微乳液相色谱测定药物的油-水分配系数的新方法研究

建立快速微乳液相色谱法(MELC)测定药物的油-水分配系数(log P)的方法. 选择5种模拟生物膜的微乳流动相体系, 以7个标准药物的log P对保留因子(log k)的回归线性方程, 计算被测药物的log P值. 并以药物文献的log P值对实验测得log k值的线性相关系数为参数, 对微乳体系的表面活性剂和油相的种类进行考察, 得到测定非同类的中性、碱性药物的油-水分配系数最佳流动相体系为6.0% Brij35-6.6%正丁醇-0.8%正辛醇-86.6%磷酸缓冲液(0.05 mol• L－1, pH 7.0), 其测得值与文献的实验值平均相差0.3个对数单位. 结果显示该方法可靠、高效、重现性好, 可用于药物的油-水分配系数log P的测定.     

Determination of the lipophilicity of Salvia miltiorrhiza Radix et Rhizoma (danshen root) ingredients by microemulsion liquid chromatography: optimization using cluster analysis and a linear solvation energy relationship‐based method

We evaluated 26 microemulsion liquid chromatography (MELC) systems for their potential as high‐throughput screening platforms capable of modeling the partitioning behaviors of drug compounds in an n‐octanol–water system, and for predicting the lipophilicity of those compounds (i.e. logP values). The MELC systems were compared by cluster analysis and a linear solvation energy relationship (LSER)‐based method, and the optimal system was identified by comparing their Euclidean distances with the LSER coefficients. The most effective MELC system had a mobile phase consisting of 6.0% (w/w) Brij35 (a detergent), 6.6% (w/w) butanol, 0.8% (w/w) cyclohexane, 86.6% (w/w) buffer solution and 8 mm cetyltrimethyl ammonium bromide. The reliability of the established platform was confirmed by the agreement between the experimental data and the predicted values. The logP values of the ingredients of danshen root (Salvia miltiorrhiza Radix et Rhizoma) were then predicted. Copyright © 2015 John Wiley & Sons, Ltd.     

微乳液相色谱法同时测定降压中成药及保健品中添加的10种化学药物

建立了微乳液相色谱(MELC)同时测定中成药及保健品中添加的10种降压药物的分析方法。考察了微乳流动相的组成成分(包括表面活性剂、助表面活性剂、油相的种类及质量分数)、pH值和固定相种类对分离效果的影响。同时利用星点设计-响应面优化法进一步优化微乳组成。实验得到的最佳分离条件:色谱柱为Agela Promosil C18(2)(250 mm×4.6 mm,5μm);微乳流动相为5.6%(质量分数)十二烷基聚乙二醇醚(Brij35)-0.17%(质量分数)十二烷基硫酸钠(SDS)-5.1%(质量分数)正丁醇-0.46%(质量分数)环己烷-水(pH 3.0);流速为1.0mL/min;检测波长为230 nm;柱温为30℃。该方法具有灵敏度高、稳定性好和环保的优点。     

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。     

Validation of an oil-in-water microemulsion liquid chromatography method for analysis of perindopril tert-butylamine and its impurities

This paper describes the development and validation of a microemulsion liquid chromatography (MELC) method for simultaneous determination of perindopril tert-butylamine and its impurities in bulk active substances and the pharmaceutical dosage form of tablets. An appropriate resolution with reasonable retention times was obtained for a microemulsion containing 0.24% (w/v) butyl acetate, 0.30% (w/v) ethyl acetate, 2% (w/v) sodium dodecyl sulfate, 7.75% (w/v) n-butanol, and 20.0 mM potassium dihydrogen phosphate, the pH of which was adjusted to 3.70 with 85% orthophosphoric acid. Separations were performed on a Nucleosil 120-5 butyl modified (C4), 250 x 4 mm, 5 microm particle size silica column at 40 degrees C, with a mobile phase flow rate of 1.25 mL/min. UV detection was performed at 254 nm. The established method was subjected to method validation, and required validation parameters were defined. Robustness testing, an important part of method validation, was performed as well. Since robustness validation can be conducted using different experimental designs, the Plackett-Burman design was applied due to its possibility of testing many factors at the same time. The validated MELC method was found to be suitable for the simultaneous determination of perindopril tert-butylamine and its impurities in pharmaceuticals.     

微乳液相色谱法对吴茱萸中辛弗林、吴茱萸碱与吴茱萸次碱含量的测定

构建了一种新的微乳体系,用于微乳液相色潜同时分析吴茱萸药材中的辛弗林、吴茱萸碱和吴茱萸次碱.用甲醇超声提取方法制备样品,通过对表面活性剂种类及浓度、酸度、添加剂等影响因素进行考察,得到最佳微乳体系的组成为:3.0%SDS-6.0%正丁醇-0.6%正辛烷-1.0%甲酸-1.2%乙腈-88.2%水.选择Diamonsil ...     

微乳液相色谱法同时测定山楂叶提取物中4种黄酮成分

建立了一种新的微乳体系,用于微乳液相色谱同时分析山楂叶提取物中牡荆素鼠李糖苷、芦丁、牡荆素及金丝桃苷4种黄酮成分.通过对影响分离选择性的主要因素进行考察,得到最佳微乳体系的组成为: 1.0%(w/w) 聚氧乙烯月桂醇(Brij35)-1.1%(w/w)正丁醇-0.1%(w/w)正辛醇-0.3%三乙胺(V/V)(H_3PO_4调节至pH 2.5).在此微乳体系中,表面活性剂类型和浓度、油相种类、添加剂三乙胺、流动相的pH值对4种黄酮成分的分离起到了重要的作用.选择Venusil ASB C_(18)色谱柱(150 mm×4.6 mm, 5μm),流速为0.8 mL/min,检测波长为360 nm,柱温为35 ℃.结果显示,4种黄酮成分在27 min内达到基线分离,在0.95～140.8 mg/L范围内,4个黄酮成分的线性相关系数r≥0.9995,平均回收率98.6%～101.6%.本方法可应用于山楂叶提取物中4种主要黄酮成分的质量分析.     

微乳液相色谱法及其应用进展

微乳液相色谱法是使用普通的正相或反相色谱柱,分别以油包水或水包油型微乳为流动相,用常规检测器进行样品分析的液相色谱法。该法具有独特的选择性,能够同时分离极性范围很广的化合物,流动相中的有机溶剂用量少,调节参数多,以及血浆样品可直接进样,梯度洗脱节省再平衡时间等优点,在复杂组分分离方面具有显著的优势。本文对微乳液相色谱法中常用微乳的组成和结构,各组成成分对分离的影响,以及该法的应用进展进行了综述。     

微乳液相色谱法测定人血浆中丙泊酚浓度

建立了基于微乳液相色谱(MELC)的人血浆中丙泊酚浓度的测定方法。采用Hypersil BDS C18色谱柱分离,并考察了微乳流动相中各组分对溶质洗脱的影响。优化的色谱条件: 以0.5%醋酸（含有3.0%十二烷基硫酸钠（SDS）,0.8%正庚烷,6.0%正丁醇）微乳为流动相,流速为1.0 mL/min,荧光检测器激发波长(λex)为274 nm、发射波长(λem)为312 nm,柱温为室温。人血浆样品用流动相稀释并离心后,直接进样分析。丙泊酚在0.25～10 μg/mL质量浓度范围内呈良好的线性关系,方法的回收率为(98.2±1.9)%～(104.6±2.2)%;日内测定峰面积的相对标准偏差(RSD)为1.42%～2.43%,日间测定峰面积的RSD为2.75%～4.79%。该方法简便可行、重复性好,可用于人血浆中丙泊酚浓度的测定。     

微乳液相色谱法同时测定4种脂溶性维生素

建立了一种新的微乳体系,并成功地应用于微乳液相色谱法(MELC)快速分析脂溶性维生素VA、VD2、VD3和VE。通过对影响分离选择性的主要因素进行考察,得到最佳微乳体系组成为98%(v/v)(50 g/L十二烷基硫酸钠(SDS)-10%(质量分数)正丁醇-1.0%(质量分数)正辛烷-84%水(质量分数))-2%(v/v)乙腈。该微乳体系中,表面活性剂类型和浓度、油相正辛烷的含量、有机添加剂乙腈对脂溶性维生素的分离起到了重要的作用。以Venusil ASB C18色谱柱(150 mm×4.6 mm, 5 μm)为分离柱,流速为0.7 mL/min,检测波长为265 nm,柱温为40 ℃, VA、VD2、VD3和VE在20 min内达到基线分离。4种脂溶性维生素的保留时间和峰面积的相对标准偏差(RSD) (n=5)分别小于2.3%和3.0%; VA、VD2、VD3和VE的线性范围分别为22.0～88.0 mg/L、20.2～81.0 mg/L、24.3～97.2 mg/L和125.0～500.0 mg/L,相应的线性相关系数r2分别为0.9996、0.9994、0.9998、0.9998;检出限(S/N=3)分别为0.37、0.34、0.41和2.12 mg/L。本方法已成功应用于多维元素片(21)中VA与VE的测定,结果令人满意。     

Migration mechanism of bases and nucleosides in oil-in-water microemulsion capillary electrophoresis.

The electrophoretic behaviors of five bases and corresponding nucleosides in the oil in water (o/w) microemulsion capillary electrophoresis, microemulsion electrokinetic chromatography (MEEKC), were examined in comparison with those in normal capillary zone electrophoresis (CZE). The microemulsion systems were composed of heptane, sodium dodecyl sulfate (SDS), 1-butanol and 10 mM phosphate buffer (pH 7.0) or toluene, SDS, 1-butanol and 5 mM carbonate buffer (pH 10.0). CZE was carried out in the range of pH 9.7-10.9, and the dissociation constants, pKa, of the bases and nucleosides and the electrophoretic mobilities of the anionic forms were determined. The electrophoretic behaviors of the solutes in the microemulsion systems were analyzed from their pKa, the electrophoretic mobilities of the anions determined by CZE, and the distribution constants, K(D), of the neutral forms between the microemulsion droplets and the outer aqueous phase. The importance of adsorption mechanism in MEEKC system was suggested from the correlation between log K(D) and log P.     

Alkane effect in the Arizona liquid systems used in countercurrent chromatography

Countercurrent chromatography (CCC) is a separation technique that uses a biphasic liquid system; one liquid phase is the mobile phase, the other liquid phase is the stationary phase. Selection of the appropriate liquid system can be a problem in CCC, since it is necessary to select both the “column” and the mobile phase at the same time as the first is completely dependent on the second. A range of systems with various proportions of solvents were developed to ease this choice; 23 variations of the heptane/ethyl acetate/methanol/water biphasic liquid system were labeled A to Z. This range proved to be extremely useful and became the popular Arizona (AZ) liquid system. However, authors often replace the heptane with hexane. In this work, the chemical compositions of the upper phases and the lower phases of 55 Arizona systems made with various alkanes (pentane, hexane, heptane, isooctane and cyclohexane) were determined by gas chromatography and Karl Fischer titration. The test mixture separated consisted of five steroid compounds. The lower phases were found to have similar compositions when different alkanes were used, but the upper phases were found to change. Exchanging heptane for hexane or isooctane produced minimal changes in the CCC chromatogram, while changing the proportions of the solvents resulted in an exponential change in the retention volumes. The high density of cyclohexane made liquid stationary phase retention difficult. All Arizona systems equilibrated within 30 min, but were not stable: water slowly hydrolyzed the ethyl acetate (as shown by a continuous decrease in the pH of the lower aqueous phase), especially in the water-rich systems (early alphabet letters).     

Hydrophobicity of ionisable compounds studied by countercurrent chromatography

Countercurrent chromatography (CCC) is a liquid chromatography technique in which the stationary phase is also a liquid. The main chemical process involved in solute separation is partitioning between the two immiscible liquid phases: the mobile phase and the support-free liquid stationary phase. The octanol-water partition coefficients (P(o/w)) is the accepted parameter measuring the hydrophobicity of molecules. It is considered to estimate active principle partitioning over a biomembrane. It was related to the substance biological activity. CCC is able to work with an octanol stationary phase and an aqueous mobile phase. In this configuration, CCC is a useful and easy alternative to measure directly the P(o/w) of the molecules compared to other methods including the classical and tedious shake-flask method. Three ketones are used as model compounds to illustrate the CCC protocol of P(o/w) measurement. The focus of this work is put on ionisable molecules whose apparent P(o/w) is completely changed by ionization. β-Blockers, diuretics and sulfonamides are compound classes that were studied. Some of the experimentally determined P(o/w) coefficients of the molecular forms disagreed with calculated and experimental values available in the literature. The P(o/w) coefficients of the ionic forms and the acidity constants were also calculated using a theoretical model. Relationships between biological properties and hydrophobicity are also discussed.     

Micellar chromatographic determination of partition coefficients and associated thermodynamic data for pharmaceutical compounds

Micelle/water partition coefficients were obtained for procaine hydrochloride using micellar liquid chromatography (MLC) to illustrate the potential application of this technique to compounds of pharmaceutical interest. Experiments were conducted over a temperature range which led to calculation of a Gibbs free energy, enthalpy and entropy of transfer for procaine hydrochloride. Successful application of this technique was confirmed using a second compound over a range of temperatures, namely caffeine. Overall, this work confirms that MLC can be used to determine precise and accurate partition coefficients that possibly more closely mimic biological membranes than traditional in vitro systems, namely octanol/water.     

Limonene in Arizona liquid systems used in countercurrent chromatography. I Physicochemical properties

Limonene is a biorenewable cycloterpene solvent derived from orange peel waste. Its potential as a “green” solvent to replace heptane was recently evaluated. Countercurrent chromatography (CCC) is a preparative separation technique using biphasic liquid systems. One liquid phase is the mobile phase; the other liquid phase is the stationary phase held in place by centrifugal fields. A particular range of special proportions of the heptane/ethyl acetate/methanol/water system is called the Arizona (AZ) liquid system when the heptane/ethyl acetate ratio is exactly the same as the methanol/water ratio. A continuous polarity decrease is obtained between the most polar A composition (ethyl acetate/water or 0/1/0/1 v/v) and the least polar Z composition (heptane/methanol or 1/0/1/0 v/v), replacing heptane by limonene and methanol by ethanol produce biphasic liquid systems much more environmentallyfriendly than the original AZ compositions. The chemical compositions of the two liquid phases of 12 AZ limonene/ethyl acetate/ethanol/water proportions were fully determined by Karl-Fisher titration of water and by gas chromatography for the organic solvents. The results were compared with the compositions of the corresponding AZ mixtures containing heptane and methanol. Significant differences in ethyl acetate and ethanol distribution between phases of the two systems with identical volume proportions were established. The ratio of the upper phase over the lower phase volumes and the phase density difference are important in CCC, there are also significant differences between the classic and “green” AZ systems that are discussed.     

Comparison of HPLC and MEEKC for Miconazole Nitrate Determination in Pharmaceutical Formulation

A simple solid phase extraction (SPE) method coupled with high performance liquid chromatography (HPLC) using UV detector and microemulsion electrokinetic chromatography (MEEKC) has been developed and compared for the quantitative determination of miconazole nitrate in pharmaceutical formulation. For HPLC method, two parameters were optimized, namely, the wavelength and the mobile phases. The optimized condition was at the 225 nm wavelength and the mobile phase of ACN:MeOH (90:10 v/v). There are seven MEEKC parameters that were optimized, in this research, which were applied to voltage, temperature, wavelength, sodium dodecyl sulfate (SDS) concentration, buffer pH, buffer concentration and butan-1-ol concentration. The optimum MEEKC condition was obtained using 86.35 % (w/w) 2.5 mM borate buffer pH 9, 0.25 % (w/w) SDS, 0.8 % (w/w) ethyl acetate, 6.6 % w/w butan-1-ol and 6.0 % (w/w) acetonitrile. The combination of SPE using a diol column with HPLC–UV and the MEEKC methods were successfully applied for the determination of miconazole nitrate in a pharmaceutical formulation with the recovery percentage of 98.35 and 92.50 %, respectively.

     

Comparison of HPLC and MEEKC for Miconazole Nitrate Determination in Pharmaceutical Formulation

A simple solid phase extraction (SPE) method coupled with high performance liquid chromatography (HPLC) using UV detector and microemulsion electrokinetic chromatography (MEEKC) has been developed and compared for the quantitative determination of miconazole nitrate in pharmaceutical formulation. For HPLC method, two parameters were optimized, namely, the wavelength and the mobile phases. The optimized condition was at the 225 nm wavelength and the mobile phase of ACN:MeOH (90:10 v/v). There are seven MEEKC parameters that were optimized, in this research, which were applied to voltage, temperature, wavelength, sodium dodecyl sulfate (SDS) concentration, buffer pH, buffer concentration and butan-1-ol concentration. The optimum MEEKC condition was obtained using 86.35 % (w/w) 2.5 mM borate buffer pH 9, 0.25 % (w/w) SDS, 0.8 % (w/w) ethyl acetate, 6.6 % w/w butan-1-ol and 6.0 % (w/w) acetonitrile. The combination of SPE using a diol column with HPLC–UV and the MEEKC methods were successfully applied for the determination of miconazole nitrate in a pharmaceutical formulation with the recovery percentage of 98.35 and 92.50 %, respectively.     

New Perspectives in Micellar Liquid Chromatography

Abstract

This paper will summarize several new findings obtained in our laboratory on the use of micellar mobile phases in liquid chromatography. The topics to be addressed include (i) stationary phase modification by the mobile phase surfactant in micellar liquid chromatography, (ii) investigation of the retention mechanism in micellar liquid chromatography (MLC) using an alkyl-benzene homologous series, (iii) evaluation of the effects of organic additives upon retention and efficiency in MLC, and (iv) preliminary characterization of several new classes of surfactant molecules for use in MLC. The information gained from these studies provides new insights into the dynamics of MLC and demonstrates their potential usefulness in several new separation applications including the resolution of optical isomers.     

Distribution ratio, distribution constant and partition coefficient. Countercurrent chromatography retention of benzoic acid

There is some confusion in chromatography between terms such as solute distribution ratio, distribution constant and partition coefficient. These terms are very precisely defined in the field of liquid-liquid systems and liquid-liquid extraction as well as in the field of chromatography with sometimes conflicting definitions. Countercurrent chromatography (CCC) is a chromatographic technique in which the stationary phase is a support-free liquid. Since the mobile phase is also liquid, biphasic liquid systems are used. This work focuses on the exact meaning of the terms since there are consequences on experimental results. The retention volumes of solutes in CCC are linearly related to their distribution ratios. The partition coefficient that should be termed (IUPAC recommendation) distribution constant is linked to a single definite species. Using benzoic acid that can dimerize in heptane and ionize in aqueous phase and an 18 mL hydrodynamic CCC column, the role and relationships between parameters and the consequences on experimental peak position and shape are discussed. If the heptane/water distribution constant (marginally accepted to be called partition coefficient) of benzoic acid is 0.2 at 20 °C and can be tabulated in books, its CCC measured distribution ratio or distribution coefficient can change between zero (basic aqueous mobile phase) and more than 25 (acidic aqueous mobile phase and elevated concentration). Benzoic acid distribution ratio and partition coefficient coincide only when both dimerization and ionization are quenched, i.e. at very low concentration and pH 2. It is possible to quench dimerization adding butanol in the heptane/water system. However, butanol additions also affect the partition coefficient of benzoic acid greatly by increasing it.