Simultaneous separation of 17 anions by capillary electrophoresis with the addition of an organic solvent

Seventeen inorganic and organic anions, that normally are insufficiently separated via ion chromatography, were completely separated by the addition of an organic solvent to a solution of BGE combined with an adjustment of the apparent pH via CE in combination with indirect UV absorbance detection. Methanol, ethanol, and acetonitrile were examined for their utility in manipulating the selective separation of anions. Methanol and acetonitrile were better modifiers than ethanol at enhancing the resolution of anions comigrating in an aqueous solution of BGE. Methanol was selected as the modifier that provided the largest separation window that could achieve a complete separation of the target analytes. Via the use of methanol, manipulation of the selectivity between inorganic anions and that between inorganic and organic anions was enhanced, but the separation between organic anions remained difficult when only methanol was used. By varying the apparent pH of the BGE in the presence of 10% v/v methanol, however, the separation selectivity between organic anions was substantially improved. Eventually, 7 inorganic and 10 organic anions were simultaneously separated using BGE at a pH of 6.3 in the presence of 10% v/v methanol.     

Partial least squares model and design of experiments toward the analysis of the metabolome of Jatropha gossypifolia leaves: Extraction and chromatographic fingerprint optimization

A major challenge in metabolomic studies is how to extract and analyze an entire metabolome. So far, no single method was able to clearly complete this task in an efficient and reproducible way. In this work we proposed a sequential strategy for the extraction and chromatographic separation of metabolites from leaves Jatropha gossypifolia using a design of experiments and partial least square model. The effect of 14 different solvents on extraction process was evaluated and an optimized separation condition on liquid chromatography was estimated considering mobile phase composition and analysis time. The initial conditions of extraction using methanol and separation in 30 min between 5 and 100% water/methanol (1:1 v/v) with 0.1% of acetic acid, 20 μL sample volume, 3.0 mL min^?1 flow rate and 25°C column temperature led to 107 chromatographic peaks. After the optimization strategy using i‐propanol/chloroform (1:1 v/v) for extraction, linear gradient elution of 60 min between 5 and 100% water/(acetonitrile/methanol 68:32 v/v with 0.1% of acetic acid), 30 μL sample volume, 2.0 mL min^?1 flow rate, and 30°C column temperature, we detected 140 chromatographic peaks, 30.84% more peaks compared to initial method. This is a reliable strategy using a limited number of experiments for metabolomics protocols.     

Demonstration of simultaneous cation-exchange and reversed-phase mechanisms on a strong-acid cation-exchange column.

It is demonstrated in this report that a conventional strong-acid cation-exchange column can exhibit reversed-phase chromatographic behavior simultaneously with ion-exchange. Adjusting the pH to control cation retention has no effect on the retention of neutral organic analytes. Likewise, changes in the methanol content of the mobile phase to adjust organic analyte retention causes only a small decrease in retention of metal ions in the 0 to 10% (v/v) methanol range, and no significant effect beyond that. Linear calibration behavior of both metal cations and neutral organic analytes is found on this column over three-order of magnitude. Examples of simultaneous metal cation-neutral organic separations in both the isocratic and gradient modes are shown, with conductivity detection for the metal ions and UV for the organic analytes. An isocratic separation of metal ions and neutrals in a vitamin pill is also demonstrated.     

Hydrophilic interaction chromatography separation mechanisms of tetracyclines on amino-bonded silica column

Effects of mobile-phase variations on the chromatographic separation on amino-bonded silica column in hydrophilic interaction chromatography (HILIC) were investigated for four zwitterionic tetracyclines (TCs): oxytetracycline, doxycycline, chlortetracycline, and tetracycline. A mixed-mode retention mechanism composed of partitioning, adsorption, and ion exchange interactions was proposed for the amino HILIC retention process. Buffer type and pH significantly influenced the retention of TCs, but showed similar separation selectivity for the tested analytes. Experiments varying buffer salt concentration and pH demonstrated the presence of ion exchange interactions in TCs retention. The type and concentration of organic modifier also affected the retention and selectivity of the analytes, providing direct evidence supporting the Alpert retention model for HILIC. The retention time of the analytes increased in the following order of organic modifiers: tetrahydrofuran < methanol < isopropanol < acetonitrile. The linear relationships of logk' versus %water (v/v) curve and logk' versus logarithm of %water (v/v) in the mobile phase indicated that TCs separation on the amino phase was controlled by partitioning and adsorption. The developed method was successfully utilized in the detection of TCs in both river water and wastewater samples using solid-phase extraction (SPE) for sample cleanup.     

离子色谱法同时测定化妆品中的铵和6种烷基胺

建立同时测定化妆品中铵和6种烷基胺的离子色谱(IC)分析方法。优化了色谱条件和样品前处理方法,样品经100 mmol/L乙酸-20%(v/v)乙腈溶液浸提,固相萃取(SPE)柱去除阴离子、中和氢离子后进样测定。考察了提取溶液的pH、有机溶剂和共存离子对测定结果的影响。分析方法的线性范围为0.3～15 mg/L,检出限为2.1～7.9 mg/kg,定量限为7～26 mg/kg。采用建立的分析方法测定了清洗、柔肤、祛斑、防晒、烫发、染发和育发类化妆品的加标回收率的范围在80.2%～109.2%之间,相对标准偏差(RSD)的范围为0.5%～3.1%。方法选择性好,灵敏度高,抗干扰强,用于实际样品测定结果准确。     

高效液相色谱-串联质谱法同时测定血液中的15种减肥药物

建立了一种专属、灵敏的同时测定血液中咖啡因、盐酸西布曲明等15种减肥药的高效液相色谱-串联质谱(HPLC-MS/MS)分析方法。样品经乙腈沉淀后,进入HPLC-MS/MS中分析检测。以甲醇和含0.1%(v/v)冰醋酸的20 mmol/L醋酸铵溶液作为流动相,采用梯度洗脱方式,以UltimateXB-C18为色谱柱进行HPLC分析;质谱分析采用电喷雾离子源,正负离子快速切换扫描,选择反应监测模式检测。15种减肥药的定量限在0.001～0.05 mg/L内,各种药物的灵敏度较高,各成分的线性相关系数均大于0.99,精密度均小于12.3%,回收率范围为77.3%～110.8%。研究了这15种药物的质谱特征。该方法灵敏、简便、快捷、专属性强,可用于动物实验样品中减肥药物的含量测定,并且对其他药品、食品中目标减肥药物的测定具有借鉴意义。     

Cation-exchange separation of uranium in dimethylsulphoxide medium

Cation-exchange chromatography in a dimethylsulphoxide (DMSO) medium is a suitable means for separating uranium from metal ions, including copper, iron, nickel and molybdenum. Quantitative separations of uranium from 26 elements can best be effected on a column of Dowex 50W-X8 (200-400 mesh), using as the eluent a 20% v/v DMSO solution which is 0.6Min hydrochloric acid and 0.25M in sodium acetate. Only calcium is eluted with the uranium and all other elements studied are eluted either before or after uranium. The elution characteristics of uranium and of other metal ions were investigated with respect to changes in eluent and resin compositions. Separations were much less effective at higher concentrations of sodium ion or DMSO. None of the organic solvents methanol, ethanol, methyl glycol, acetone, dioxan or acetic acid was found to produce favourable separation conditions. Results with Dowex 50 resins of lower or higher cross-linkage were inferior to those obtained with the X8 resin.     

Analysis of metacycline by capillary electrophoresis

The development and validation of an optimized capillary electrophoresis method for the determination of metacycline in the presence of its related substances by capillary electrophoresis is shown. The influence of methanol as organic modifier, buffer pH, buffer concentration, capillary length, column temperature, Triton X-100 and methyl-beta-cyclodextrin was investigated. A central composite design was performed in order to optimize the method. The optimal separation conditions were: uncoated fused-silica capillary (39 cm total length, 31 cm effective length, 50 microm ID); as background electrolyte a solution of 160 mM sodium carbonate and 1 mM EDTA (pH 10.35)/methanol (89:13 v/v); temperature, 15 degrees C; voltage, 12 kV. The method showed good selectivity, repeatability, linearity, and sensitivity. The limits of detection and quantitation are 0.024% and 0.06%, respectively, relative to a 2.5 mg/mL solution. Six commercial samples were analyzed quantitatively.     

Fully automated method for the liquid chromatographic-tandem mass spectrometric determination of cyproterone acetate in human plasma using restricted access material for on-line sample clean-up

A new automated method for the quantitative analysis of cyproterone acetate (CPA) in human plasma has been developed using on-line solid phase extraction (SPE) prior to the LC-MS/MS determination. The method was based on the use of a pre-column packed with internal-surface reversed-phase material (LiChrospher RP-4 ADS, 25 mm x 2 mm) for sample clean-up coupled to LC separation on an octadecyl silica stationary phase by means of a column switching system. A 30 microl plasma sample volume was injected directly onto the pre-column using a mixture of water, acetonitrile and formic acid (90:10:0.1 (v/v/v)) adjusted to pH 4.0 with diluted ammonia as washing liquid. The analyte was then eluted in the back-flush mode with the LC mobile phase consisting of water, methanol and formic acid (10:90:0.1 (v/v/v)). The dispensing flow rates of the washing liquid and the LC mobile phase were 300 microl min(-1). Medroxyprogesterone acetate (MPA) was used as internal standard. The MS ionization of the analytes was achieved using electrospray (ESI) in the positive ion mode. The pseudomolecular ionic species of CPA and MPA (417.4 and 387.5) were selected to generate daughter ions at 357.4 and 327.5, respectively. Finally, the developed method was validated according to a new approach using accuracy profiles as a decision tool. Very good results with respect to accuracy, detectability, repeatability, intermediate precision and selectivity were obtained. The LOQ of cyproterone acetate was 300 pg ml(-1).     

Separation of alpha-, beta-, gamma-, delta-tocopherols and alpha-tocopherol acetate on a pentaerythritol diacrylate monostearate-ethylene dimethacrylate monolith by capillary electrochromatography

This work reports the first use of a monolith with method development for the separation of tocopherol (TOH) compounds by CEC with UV detection. A pentaerythritol diacrylate monostearate-ethylene dimethacrylate (PEDAS-EDMA) monolithic column has been investigated for an optimised condition to separate alpha-, beta-, gamma- and delta-TOHs, and alpha-tocopherol acetate (TAc). The PEDAS-EDMA monolith showed a remarkably good selectivity for separation of the TOH isomers including the beta- and gamma-isomers which are not easily separated by standard C8 or C18 particle-packed columns. Retention studies indicated that an RP mechanism was involved in the separation on the PEDAS-EDMA column, but polar interactions with the underlying ester and hydroxyl groups enhanced the separation of the problematic beta- and gamma-isomers. Separation of all the compounds was achieved within 25 min using 3:10:87 v/v/v 100 mM Tris buffer (pH 9.3)/methanol/ACN as the mobile phase. The method was successfully applied to a pharmaceutical sample with recoveries from 93 to 99%. Intraday and interday precisions (%RSD) for peak area and retention time were less than 2.3. LODs for all four TOHs and TAc were below 1 ppm.     

Characterization and determination of chlorophacinone in plasma by ion chromatography coupled with ion trap electrospray ionization mass spectrometry

Plasmatic chlorophacinone is commonly measured with liquid chromatographic assay, which convenient but lacks sensitivity and selectivity and usually requires ion pair reagents to reduce the chromatographic tailed peak. In this paper, a novel method using eluent generator reagent‐free ion chromatography coupled with electrospray ionization ion trap mass spectrometric detection for the determination of chlorophacinone in plasma has been developed. After samples were extracted with 10% (v/v) methanol in acetonitrile and cleaned by solid‐phase extraction, chromatographic separation was performed on an IonPac^® AS11 analytical column (250 × 4.0 mm) using 40.0 mmol/L KOH containing 10% (v/v) methanol as organic modifier. Quantification was performed by negative electrospray ionization in multiple reaction monitoring mode. The transition m/z 373 → 201 was for the quantification ion; the transitions m/z 373 → 172 and m/z 373 → 145, as well as the isotope ions m/z 375 and m/z 203, were for the qualitative ions. All the method parameters were validated. It was confirmed that this method can be used in clinical diagnosis and forensic toxicology. Copyright © 2008 John Wiley & Sons, Ltd.     

纤维素键合手性固定相拆分6种萘满酮衍生物对映体

使用Chiralpak IC（纤维素-三（3,5-二氯苯基氨基甲酸酯）共价键合硅胶）手性柱,建立了采用手性固定相高效液相色谱拆分6种 α -芳基萘满酮类衍生物对映体的方法。考察了流动相中有机改性剂的种类和比例、柱温和流速对对映体分离的影响。结果显示6种化合物在异丙醇为改性剂的条件下均可获得较高的对映体分离度。热力学研究表明6种化合物对映体的手性拆分过程均受焓驱动影响,且低温有利于对映体分离。最终推荐分离化合物Ⅰ对映体的流动相是正己烷-异丙醇（90：10,v/v）;分离化合物Ⅱ、Ⅲ、Ⅳ对映体的流动相是正己烷-异丙醇（99：1,v/v）;分离化合物Ⅴ对映体的流动相是正己烷-异丙醇（85：15,v/v）;分离化合物Ⅵ对映体的流动相是正己烷-异丙醇（80：20,v/v）。柱温为25℃,流速为1.0 mL/min。6种化合物对映体均可在Chiralpak IC手性固定相上得到完全分离,证明该色谱柱对6种化合物具有较高的对映体选择性。     

Separation of transition metals in nonaqueous media with capillary electrophoresis

The separation of transition metal Ni2+, Cu2+, Co2+, Zn2+, Cd2+ and Fe3+ in methanol was investigated by using different types of organic acids as complexing agents. In pure methanol, the weaker and simpler acetic, propionic, butyric and valeric acids could enhance metal ions selectivity by increasing acid concentration and metal ions could be separated with high efficiency. However, hydroxycarboxylic acids obviously made separation efficiency worse. The effect of mixed organic acids, mixture solvent (methanol-acetonitrile, methanol-water) on metal ions separation was discussed further. The advantages of using nonaqueous solvent over aqueous for metal ions separation were shown finally.     

Focused microwave-assisted solvent extraction and HPLC determination of effective constituents in Eucommia ulmodies Oliv. (E. ulmodies)

A new focused microwave-assisted solvent extraction method using water as solvent has been developed for leaching geniposidic and chlorogenic acids from Eucommia ulmodies Oliv. The extraction procedures were optimized using a two indexes orthogonal experimental design and graphical analysis, by varying irradiation time, solvent volume, solvent composition and microwave power. The optimum extraction conditions were obtained: for geniposidic acid, 50% micorwave power, 40 s irradiation, and 80% (v/v) aqueous methanol as extraction solvent (20 ml g⁻¹ sample); and for chlorogenic acid, 50% micorwave power, 30 s irradiation, and 20% aqueous methanol (20 ml g⁻¹ sample). The composition of the extraction solvent was optimized and can be directly used as the mobile phase in the HPLC separation. Quantification of organic acids was done by HPLC at room temperature using Spherigel C₁₈ chromatographic column (, i.d. 5 μm), the methanol:water:acetic acid (20:80:1.0, v/v) mobile phase and UV detection at 240 nm. The R.S.D. of the extraction process for geniposidic and chlorogenic acid were 3.8 and 4.1%, respectively.     

Isolation and purification of oridonin from Rabdosia rubescens using upright counter-current chromatography

A preparative counter-current chromatography (CCC) method for isolation and purification of oridonin, a new cancer chemoprevention agent, from the Chinese medicinal plant Rabdosia rubescens was successfully established. The crude oridonin was obtained by elution with a light petroleum/acetone solvent mixture from ethanol extracts of R. rubescens using column chromatography on silica gel. With a two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water (1:2:1:2, v/v), 120 mg of oridonin at the purity of 97.8% was obtained from 200 mg of the crude sample in a single-step CCC separation. The structure of oridonin was identified by ESI-MS, 1H NMR, and 13C NMR.     

Ion chromatography tandem mass spectrometry for simultaneous confirmation and determination of indandione rodenticides in serum

This paper describes a simple method for the simultaneous determination and confirmation of the indandione rodenticides in serum. After samples were extracted with 10% (v/v) methanol in acetonitrile and cleaned by solid‐phase extraction, chromatographic separation was performed on an IonPac® AS11 analytical column (250 × 4.0 mm) using gradient KOH eluent with 10% (v/v) methanol as organic modifier. Confirmation was depended on the extensive fragmentation of the indandione molecule under MS/MS conditions which provides sufficient structural information. Quantification was performed by negative electrospray ionization in multiple reaction monitoring mode. All the method parameters were validated. It was confirmed that this method could be used in clinical diagnosis and forensic toxicology. Copyright © 2009 John Wiley & Sons, Ltd.     

On-line sample cleanup and enrichment chromatographic technique for the determination of ambroxol in human serum

A sensitive and efficient on-line clean up and pre-concentration method has been developed using column-switching technique and protein-coated μ-Bondapak CN silica pre-column for quantification of ambroxol (AM) in human serum. The method is performed by direct injection of serum sample onto a protein-coated μ-Bondapak CN silica pre-column, where AM is pre-concentrated and retained, while proteins and very polar constituents are washed to waste using a phosphate buffer saline (pH 7.4). The retained analyte on the pre-column is directed onto a C(18) analytical column for separation, with a mobile phase consisting of a mixture of methanol and distilled deionized water (containing 1% triethylamine adjusted to pH 3.5 with ortho-phosphoric acid) in the ratio of 50:50 (v/v). Detection is performed at 254 nm. The calibration curve is linear over the concentration range of 12-120 ng/mL (r(2) = 0.9995). The recovery, selectivity, linearity, precision, and accuracy of the method are convenient for pharmacokinetic studies or routine assays.     

Poly(methacrylic acid-ethylene glycol dimethacrylate) monolith in-tube solid-phase microextraction applied to simultaneous analysis of some amphetamine derivatives in urine by capillary zone electrophoresis

A method based on in-tube solid-phase microextraction and capillary zone electrophoresis (CZE) was proposed for simultaneously determining four amphetamines (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, and 3,4-methylenedioxymethamphetamine) in urine. A poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary column, which can provide sufficient extraction efficiency, was introduced for the extraction of amphetamines from urine samples. The hydrophobic main chains and acidic pendant groups of the monolithic column make it a superior material for extraction of basic analytes from aqueous matrix. After extraction, the samples were analyzed by CZE. The best separation was achieved using a buffer composed of 0.1 M disodium hydrogen phosphate (adjusted to pH 4.5 with 1 M hydrochloric acid) and 20% methanol v/v, with a temperature and voltage of 25 degrees C and 20 kV, respectively. By applying electrokinetic injection with field-amplified sample stacking, detection limits of 25-34 microg/L were achieved. Excellent method of reproducibility was found over a linear range of 0.1-5 mg/L. Determination of these analytes from abusers' urine sample was also demonstrated.     

Separation and purification of two taxanes and one xylosyl‐containing taxane from Taxus wallichiana Zucc.: A comparison between high‐speed countercurrent chromatography and reversed‐phase flash chromatography

10‐Deacetylbaccatin III, an important semisynthetic precursor of paclitaxel and docetaxel, can be extracted from Taxus wallichiana Zucc. A process for the isolation and purification of 10‐deacetylbaccatin III ( 1 ), baccatin III ( 2 ), and 7β‐xylosyl‐10‐deacetyltaxol ( 3 ) from the leaves and branches of Taxus wallichiana Zucc. via macroporous resin column chromatography combined with high‐speed countercurrent chromatography or reversed‐phase flash chromatography was developed in this study. After fractionation by macroporous resin column chromatography, 80% methanol fraction was selected based on high‐performance liquid chromatography and liquid chromatography with mass spectrometry qualitative analysis. A solvent system composed of n‐hexane, ethyl acetate, methanol, and water (1.6:2.5:1.6:2.5, v/v/v/v) was used for the high‐speed countercurrent chromatography separation at a flow rate of 2.5 mL/min. The reversed‐phase flash chromatography separation was performed using methanol/water as the mobile phase at a flow rate of 3 mL/min. The high‐speed countercurrent chromatography separation produced compounds 1 (10.2 mg, 94.4%), 2 (2.1 mg, 98.0%), and 3 (4.6 mg, 98.8%) from 100 mg of sample within 110 min, while the reversed‐phase flash chromatography separation purified compounds 1 (9.8 mg, 95.6%) and 3 (4.9 mg, 97.9%) from 100 mg of sample within 120 min.     

Determination of tetraethyl ammonium by ion-pair chromatography with indirect ultraviolet detection using 4-aminophenol hydrochloride as background ultraviolet absorbing reagent

A method was developed for the determination of tetraethyl ammonium (TEA) by reversed-phase ionpair chromatography with indirect ultraviolet detection. Chromatographic separation was achieved on a reversed-phase C18 column using background ultraviolet absorbing reagent - ion-pair reagent - organic solvent as mobile phase. The effects of the background ultraviolet absorbing reagents, detection wavelength, ion-pair reagents, organic solvents and column temperature on the determination method were investigated and the retention rules discussed. Results found that TEA could be successfully analyzed by using 0.7 μmol/L 4-aminophenol hydrochloride and 0.15 μmol/L 1-heptanesulfonic acid sodium mixed with 20% (v/v) methanol asmobile phase at a UV detection wavelength of 230 nm. Under these conditions, the retention time of tetraethyl ammonium was 2.85 min. The detection limit (S/N = 3) for TEA was 0.06 mg/L. The relative standard deviations (n = 5) for peak area and retention time were 0.35% and 0.02%, respectively. The method has been successfully applied to the determination of synthesized tetraethyl ammonium bromide. Recovery of tetraethyl ammonium after spiking was 99.1%.