11种氨基苯酚

建立了高效液相色谱测定直接型与氧化型染发剂中11种氨基苯酚含量的分析方法。样品在亚硫酸氢钠的保护下用甲醇提取,高速冷冻离心净化;采用Agilent Zorbax SB C18色谱柱分离,流动相为5 mmol/L庚烷磺酸钠-40 mmol/L pH 2.8磷酸盐缓冲液和乙腈,梯度洗脱,11种氨基苯酚分别于230、270和400 nm下检测。11种氨基苯酚在0.05～500 mg/L线性范围内相关系数不小于0.9992,4-氨基-3-硝基苯酚、2-氨基-5-硝基苯酚、3-硝基-4-(2-羟乙基)氨基苯酚的方法定量限为5 mg/kg,其他8种氨基苯酚包括对氨基苯酚、对甲氨基苯酚、间氨基苯酚、邻氨基苯酚、5-氨基-2-甲基苯酚、4-氨基-3-甲基苯酚、2-甲基-5-(2-羟乙基)氨基苯酚和2-氨基-4-硝基苯酚定量限为20 mg/kg,方法回收率为88.5%～109.5%,相对标准偏差为2.2%～8.3%。测定了多种市售染发剂,结果表明该方法操作简单、测定结果准确,可用于直接型与氧化型染发剂中11种氨基苯酚的测定。     

高效液相色谱法同时测定氧化型染发产品中33种染发剂

建立了高效液相色谱同时测定氧化型染发产品中33种禁限用染发剂含量的分析方法。采用Waters Atlantis^® T3 MV Kit色谱柱（250 mm×4.6 mm，5 μm），以磷酸盐缓冲液-乙腈为流动相，梯度洗脱，流速为1.0 mL/min，柱温为25℃；采用二极管阵列检测器（DAD），检测波长为235及280 nm，进样量为5 μL。结果表明，各成分在线性范围内线性关系良好，相关系数均高于0.999，各成分精密度实验相对标准偏差（RSDs）均小于2%；四氨基嘧啶硫酸盐和2，4-二氨基苯氧基乙醇盐酸盐在12 h内稳定性实验RSDs小于5%，其余31种成分在24 h内稳定性实验RSDs均小于5%；各成分在3个浓度水平下的加标回收率为77.6%~116.3%。该方法简便、快速、准确，适用于氧化型染发产品中多种染发剂的检测。     

离子液体作高效液相色谱流动相添加剂分离测定芳香胺

建立了以离子液体作反相高效液相色谱流动相添加剂分离测定邻苯二胺、苯胺和对甲苯胺3种芳香胺的方法。实验以C18反相色谱柱为分离柱,采用紫外检测方法,考察了检测波长、甲醇含量、咪唑离子液体烷基链长度、离子液体溶液浓度等条件对分离和测定的影响,并与其它分离测定芳香胺的方法进行了比较。优化的色谱条件为:以甲醇/1-丁基-3-甲基咪唑四氟硼酸盐水溶液(3.0mmol/L,乙酸调节pH 3.5)=30/70(V/V)为流动相;检测波长254 nm;流速1.0mL/min;柱温30℃。在此条件下,3种芳香胺达到基线分离,在6.5 min之内分离完全;在1～40 mg/L范围内,线性回归方程的相关系数达到0.99以上;检出限为0.07～0.41 mg/L。将本方法应用于废水的测定,加标回收率在92.3%～96.7%之间,相对标准偏差小于3.5%。     

Analytical approach to determine biogenic amines in urine using microextraction in packed syringe and liquid chromatography coupled to electrochemical detection

The goal of this work was to develop and validate an analytical method for the detection and quantification of the biogenic amines serotonin (5‐HT), dopamine (DA) and norepinephrine (NE), using microextraction in packed syringe (MEPS) and liquid chromatography coupled to electrochemical detection (HPLC‐ED) in urine. The method was validated according to internationally accepted guidelines from the Food and Drug Administration. Linearity was established between 50 and 1000 ng/mL for 5‐HT and between 5 and 1000 ng/mL for DA and NE, with determination coefficients (R²) >0.99 for all compounds. The limits of quantification and detection were respectively 50 and 20 ng/mL for 5‐HT, and 5 and 2 ng/mL for DA and NE. Within‐ and between‐run precision ranged from 0.84 to 9.41%, while accuracy ranged from 0.79 to 12.76% for all compounds. The intermediate precision and accuracy were 1.50–8.36 and 0.54–13.51%, respectively. The method was found suitable for clinical routine analysis of the studied compounds, using a sample volume of 0.5 mL. This is the first study employing a commercially available MEPS column for the simultaneous detection and quantification of 5‐HT, DA and NE in urine by coulometric detection. Copyright © 2012 John Wiley & Sons, Ltd.     

Gunshot residue determination by high-performance liquid chromatography with electrochemical detection

A procedure for the detection of gunshot residue via the organic constituent diphenylamine is described. The method incorporates high-performance liquid chromatography with electrochemical detection.     

Amine oxidase amperometric biosensor coupled to liquid chromatography for biogenic amines determination

A selective and sensitive method is presented for biogenic amines (BA) determination. The novelty consists in coupling a highly selective electrochemical biosensor to a weak acid cation-exchange column for online detection of amines. A bienzyme design, based on a recently isolated amine oxidase from grass pea and commercial horseradish peroxidase, was used for the biosensor construction. The enzymes were co-immobilized on the surface of a graphite electrode together with the electrochemical mediator (Os-redox polymer). The electrochemical detection was performed at a low applied potential (?50 mV vs. Ag/AgCl, KCl_0.1 M), where biases from interferences are minimal. The separation and determination of six BA, with relevance in food analysis (tyramine, putrescine, cadaverine, histamine, agmatine and spermidine), were investigated. Irrespective of the BA nature, the amine oxidase-based biosensor showed a linear response up to 5 mM, and its sensitivity decreases in the following order: cadaverine, putrescine, spermidine, agmatine, histamine and tyramine. The approach was used to estimate the BA content in fish samples, after their extraction with methanesulfonic acid.     

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 dye precursors in hair coloring products by liquid chromatography with electrochemical detection

The simultaneous determination of seven aminophenols, resorcinol and p-phenylenediamine in hair coloring products was performed by liquid chromatography (HPLC) with amperometric detection (ED). The aminophenols were separated on a ODS C18 reversed-phase column by isocratic elution with a mobile phase based on 0.1 M acetate buffer pH 4.5-methanol (90:10%, v/v) at a flow rate 0.8 mL min⁻¹. The limit of detection (S/N = 3) for the aminophenols was in the 15-40 pg (injected mass) range at an applied potential of 0.950 V versus Ag/AgCl. Peak heights for the aminophenols and the two others compounds were found to be linearly related to the amount injected, from 0.3 to 300 ng (r > 0.994-0.999).The relative standard deviation (R.S.D., n = 10) for 1 ng injected was comprised in the range from 2.5 to 6.2%, depending on the aminophenol tested. The present method minimizes troublesome and time-consuming pretreatment procedures and it was applied to the determination of aminophenols, resorcinol and phenylenediamine in hair coloring formulations.     

Attomole catechins determination by capillary liquid chromatography with electrochemical detection.

Attomole quantities of catechins were determined by a capillary liquid chromatography system with electrochemical detection (CLC-ECD) and the system is applied to the determination of catechins in human plasma. The eight catechins: catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg), and epigallocatechin gallate (EGCg), were separated within 10 min using a capillary column (0.2 mm i.d.) and a mobile phase of phosphoric acid (85%)-methanol-water (0.5:27.5:72.5, v/v/v), and were detected at +0.85 V vs. Ag/AgCl. Peak heights were found to be linearly related to the amount of catechins injected, from 200 amol to 500 fmol (r > 0.998). The detection limits of the catechins were 61 amol for EGC, 75 amol for EC, 54 amol for GC, 61 amol for C, 67 amol for GCg, 75 amol for EGCg, 75 amol for ECg and 89 amol for Cg (S/N = 3). Because the present method is highly sensitive and allows facile pretreatment for plasma sample, the time courses of concentrations of catechins (GCg, EC, EGCg, ECg, and Cg) and their conjugates in human plasma obtained from a 10 microl plasma sample after ingestion of green tea could be determined.     

Solid-phase microextraction coupled with high-performance liquid chromatography for the analysis of heterocyclic aromatic amines

Solid-phase microextraction (SPME) coupled with high-performance liquid chromatography (HPLC) with UV diode array detection (DAD) for the analysis of heterocyclic aromatic amines (HAs) is described. Four kinds of fiber coatings: Carbowax-templated resin (CW-TPR), Carbowax-divinylbenzene (CW-DVB), poly(dimethylsiloxane)-divinylbenzene (PDMS-DVB) and polyacrylate (PA) were evaluated for extraction of nine most biologically active heterocyclic aromatic amines. Different parameters affecting to the microextraction and determination of HAs were studied, such as absorption and desorption time, desorption mode, composition of the solvent for desorption, pH, ionic strength, and percentage of methanol in the sample. To determine these amines in food samples a new simplified procedure is proposed, consisting of treatment of the sample with methanolic NaOH prior microextraction by CW-TPR fiber coating and HPLC-DAD determination. The advantages of this new method are the reduced amounts of time and organic solvents required.     

Analysis of biogenic amines by solid-phase microextraction and high-performance liquid chromatography with electrochemical detection

We investigated the new solid-phase microextraction method by high-performance liquid chromatography with electrochemical detection for the analysis of biogenic amines. The Carbowax–Templated Resin 50 μm (purple) fibre coating offers good performances for dopamine and serotonin separation, i.e., good selectivity and high sensibility (0.1 μg l⁻¹). We also tested this fibre for biogenic amines quantification of rat striatum. The coating seems to be selective towards the amines and has low affinity for the metabolites, allowing a good separation and preventing drawbacks from the biological matrix. These first results obtained using this original separation method offer large perspectives of application to many biological samples.     

Sensitive determination of aliphatic amines in water by high-performance liquid chromatography with chemiluminescence detection

A sensitive method has been developed for liquid chromatographic determination of short aliphatic amines in water samples. Analytes are preconcentrated and dansylated on solid sorbents (C18 solid-phase extraction cartridges). The dansyl derivatives are chromatographed and post-column mixed with peroxyoxalate (TCPO) and H2O2 in order to perform chemiluminescence detection. Optimal results have been obtained using a sample volume of 5 ml. The method has been applied to the quantification or screening of several aliphatic amines: methylamine, ethylamine, butylamine, diethylamine, pentylamine and hexylamine. The screening procedure has been developed including also polyamines (putrescine, cadaverine, spermidine and spermine). The results obtained by using chemiluminescence (CL) detection have been compared with other detection systems (fluorescence and UV). The sensitivity can increase from 3 to 75 times respect UV detection and from 2 to 10 times respect fluorescence detection depending on the amine. The detection limits achieved were between 0.15 and 0.9 microg/l.     

Simultaneous determination of dihydroxybenzenes, aminophenols and phenylenediamines in hair dyes by high-performance liquid chromatography on hypercross-linked polystyrene

The retention of polar organic molecules such as dihydroxybenzenes, aminophenols and phenylenediamines on a 250 x 4.6 mm id column packed with 5 microns hypercross-linked polystyrene Chromalite 5HGN (Purolite) was studied. The influence of separation parameters such as concentration of acetonitrile, buffer (citrate, phosphate) concentration, ionic strength and pH of the eluent on their retention was investigated. Under optimum conditions [acetonitrile-0.3 mol l-1 ammonium phosphate, pH 5.15 (30:70 v/v)], eight substances generally used as dye intermediates in hair colouring compositions could be separated within 20 min. An HPLC method with spectrophotometric detection is proposed for the simultaneous determination of pyrocatechol, resorcinol, hydroquinone, o-, m- and p-aminophenols and p-phenylenediamine in commercial haircolour products. The detection limits of these compounds are in the range 0.05-0.16 microgram ml-1. The suitability of the method is demonstrated by the analysis of three different permanent hair dyes.     

超高效液相色谱-三重四极杆质谱法同时测定美术颜料中33种游离态初级芳香胺

建立了超高效液相色谱-三重四极杆质谱法(UPLC-MS/MS)同时测定水粉画颜料、油画颜料、丙烯画颜料等美术颜料中33种初级芳香胺(PAAs)的检测方法。样品中的初级芳香胺用乙腈提取,经离心分离、氮吹浓缩后,以甲醇-水(1:9, v/v)定容至2 mL, 0.22 μm膜过滤后上机检测。采用BEH Phenyl柱(100 mm×2.1 mm, 1.7 μm),以含0.07%(v/v)甲酸的甲醇溶液-水(1:9, v/v)为流动相,梯度洗脱分离,UPLC-MS/MS多反应监测模式(MRM)检测,同位素内标法定量。方法优化了色谱分离条件、质谱碎裂电压、碰撞能量等,并考察了提取时间、提取溶剂、浓缩方式等对回收率的影响。33种初级芳香胺的方法检出限为5~50 μg/kg,定量限为15~150 μg/kg, 3种不同基质样品在3个添加水平的平均回收率为70.1%~115.8%,相对标准偏差(RSD)为2.1%~15%。本方法操作简便、快速、准确、灵敏度高,能满足相关测定的要求。     

Trace determination of nitrated polycyclic aromatic hydrocarbons using liquid chromatography with on-line electrochemical reduction and fluorescence detection

An efficient clean-up procedure coupled with a high performance liquid chromatography (HPLC) with on-line electrochemical (EC) reduction and fluorescence detection (FLD) was developed to quantify nitrated polycyclic aromatic hydrocarbons (NPAHs) in the airborne particulate. In this process, NPAHs were extracted ultrasonically followed by analysis by using a reversed phase column with an aqueous eluent containing 70% aqueous acetonitrile and sodium monocholoroacetate as a buffer solution. The extraction efficiencies were above 83% for 1-nitropyrene and 1,3-dinitropyrene (1,3-DNP) 1,6-DNP, and 1,8-DNP, and calibration graphs were linear with very good correlation coefficients (r>0.999) and the detection limits were in the range of 1.0-2.2 pg for dinitropyrenes and nitropyrene. The proposed method provides a relatively simple and convenient procedure for determining the NPAHs samples in airborne particulate.     

Separation and determination of aminohalogenbenzophenones by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method with electrochemical detection has been developed for the determination of three aminohalogenbenzophenones: 2-amino-2',5-dichlorobenzophenone, 2-amino-5-chlorobenzophenone and 2-amino-5-bromo-2'-fluorobenzophenone, metabolites of benzodiazepinooxazoles and other psychotropic drugs. A mobile phase of methanol-water (65:35), containing 5 mM KH2PO4 appeared to be the optimal when a 4-microns, 60-A Nova-Pak C18 column and a flow-rate of 0.75 ml/min (130 bar) were used. The temperature was optimized at 30 degrees C. The amperometric detector, equipped with glassy carbon electrode, was operated at 1.3 V versus Ag/AgCl in the DC mode. The method was applied to the determination of these compounds at two concentration levels: ppm and ppb (ng/cm3) using 2-amino-5-chlorobenzophenone as internal standard. The limit of determination was 750 pg/ml of biological fluid for each compound, and recoveries greater than 97% were obtained for spiked samples of urine and serum, using C18 Sep-Pak cartridges in the sample clean-up procedure.