On-line coupling of pressurized capillary electrochromatography with end-column amperometric detection for analysis of estrogens

An improved technique, pressurized capillary electrochromatography (pCEC) coupling with end-column amperometric detection (AD), was developed and used for the separation and determination of estrogens. The effects of pH value, composition of mobile phase, concentration of the surfactant sodium dodecyl sulfate (SDS) and applied voltage on separation were investigated. The electrochemical oxidation of diethylstilbestrol (DES), dienestrol (DE), and hexestrol (HEX) could be reliably monitored with a carbon electrode at 0.9 V (vs. Ag/AgCl). The pCEC analyses were performed on a capillary separation column packed with 3 microm C18 particles with an acetonitrile/water (31%: 69%) mobile phase containing Tris buffer (5 mmol/L, pH 4.5) and 4 mmol/L SDS. High voltage up to 12 kV reduced the retention time dramatically and still provided a baseline resolution. In addition, supplementary pressure prevented bubble formation and provided reliability and reproducibility of the pCEC performance. The detection limits for the three estrogens ranged from 1.2 to 2.2x10(-7) mol/L, about 10 20-fold lower than those obtained with pCEC-UV detection. To evaluate the feasibility and reliability of this system, the proposed pCEC-AD method was further demonstrated with fish muscle samples spiked with estrogens.     

Separation of structurally related estrogens using isocratic elution pressurized capillary electrochromatography

In this paper, the pressurized capillary electrochromatography (pCEC) with UV detection was utilized for the separation and determination of three structurally related estrogens, such as diethylstilbestrol (DES), hexestrol (HEX) and dienestrol (DE), which were difficult to be separated by capillary electrophoresis (CE) and HPLC due to their similarity in the structure and charge-to-mass ratios. Experiments were carried out in a commercially available pCEC instrument using a capillary column packed with 3 microm octadecyl silica (ODS). Surfactant sodium dodecyl sulfate (SDS) was introduced in the mobile phase to enhance the speed of analysis. The effective factors on the retention time and separation resolution, such as the applied voltage, supplementary pressure, the pH and the concentration of the buffer solution, the concentration of SDS, and the content of acetonitrile in the mobile phase, were evaluated. Based on the investigation, 31% (v/v) acetonitrile and 69% (v/v) of 10 mmol/L phosphate buffer (pH 6.5) containing 1.0 mmol/L SDS at an applied voltage of -12 kV and a supplementary pressure of 1000 psi were found to be the optimal conditions for pCEC to separate the three estrogens. The method also had been applied to the analysis of fish muscle samples spiked with estrogens.     

Pressure-assisted capillary electrochromatography with electrospray ionization-mass spectrometry based on silica-based monolithic column for rapid analysis of narcotics

A pressure-assisted CEC (pCEC) with ESI-MS based on silica-based monolithic column was developed for rapid analysis of narcotics. Combining the extremely high permeability and separation efficiency of silica-based monolithic column with the high selectivity and sensitivity of pCEC-ESI-MS, the developed system exhibited its prominent advantages in separation and detection. A systematic investigation of the pCEC separation and ESI-MS detection parameters was performed. Experiment results showed that the optimized separation efficiency could be obtained at 8 bar assisted pressure with 25 kV separation voltage, using the solution containing 65% ACN v/v and 20 mmol/L ammonium acetate with pH 6.0 as running buffer. 3 microL/min of sheath liquid was considered as the optimized flow rate since it could provide the maximum signal intensity. Under the optimum conditions, the tested five narcotics could be completely separated within 10 min with the detection limit in the range of 2.0-80 nmol/L. The proposed method has been successfully used for detection of narcotics in real urine samples.     

Rapid separation and sensitive detection method for beta-blockers by pressure-assisted capillary electrochromatography-electrospray ionization mass spectrometry

A new method for rapid separation and sensitive detection of beta-blockers by pressure-assisted capillary electrochromatography (pCEC) with electrospray ionization mass spectrometry (ESI-MS) using silica-based monolithic column was studied in this paper. The proposed method has been confirmed to be very powerful since the fast mass transfer property and good permeability of silica monolithic column was used in this pCEC-ESI-MS system. In this work, a silica monolithic column was prepared with sol-gel method for simultaneous fast separation of beta-blockers. Furthermore, in order to obtain the highly selective and sensitive result of pCEC-ESI-MS, both the CEC separation and MS detection parameters were optimized in detail. Under the optimized conditions, namely 80% acetonitrile and 20% 20 mmol/L ammonium acetate (pH 6.0) as the mobile phase, 20 kV and 8 bar as the separation voltage and the assisted pressure, isopropanol/water (1:1, v/v) containing 7.5 mmol/L acetic acid as the sheath liquid, and 3 microL/min as the flow rate of sheath liquid, seven beta-blockers were well separated within 11 min with detection limits in the range of 0.15-0.80 ng/mL (defined as S/N=3). The recoveries of spiked urine samples of these beta-blockers were between 86.3 and 103% with the RSDs lower than 8.0%. The real samples from some male volunteers were successfully analyzed and confirmed with the proposed method. Comparing with GC-MS or LC-MS, the new method has some superiority (such as fast analysis capacity and simple pretreatment) in clinical practice and doping control.     

加压毛细管电色谱-紫外检测法分析糖皮质激素及其在头发检测中的应用

采用反相加压毛细管电色谱与紫外检测联用技术,建立了一种高效、简便的糖皮质激素分析方法,适用于头发中糖皮质激素的检测。使用C18反相色谱柱,流动相为pH 8.0, 1.5 mmol/L的Tris-乙腈(65:35, v/v),检测波长为245 nm、分离电压为~10 kV、反压为10.5 MPa、泵流速为0.05 mL/min,进行等度洗脱,倍他米松、地塞米松、泼尼松、泼尼松龙、醋酸泼尼松龙、醋酸氢化可的松、醋酸可的松、皮质脂酮等8种激素在20 min内实现快速分离。各组分的质量浓度线性范围达到3个数量级,检出限(S/N=3)在μg/g水平,迁移时间和峰面积的相对标准偏差(RSD)分别小于4.8%和7.4%。将所建立方法应用于头发样品分析,检测前采用蛋白酶水解提取和净化处理样品,不同浓度糖皮质激素的回收率为71%～85%。该研究为糖皮质激素药物暴露监测以及压力检测提供了新手段,有望用于滥用药物的控制和临床诊断。     

亲水作用毛细管整体柱的制备及其用于奶制品中三聚氰胺的加压毛细管电色谱分析

以甲基丙烯酸丁酯(BMA)和3-［N,N-二甲基-［2-(2-甲基丙-2-烯酰氧基)乙基］铵］丙烷-1-磺酸内盐(SPE)为单体,制备了新型的亲水作用毛细管整体柱,并通过三聚氰胺在此柱上的保留行为证明其具有亲水性。以加压毛细管电色谱(pCEC)技术为平台,优化了整体柱基于亲水作用分离分析奶制品中三聚氰胺的色谱条件。当流动相中乙腈与10 mmol/L磷酸盐缓冲液的体积比为80:20, pH为3.0,电压为3 kV,检测波长为215 nm时,三聚氰胺能获得很好的分离。方法学考察结果表明,合成的亲水整体柱具有良好的重现性和渗透性,建立的pCEC分析方法的检出限为0.05 mg/L。该方法简单方便,回收率较高,而且流动相中无需添加离子对试剂,适合于奶制品中三聚氰胺的定量测定。     

Development of a new method for analysis of Sudan dyes by pressurized CEC with amperometric detection

A new analytical method, pressurized CEC (pCEC) with amperometric detection (AD) using 1.5 microm RP nonporous silica packed columns has been developed for the rapid separation and determination of four Sudan dyes in hot chilli. The influence of several experimental parameters on the retention behavior has been investigated. The electrochemical oxidation of Sudans I-IV separated by pCEC can be reliably monitored with a carbon electrode at +0.95 V (vs. Ag/AgCl). Fast and efficient separation of the analytes was achieved within 7 min by pCEC under the optimum conditions with an ACN/water (95:5%) mobile phase containing formic acid (pH 4.3), 5% acetone and 0.002% triethylamine using a separation voltage of 12 kV. The detection limits for four Sudan dyes ranged from 8.0 x 10(-7) to 1.2 x 10(-6) mol/L. To evaluate the feasibility and reliability of this method, the proposed pCEC-AD method was further demonstrated with hot chilli samples spiked with Sudan dyes.     

亚微米C18键合硅胶固定相加压毛细管电色谱法同时拆分3种手性三唑类农药

采用改良Stöber法制备420 nm亚微米单分散二氧化硅微球,采用C18硅烷化修饰后装填成毛细管色谱柱。采用该色谱柱,在加压毛细管电色谱平台上成功地实现了3对手性三唑类农药烯效唑、烯唑醇和丙环唑的同时拆分和分离。考察了各因素对手性分离效果的影响,优化后的色谱条件为：流动相为乙腈-20 mmol/L磷酸盐缓冲液（pH=6.8）（45：55,v/v）,其中缓冲液中含20 mmol/L羟丙基-γ-环糊精（HP-γ-CD）;泵流速为0.04 mL/min;施加电压-9.4 kV;检测波长220 nm。在上述条件下,烯效唑、烯唑醇和丙环唑3种对映体同时得到拆分和分离,相邻两峰之间的分离度依次为4.20、12.9、4.41、4.09、1.70,分离时间仅为12 min,柱效最高达到310000 plates/m。该研究为手性三唑类农药的同时分离提供了新的分离分析思路。     

Sequential injection-bead injection-lab-on-valve coupled to high-performance liquid chromatography for online renewable micro-solid-phase extraction of carbamate residues in food and environmental samples

A sequential injection‐bead injection‐lab‐on‐valve system was hyphenated to HPLC for online renewable micro‐solid‐phase extraction of carbamate insecticides. The carbamates studied were isoprocarb, methomyl, carbaryl, carbofuran, methiocarb, promecarb, and propoxur. LiChroprep^® RP‐18 beads (25–40 μm) were employed as renewable sorbent packing in a microcolumn situated inside the LOV platform mounted above the multiposition valve of the sequential injection system. The analytes sorbed by the microcolumn were eluted using 80% acetonitrile in 0.1% acetic acid before online introduction to the HPLC system. Separation was performed on an Atlantis C‐18 column (4.6×150 mm, 5 μm) utilizing gradient elution with a flow rate of 1.0 mL/min and a detection wavelength at 270 nm. The sequential injection system offers the means of performing automated handling of sample preconcentration and matrix removal. The enrichment factors ranged between 20 and 125, leading to limits of detection (LODs) in the range of 1–20 μg/L. Good reproducibility was obtained with relative standard deviations of <0.7 and 5.4% for retention time and peak area, respectively. The developed method has been successfully applied to the determination of carbamate residues in fruit, vegetable, and water samples.     

Separation of seven fluoroquinolones by microemulsion electrokinetic chromatography and application to ciprofloxacin, lomefloxacin determination in urine

A simple, reliable microemulsion electrokinetic chromatography (MEEKC) method is developed for the simultaneous separation of seven fluoroquinolones (FQs). The best separation is achieved in a carrier electrolyte containing 1% (v/v) heptane, 100 mmol/L sodium dodecyl sulfate (SDS), 10% (v/v) 1-butanol, and 8 mmol/L phosphate-sodium tetraborate buffer at pH 7.30. The proposed method was directly applied to the determination of ciprofloxacin (CPF) and lomefloxacin (LMF) in urine samples of subjects administered either with CPF or LMF.     

Preparation and evaluation of C18-bonded 1-microm silica particles for pressurized capillary electrochromatography

Nonporous silica spheres (1 microm) were synthesized and bonded with octadecylsilane functionality. These stationary phase particles were packed electrokinetically into fused-silica capillaries with 100 microm id for a length of 20 cm, which was evaluated by using pressurized CEC (pCEC). The efficiency of the C18 RP column was characterized through the theoretical plates of thiourea, benzyl alcohol, toluene, styrene, and naphthalene. The effects of experimental parameters such as the applied voltage, sample size, pump flow rate, pH value and the concentration of the buffer solution, and the content of methanol in the mobile phase, on-column efficiency were evaluated. Column efficiency as high as 200 000 theoretical plates per meter for naphthalene was obtained with the optimal condition of 70% v/v methanol and 30% v/v of 10 mmol/L phosphate buffer (pH 7.8) at an applied voltage of 10 kV and a supplementary pressure of 500 psi.     

Pressurized gradient capillary electrochromatographic separation of eighteen amino acid derivatives

A pressurized gradient capillary electrochromatography (pCEC) instrument was developed to separate 18 amino acid derivatives. A reversed-phase C18 column (3 microm, 130 mm x 75 microm I.D.) and an acetate buffer (50 mmol/l NaAc, pH 6.4) with an ion-pair reagent (1% N,N-dimethylformamide) were used to separate derivatized amino acids from a standard solution (2 microg/ml), and the wavelength of the UV-Vis detector was 360 nm. The pressure on the capillary column was kept at approx. 70 Pa and 3 kV positive voltage was added on the outlet end of column. The effect of voltage on the eluting order of amino acids and the resolution of separation were studied, and it was found that when the voltage was higher than 3 kV, the adsorption of amino acids in the porous C18 column occurred. The effect of salt concentration, injection volume, and column length on the separation of amino acids was determined. The amino acid sample was separated by pCEC, and RSDs of the migration times of each amino acid were all less than 2.5%.     

毛细管胶束电动色谱法分析PTH氨基酸

建立了用毛细管胶束电动色谱法（MEKC）分离19种PTH氨基酸的方法,并探讨了电压、pH值、温度、胶束浓度对氨基酸迁移时间的影响。方法具有速度快、灵敏度高、样品用量少的优点。     

On-line pretreatment and determination of parabens in cosmetic products by combination of flow injection analysis, solid-phase extraction and micellar electrokinetic chromatography

A convenient and automated method for on-line pretreatment and determination of three parabens (i.e. methyl, ethyl and propyl p-hydroxybenzoate) in cosmetic products is proposed by using flow injection analysis (FIA), solid-phase extraction (SPE) and micellar electrokinetic chromatography (MEKC). An improved split–flow interface is used to couple SPE on C₈-bonded silica with MEKC separation, which can avoid running buffer contamination and reduce buffer consumption, especially, containing expensive reagents. The analytes are loaded onto a C₈ column at 0.6 mL/min for 60 s and eluted with a mixed eluent of 40% (v/v) 10 mmol/L sodium tetraborate buffer (pH 9.3) and 60% (v/v) ethanol at 0.75 mL/min. The MEKC separation is accomplished with a running buffer of 20 mmol/L sodium tetraborate (pH 9.3) containing 100 mmol/L sodium dodecyl sulfate (SDS) at 15 kV. For butyl p-hydroxybenzoate did not be detected in the cosmetic products, it was used as an internal standard (IS) added into the real samples. This FIA–SPE–MEKC method using IS allows the sample separation within 12 min and the sample throughput of five samples per hour with the relative standard deviation (R.S.D.) less than 2.3% (n = 5). The limits of detection (LOD) are in the range from 0.07 to 0.1 μg/mL (S/N = 3 and n = 11). The proposed method has been used to determine three parabens in real cosmetic products satisfactorily.     

Monitoring disopyramide, lidocaine, and quinidine by micellar liquid chromatography

A micellar liquid chromatography (MLC) method using a C18 column was developed to determine three antiarrhythmic drugs--disopyramide, lidocaine, and quinidine--that are most usually monitored in serum samples. After the application of an interpretative strategy for optimization of sodium dodecyl sulfate (SDS) and modifier concentrations in order to ensure the minimum analysis time, maximum sensitivity, and good resolution, the optimum chromatographic conditions for the determination of the three antiarrhythmics were flow rate, 1 mL/min; injection volume, 20 microL; separation temperature, 25 degrees C; mobile phase, 150 mmol/L SDS-7% (v/v) butanol-phosphate buffer, 10 mmol/L, pH 7-0.9% (w/v) NaCl; and detection at 214 nm. The calibration curves for the drugs were linear (r2 > 0.999). The intraday and interday precisions were lower than 3.9% (CV). Recoveries were 100 +/- 0.6% when the method was applied to both serum samples spiked with the antiarrhythmics (n = 10) and real serum samples. In all cases, the results were similar to those obtained using the reference method (fluorescence polarization immunoassay) usually used in the Spanish hospital. The proposed method is useful for hospital monitoring of the antiarrhythmics by direct injection into the chromatograph.     

胶束电动毛细管色谱法同时测定西洋参中人参皂苷Rg1、Re和Rb1

建立了西洋参中人参皂苷Rg1、Re及Rb1同时分离测定的胶束电动毛细管色谱新方法,以解决西洋参样品中难溶于水的3种人参皂苷的准确定量问题。以40.2 cm(有效长度30 cm)×50 μm的熔融石英毛细管柱为分离柱,分离缓冲液的组成为V(15 mmol/L Na2B4O7+30 mmol/L H3BO3 (pH 9.0)+100 mmol/L十二烷基硫酸钠(SDS)+30 g/L聚乙二醇35000):V(甲醇):V(异丙醇)=2:1:1,于214 nm下检测。详细研究了影响分离的因素。Rg1、Re及Rb1检出限(信噪比(S/N)为3)分别为30、40及30 mg/L,定量限(S/N=9)分别为90、120及90 mg/L,加标回收率为87.4%～95.2%。用该法测定了西洋参标准物质,并与高效液相色谱法的检测结果进行了比对,结果吻合。应用该方法分别测定了中国、加拿大及美国的西洋参,获得满意的结果。     

Sensitive amperometric detection for capillary electrophoresis of phenol carbamates with in‐line thermal hydrolysis strategy

A strategy on amperometric detection for CZE of phenol carbamates as model analytes with a facile in‐line thermal hydrolysis was presented, in which a thermal hydrolysis, subsequent CZE separation and final column‐end amperometric detection were accomplished in an intact capillary. Key parameters of hydrolysis dynamics of carbamates and electrochemical detection of the hydrolysates were studied, as well as electrophoretic conditions. Under the optimal conditions, the capillary was utilized as chambers for in situ hydrolysis, CZE separation, and electrochemical detection. The successive separation of hydrolysates of five carbamates (propoxur, carbofuran, 3‐OH‐carbofuran, carbaryl and bendiocarb) were achieved within 17 min. Applied to vegetable samples, the recoveries of carbamates fortified at 0.02 and 0.05 mg/kg were ranging in 88–107.2 and 86.3–107.3%, respectively. The success in the implementation of such a scheme resulted in a simple instrument as compared with those current analytical methods with post‐column derivization or pre‐column hydrolysis, or online enrichment in chip, respectively. This protocol might possess a potential utility for the sensitive amperometric detection of phenol carbamates.     

Online trace enrichment to determine pyrethroids in river water by HPLC with column switching and photochemical induced fluorescence detection

The potential of online trace enrichment on a highly apolar short column in LC was evaluated for the determination of pyrethroids in river water. Twelve millilitres of water samples, modified with 8 mL ACN (ACN/water 40:60, v/v), were passed through 50 x 4.6 mm ID first separation column packed with 5 microm Hypersil Elite C18. Pesticides were preconcentrated in this column while the matrix background was eluted to waste. Separation of pesticides was performed on a 3.5 microm symmetric C18 column (250 x 4.6 mm ID) with an ACN step gradient as mobile phase and fluorescence detection was used after postcolumn derivatization by using UV light. The use of photochemically induced fluorescence for detection improved sensitivity and selectivity. Quantification limits ranged from 0.05 to 0.1 microg/L and pesticide recoveries at two concentration levels (0.1 and 0.5 microg/L) were between 93.1 and 118.6%, with RSD between 2.5 and 7.5% (n = 3) in river water samples. No matrix effect was detected.     

Analysis of urinary metabolites for metabolomic study by pressurized CEC

A new approach for the metabolomic study of urinary samples using pressurized CEC (pCEC) with gradient elution is proposed as an alternative chromatographic separation tool with higher degree of resolution, selectivity, sensitivity, and efficiency. The pCEC separation of urinary samples was performed on a RP column packed with C(18), 5 microm particles with an ACN/water mobile phase containing TFA. The effects of the acid modifiers, applied voltage, mobile phase, and detection wavelength were systematically evaluated using eight spiked standards, as well as urine samples. A typical analytical trial of urine samples from Sprague Dawley (S.D.) rats exposed to high-energy diet was carried out following sample pretreatment. Significant differences in urinary metabolic profiles were observed between the high energy diet-induced obesity rats and the healthy control rats at the 6th wk postdose. Multivariate statistical analysis revealed the differential metabolites in response to the diet, which were partially validated with the putative standards. This work suggests that such a pCEC-based separation and analysis method may provide a new and cost-effective platform for metabolomic study uniquely positioned between the conventional chromatographic tools such as HPLC, and hyphenated analytical techniques such as LC-MS.     

在线推扫富集-胶束电动毛细管色谱检测旋覆花素中和大鼠血浆中的旋覆花内酯

采用熔融石英毛细管,以含有50 mmol/L 十二烷基硫酸钠的50 mmol/L硼酸盐缓冲液为电极缓冲液,以10 mmol/L硼酸盐缓冲液为上样缓冲液,经过对分离条件的优化,成功地建立了胶束电动毛细管色谱结合在线sweeping(推扫)富集技术检测中性脂溶性物质旋覆花内酯(acetylbritannilactone,ABL)的实验方法。所建方法的批内、批间测定值的相对标准偏差均小于5%,灵敏度为0.005 g/L,回收率大于92%;被检测样品的含量与峰面积呈良好的线性关系,相关系数为0.9975。用所建立的方法检测了旋覆花素中ABL的含量及其在体内的动态变化,结果表明胶束电动毛细管色谱结合在线sweeping样品富集技术可显著提高检测的灵敏度。该方法具有操作简单、进样量小(nL级)、检测速度快等特点,弥补了毛细管电泳在测定痕量组分方面的不足。