Mixture-designed electrolytes for the MEKC separation of natural and synthetic steroids

In this work, the separation of 11 natural and synthetic steroids was studied using MEKC electrolytes modified by property-selected organic solvents: ethanol, ACN, and THF. The interplay between electrophoretic behavior and structural features was disclosed and the effects of organic modifiers to modulate retention and to alter selectivity were discussed in terms of system linear solvation energy relationships (LSER). The LSER indicated the total organic solvent percentage in the electrolyte as a major parameter to control retention and as a minor contribution, the hydrogen bond acidity. By evaluating the electropherograms obtained from mixture-designed electrolytes, a favorable separation condition for all solutes was achieved in ca. 25 min with an electrolyte composed of 20 mmol/L sodium tetraborate at pH 9.4, 20 mmol/L SDS and 20% EtOH (0.80% CV for migration time and 2.5% CV for peak area, n = five consecutive injections). The applicability of the proposed separation condition was demonstrated by the inspection of estrogens in urine sample (puberty stage).     

Liquid chromatographic method development for steroids determination (corticoids and anabolics). Application to animal feed samples

A LC isocratic separation study of a complex mixture containing 18 steroids (corticoids and anabolics), used potentially as growth promoters, was carried out. For this purpose, using a Hypersil ODS column at controlled temperature, mobile phases (from binary to quaternary) prepared from water and MeOH, ACN or THF as organic modifiers and UV detection at 245 nm, were employed (dehydroepiandrosterone was detected at 200 nm). The optimum separation was achieved using water/acetonitrile (65:35, v/v) as mobile phase at 30 degrees C, allowing the separation of 16 out of 18 steroids in about 30 min. The retention scale using optimized binary mobile phases was related with steroids hydrophobicity and structure, allowing a classification into three groups for these compounds. To improve the separation several alkyl-silica packings were tested: Type A (Lichrospher C8) and Type B (Luna C18, Kromasil C18, Purospher C18 and Synergy C12). Taking into account resolution, number of separated compounds and run time analysis the Hypersil column was selected as the best choice for further applications. Calibration graphs were obtained using fluorocortisone, fluoxymesterone or methylprednisolone as internal standard. The optimized separation was applied to the analysis of piglet feed samples spiked with steroids. The sample preparation process included solvent extraction using diethyleter and solid phase extraction using silica cartridges. The recoveries were in the range 70-92%. Decision limits and detection capability were in the range 34-198 and 41-249 microg/kg, respectively. Repeatability was also evaluated.     

甲氧苄氨嘧啶纯度的反相高效液相色谱分析

本工作建立了抗菌药物甲氧苄氨嘧啶中少量杂质的反相高效液相色谱分析方法。方法操作简便，分析快。同时考察了有机改进剂对该药物分离的影响。     

Method development for the separation of steroids by capillary electrochromatography

A rapid capillary electrochromatography (CEC) method was developed to separate five structurally related steroid compounds from the production line of steroid hormones. The separation was performed on a Hypersil C8 MOS and Unimicro C18 stationary phases using acetonitrile (ACN), methanol (MeOH), and tetrahydrofuran (THF) as organic modifiers and tris(hydroxymethyl)aminomethane (Tris) as buffer additive. The Hypersil C8 MOS stationary phase performed best together with ACN as organic modifier and Tris buffer. The method was extensively tested for ruggedness with respect to sensitivity to temperature, ACN composition, pH change, concentration of Tris buffer, injected plug length, and run‐to‐run and day‐to‐day repeatability. The minimal detectable concentration and amount were investigated for quantification purposes. The developed CEC method was shown to be fast, rugged, and well suited for quantification of the steroids under study.     

Effects of organic modifiers on the chiral recognition by different types of silica-immobilized bovine serum albumin

We prepared three columns containing bovine serum albumin immobilized on silica by different means and the effects of organic modifiers in the eluent on chiral separation were studied using N-substituted amino acids. Adsorption on silica, covalent immobilization to diol-silica with carbonyldiimidazole (CSP-II) and covalent immobilization to amino-silica with glutaraldehyde were studied. CSP-II had the highest stereoselectivity and was the most affected by organic modifiers in the eluent. The hydrophobicity of amino acid moiety affected the chiral recognition of N-benzoylamino acids and the aromaticity of the N-substituted group was important.     

固定化有机改进剂RP－HPLC固定相的研究

通过在ＲＰＨＰＬＣ固定相Ｃ１８表面键合氨基硅烷，引入氨基基团，将有机改进剂固定至硅胶键合相表面，克服了固定相表面硅羟基对含氮有机化合物的不可逆吸附。以最简单的ＣＨ３ＯＨ－Ｈ２Ｏ洗脱体系即能获得含氮有机物的最佳分离，从而可简化分离操作，提高柱寿命，降低分离成本。     

有机改性剂对氯代酚毛细管区带电泳分离的影响

在分离19种氯代酚的过程中,考察了不同的有机添加剂对其毛细管区带电泳分离的影响,发现除了缓冲溶液的pH值外,缓冲溶液添加剂对氯代酚的电泳分离也有较大影响。这种影响与添加剂和氯代酚形成氢键的能力有关。     

固定化有机改进剂RP－HPLC固定相的研究

通过在ＲＰＨＰＬＣ固定相Ｃ１８表面键合氨基硅烷，引入氨基基团，将有机改进剂固定至硅胶键合相表面，克服了固定相表面硅羟基对含氮有机化合物的不可逆吸附。以最简单的ＣＨ３ＯＨ－Ｈ２Ｏ洗脱体系即能获得含氮有机和的最佳分离，从而可简化分离操作，提高柱寿命，降低分离成本。     

Separation of imidacloprid and its degradation products using reversed phase liquid chromatography with water rich mobile phases

Water rich mobile phases in RPLC are not generally used because of the longer retention times involved when organic modifiers such as methanol or acetonitrile are used. The problem of longer retention times can be addressed using hydrophobic alcohols such as pentanol in low quantities (less than 1%) as organic modifiers. The advantages of using these mobile phases in RPLC for the separation of water soluble and weakly retained congeners is demonstrated through the separation of imidacloprid and its degradation products using a 0.4% pentanol in water mobile phase with 0.2% acetic acid.     

Designing polymer matrix for microchip-based double-stranded DNA capillary electrophoresis

An optimization strategy for ternary solvent-strength gradient elution RP chromatography is described in which a two-dimensional model of gradient time (2 levels) against ternary proportions of organic modifiers (4 levels) was constructed. From the resolution surface the optimum ratio of organic modifiers could be selected. Excellent retention time and acceptable peak width and resolution simulations were obtained. The separation could be further optimized from the same input data by using a standard one-dimensional model in order to optimize for gradient slope, duration and shape. Excellent retention time and acceptable peak width and resolution simulations were obtained (< 1, 2 and 6% error, respectively).     

含磷手性化合物在多聚糖类手性固定相上的手性分离

在纤维素 三(3,5 二甲基苯基氨基甲酸酯)(ChiralcelOD)和直链淀粉 三(3,5 二甲基苯基氨基甲酸酯)(ChiralpakAD H)手性固定相上,采用高效液相色谱正相条件,分离了系列含磷手性化合物。考察了流动相中有机改性剂的种类及浓度对手性分离的影响;研究了化合物的结构与保留及对映体选择性的关系;并探讨了手性识别机理。     

Effect of organic modifiers on the capacity factor in micellar electrokinetic chromatography

Organic modifiers were effective both to extend the migration time window and to improve the separation of very hydrophobic compounds in MEKC.An iteration method was used to determine the migration time of micelles.The quantitative relationship between the capacity factor k' and the concentration of organic modifiers was derived,which was investigated experimentally.The linear solvation energy relationships (LSER) methodology was applied to MEKC,and good linear relationships between Ink' and solvatochromic parameters of 15 solutes were obtained in the presence of organic modifier in different concentrations,which indicated a new access in MEKC to predict k' from the structural parameters of solutes.The effect of column temperature T on k' was also investigated.     

Enantiomeric resolution of bifonazole by supercritical fluid chromatography

The enantiomeric separation of bifonazole by supercritical fluid chromatography on Chiralpak AD has been studied. The effect of different modifiers (methanol, ethanol, 2-propanol, and acetonitrile) was examined. Enantioseparation was possible with all of them, but the best results were provided by the alcohol-type ones. The resolution was higher than 5 in all cases. The isoelution temperatures Tiso were calculated from the study of the temperature effect for the different organic modifiers. The value of Tiso was below the working temperature range on using methanol, but above it with ethanol or 2-propanol.     

卡那霉素作为手性选择剂的毛细管电泳手性药物分离研究

建立了一种以天然易得的卡那霉素为手性添加剂，用毛细管区带电泳法快速分离市售对乙肝有良好治疗效果的药物联苯双脂衍生物的方法，拓宽了毛细管电泳中手性选择剂的范围，通过实验研究了卡那霉素、甲醇 含量PH值，磷酸盐缓冲体系和硼硝缓冲体系对手性分离的影响，以及三种有机溶剂（甲醇、乙晴、异丙醇）添加剂对手性分离的影响，结果表明，在含有3％卡那霉素，30mol/L，硼砂缓冲体系（PH＝8．0）添加30％异丙醇是最佳的分离条件。     

Effects of organic modifiers on retention mechanism and selectivity in micellar electrokinetic capillary chromatography studied by linear solvation energy relationships

The effects of six organic modifiers (urea, methanol, dioxane, tetrahydrofuran, acetonitrile and 2-propanol) on the retention mechanism and separation selectivity of the bulk buffer in micellar electrokinetic capillary chromatography (MECC) with sodium dodecyl sulfate (SDS) micelles as pseudo-stationary phase have been investigated through linear solvation energy relationships (LSERs). It is found that the retention value in MECC systems with or without organic modifier is primarily dependent on the solvophobic interaction and the hydrogen bonding interaction with the solute as proton acceptor, while the dipolar interaction and the hydrogen bonding interaction with the solute as proton donor play minor roles. The effects of the organic modifiers on the solvophobic, dipolar and hydrogen bonding interactions are evaluated in terms of the relationship between regression coefficient of the LSER equations and the modifier concentration. The variations of the solvophobic interaction and the dipolar interaction with change of the modifier concentration can be approximately explained using the solubility parameter and the dipolarity/polarizability parameter of the organic modifier, respectively. However, the relationships between the hydrogen bond acidity and basicity of the bulk buffer and the organic modifiers are rather complicated. Those results may be caused from the displacement of organic modifiers to the water adsorbed on the micellar surface as well as changes in the acidity and basicity of the bulk buffer with the addition of organic modifiers. In addition, it is found that the phase ratio is influenced significantly by the use of organic modifier.     

Separation of naphthalene-2,3-dicarboxaldehyde-derivatized-substance P and its metabolites by micellar electrokinetic chromatography

This paper describes the development of analytical methodology for the separation of naphthalene-2,3-dicarboxaldehyde (NDA)-derivatized substance P (CBI-SP) and five lysine-containing metabolites by micellar electrokinetic chromatography (MEKC). The effect of surfactant composition and organic modifiers on the separation was investigated. The final separation buffer consisted of 80 mM sodium cholate in 50 mM N-tris (hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), pH 7. All six lysine-containing peptides were separated under these conditions.     

High Performance Liquid Chromatography of Uroporphyrin Isomers

Abstract

The separation of uroporphyrin I and III isomers by reversed-phase high performance liquid chromatography on ODS-Hypersil with ammonium acetate buffer-acetonitrile solvent systems is described. The effects of buffer concentration, pH, organic modifier proportion and different organic modifiers on the resolution are studied. The optimum conditions for the separation were 12–13% acetonitrile in 1M ammonium acetate buffer pH 5.10–5.20. The method also separated uroporphyrin I and III from the II isomers but the resolution of uroporphyrin III and IV isomers was not achieved.     

水包油型微乳液相色谱分离激素类药物的影响因素

采用水包油型微乳液相色谱(MELC)分离了6种激素类药物(醋酸可的松、泼尼松龙、己烯雌酚、炔雌醇、醋酸氟轻松及黄体酮)。考察了微乳流动相的组成成分(包括表面活性剂的浓度、油相种类、有机添加剂种类)及固定相孔径等对分离的影响。实验得到的最佳分离条件: 色谱柱为Venusil ASB C18 (T)(粒径5 μm,孔径30 nm,250 mm×4.6 mm),微乳流动相为30 g/L十二烷基硫酸钠(SDS)-0.8%正辛烷-6.6%正丁醇,流速为0.8 mL/min,检测波长为254 nm,柱温为35 ℃。该方法可用于甾体药物及其制剂的分离鉴别以及快速测定。     

Rapid Supercritical Fluid Chromatography Method for Separation of Chlorthalidone Enantiomers

Supercritical fluid chromatography employing chiral stationary phases is a popular separation technique to perform enantioselective separations. The main advantages of supercritical fluid chromatography are low analysis time, low consumption of organic modifiers, and therefore lower costs and higher environmental friendliness. A novel method for the separation of chlorthalidone enantiomers, widely used diuretic drug, is reported that clearly demonstrates the advantages of supercritical fluid chromatography. The effects of the amount and type of organic modifiers, temperature, and back pressure on enantioselectivity and resolution of the enantiomers were evaluated. The baseline separation was achieved in less than 2.5 min in the optimized system composed of Chiralpak AD column, mobile phase CO₂/MeOH 50/50 (v/v), temperature 40°C, a flow rate of 4.0 mL/min, and 120 bar back pressure. Moreover, enantiomers of chlorthalidone were determined in two commercially available pharmaceuticals. The proposed method may be easily transferred to a semi-preparative scale.     

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.