Effects of organic modifiers on solute retention and electrokinetic migrations in micellar electrokinetic capillary chromatography

Influences of seven organic modifiers, including urea, methanol (MeOH), dioxane (DIO), tetrahydrofuran (THF), acetonitrile (ACN), 1-propanol (1-PrOH) and 2-propanol (2-PrOH), on the solute retention and the electrokinetic migrations in micellar electrokinetic capillary chromatography (MEKC) are investigated with sodium dodecyl sulfate (SDS) micelle as pseudostationary phase. It is observed that in the limited concentration ranges used in the MEKC systems the effect of organic modifier concentration on the retention can be described by the equation logk'=logk'(w)-SC for most binary aqueous-organic buffer, but deviations from this retention equation are observed at ACN and particularly THF as organic modifiers. With parameter S as a measure of the elutropic strength, the elutropic strength of the organic modifiers is found to follow a general order urea

Multivariate optimization in micellar electrokinetic capillary chromatography

The optimization of the organic modifier concentration in micellar electrokinetic capillary chromatography (MECC) has been achieved by a uniform design and iterative optimization method, which has been developed for the optimization of composition of the mobile phase in high performance liquid chromatography. According to the proposed method, the uniform design technique has been applied to design the starting experiments, which can reduce the number of experiments compared with traditional simultaneous methods, such as the orthano design. The hierarchical chromatographic response function has been modified to evaluate the separation quality of a chromatogram in MECC. An iterative procedure has been adopted to search the optimal concentration of organic modifiers for improving the accuracy of retention predicted and the quality of the chromatogram. Validity of the optimization method has been proved by the separation of 31 aromatic compounds in MECC. (C) 2000 John Wiley & Sons, Inc.

Analysis of five pharmacologically active compounds from the Tibetan medicine Elsholtzia with micellar electrokinetic capillary chromatography

A high performance capillary electrophoresis method with diode array detector detection for the determination of five bioactive ingredients in Tibetan medicine Elsholtzia, namely quercetin, rutin, saussurenoside, kaempferol, and oleanolic acid, has been developed. The effects of several factors, such as the acidity, concentration of running buffer, separation voltage, temperature, and SDS concentration were investigated. The optimal conditions were 44 mmol/L boric acid running buffer (pH 8.5), 45 mmol/L SDS, 16 KV voltage, 20 degrees C, and 10.0% (V/V) of acetonitrile. Under the optimum conditions, five components could be separated with a good baseline resolution within 17 min. The calibration curves showed good linear relationship over the concentration range of 5 x 10(-4)similar to 0.1 mg/mL for quercetin, rutin, saussurenoside, kaempferol, and 1 x 10(-3) similar to 0.1 mg/mL for oleanolic acid. The average recoveries of the method and RSD were ( 99.2%, 3.2%) for quercetin, (102.1%, 2.1%) for rutin, (99.4%, 1.5%) for saussurenoside, (98.9%, 1.8%) for kaempferol, and (99.0%, 2.9%) for oleanolic acid, respectively. The detection limits (S/N = 3) were 1.1 x 10(-4) mg/mL for quercetin, 2.6 x 10(-4) mg/mL for rutin, 1.8 x 10(-4) mg/mL for saussurenoside, 2.9 x 10(-4) mg/mL for kaempferol, and 6.3 x 10(-4) mg/mL for oleanolic acid, respectively. The method was simple, rapid, and reproducible and could be applied for the determination of quercetin, rutin, saussurenoside, kaempferol, and oleanolic acid in Tibetan medicine Elsholtzia, and the assay results were satisfactory.

Therapeutic drug monitoring of cefepime with micellar electrokinetic capillary chromatography: Assay improvement, quality assurance, and impact on patient drug levels

The improvement and performance of a micellar electrokinetic capillary chromatography assay for cefepime in human serum and plasma with a 50 μm id fused-silica capillary elongated from 40 to 60 cm is reported. Sample preparation with dodecylsulfate protein precipitation at pH 4.5, the pH 9.1 separation medium and the applied voltage were as reported previously[16]. The change resulted in a significant lower current, higher resolution and increased detection time intervals. The performance of the assay with multi-level internal calibration was assessed with calibration and control samples. Quality assurance data of a two year period assessed under the new conditions demonstrated the robustness of the assay. In serum samples of patients who received both cefepime and sulfamethoxazole, cefepime could not be detected due to the inseparability of the two compounds. The presence of an interference can be recognized by an increased peak width (width > 0.2 min), the appearance of a shoulder or an unresolved double peak. The patient data gathered during a three year period reveal that introduction of therapeutic drug monitoring led to a 50% reduction of the median drug level. The data suggest that therapeutic drug monitoring can help to minimize the risk of major adverse reactions and to increase drug safety on an individual basis. This article is protected by copyright. All rights reserved.

Determination of voriconazole in human serum and plasma by micellar electrokinetic chromatography

The micellar electrokinetic capillary chromatography (MEKC) separation and analysis of voriconazole and UK 115794 (internal standard) were examined and an assay for determination of voriconazole in human plasma and serum was developed. The MEKC medium comprises a 2:15 (v/v) mixture of methanol and a pH 9.3 buffer composed of 5mM Na(2)B(4)O(7), 7 mM Na(2)HPO(4) and 54 mM SDS. Sample preparation is based upon liquid/liquid extraction with ethylacetate and dichloromethane (75%/25%) at physiological pH. Using this approach with 250 microl serum or plasma and reconstitution of the dried extract into 100 microl of a buffer composed of 0.5mM Na(2)B(4)O(7) and 0.7 mM Na(2)HPO(4) (pH 9.3), the detection and quantitation limits were determined to be 0.1 and 0.2 microg/ml, respectively, a sensitivity that is suitable for therapeutic drug monitoring of voriconazole (provisional therapeutic range: 1-6 microg/ml) in human plasma and serum samples. The method was validated and compared to an HPLC method, showing excellent agreement between the two for a set of 91 samples that stemmed from patients being treated with voriconazole. The MEKC assay is also demonstrated to be suitable to explore pharmacokinetic data of voriconazole.

Determination of 2,4-D and dicamba in food crops by MEKC

The determination of 2,4-D (2,4-dichlorophenoxyacetic acid) and Dicamba (2-methoxy-3,6-dichlorobenzoic acid) residues in sugar cane, rice and corn was performed by a supercritical fluid extraction (SFE) method using CO2/acetone as extraction mix and an SFE apparatus developed in our laboratory. The extracts were cleaned up after extraction by both liquid- liquid partition and a Florisil column. Micellar electrokinetic capillary chromatography (MEKC) coupled with ultraviolet on-column detection was used for the analysis of these pesticides. The detection limits were improved by the preparation of a special detection cell with an increased pathlength that gave detection limits of ca. 0.6 pg for 2,4-D and Dicamba. Our results demonstrated that capillary electrophoresis can be a powerful new analytical tool for pesticide residue analysis.

Studying Electrophoretic Separations Using Cholate Micelles: Effects Of Ph, Temperature, And Composition

Micelle-forming bile salts have previously been shown to be effective pseudo-stationary phases for separating the chiral isomers of binaphthyl compounds with micellar electrokinetic capillary chromatography (MEKC). Here, cholate micelles are systematically investigated via electrophoretic separations and NMR using R, S-1, 1¿- binaphthyl- 2, 2¿-diylhydrogenphosphate (BNDHP) as a model chiral analyte. The pH, temperature, and concentration of BNDHP were systematically varied while monitoring the chiral resolution obtained with MEKC and the chemical shift of various protons in NMR. NMR data for each proton on BNDHP is monitored as a function of cholate concentration: as cholate monomers begin to aggregate and the analyte molecules begin to sample the micelle aggregate we observe changes in the cholate methyl and S-BNDHP proton chemical shifts. From such NMR data, the apparent CMC of cholate at pH 12 is found to be about 13-14 mM, but this value decreases at higher pH, suggesting that more extreme pHs may give rise to more effective separations. In general, CMCs increase with temperature indicating that one may be able to obtain better separations at lower temperatures. S-BNDHP concentrations ranging from 50 ¿M to 400 ¿M (pH 12.8) gave rise to apparent cholate CMC values from 10 mM to 8 mM, respectively, indicating that S-BNDHP, the chiral analyte molecule, may play an active role in stabilizing cholate aggregates. In all, these data show that NMR can be used to systematically investigate a complex multi-variable landscape of potential optimizations of chiral separations.

Understanding Bile Micelle Chiral Interactions

Bile salts are known to aggregate into micelles in biological systems; however, the fundamental structure and dynamics of bile molecule micelle formation are poorly understood. Previous studies have established that the bile salt cholate is capable of performing chirally selective micellar electrokinetic capillary chromatography (MEKC) separations of model racemic binaphthyl compounds 1,1¿-binaphthyl-2,2¿-diyl hydrogen phosphate (R,S-BNDHP) and 1,1¿-bi-2-naphthol (R,S-BN). Nuclear magnetic resonance (NMR) has been established as a complementary technique for understanding chiral selectivity and micelle formation events based on changes in proton chemical shifts of the probe molecules BNDHP and BN as well as of cholate. This work investigated the effects of the probe molecule, the alkali cation identity and temperature on cholate micelle aggregation and MEKC separations of R,S-BN and R,S-BNDHP. The probe molecule was found to mediate micelle formation by MEKC and proton NMR. A low (0.1 mM) concentration of probe was found to have minimal effects on micellization events detected by proton NMR while higher probe concentration (2.5 mM) was found to mediate micellization causing micellization events to occur at lower cholate concentrations. This work also investigated the effects of alkali counterion on chiral separation. Generally, counterions with larger crystal cationic radius were found to cause greater chiral separation power. NMR data suggest that protons near the surface of the cholate micelle are most sensitive to the cation identity, suggesting a model of improved separation based on the cation sterically inhibiting binding of one isomer. Finally, the effect of temperature on MEKC separation was investigated. Separation power of R,S-BN and R,S-BNDHP appeared to increase linearly with temperature for 22.0 mM to 50.0 mM pH 12.0 cholate. In total, these results indicate that cholate aggregation is dependent on multiple conditions. Understanding the roles that these factors play in influencing cholate micellization can inform better separation in MEKC.

Analysis of N-nitrosames in water using micellar electrokinetic chromatography (MEKC) and development of online and offline methods of extraction and enrichment of neutral polar analytes

Evaluation of the quality of the environment is essential for human wellness as pollutants in trace amounts can cause serious health problem. Nitrosamines are a group of compounds that are considered potential carcinogens and can be found in drinking water (as disinfection byproducts), foods, beverages and cosmetics. To monitor the level of these compounds to minimize daily intakes, fast and reliable analytical techniques are required. As these compounds are relatively highly polar, extraction and enrichment from environmental samples (aqueous) are challenging. Also, the trend of analytical techniques toward the reduction of sample size and minimization of organic solvent use demands new methods of analysis. In light of fulfilling these requirements, a new method of online preconcentration tailored to an electrokinetic chromatography is introduced. In this method, electroosmotic flow (EOF) was suppressed to increase the interaction time between analyte and micellar phase, therefore the only force to mobilize the neutral analytes is the interaction of analyte with moving micelles. In absence of EOF, polarity of applied potential was switched (negative or positive) to force (anionic or cationic) micelles to move toward the detector. To avoid the excessive band broadening due to longer analysis time caused by slow moving micelles, auxiliary pressure was introduced to boost the micelle movement toward the detector using an in house designed and built apparatus. Applying the external auxiliary pressure significantly reduced the analysis times without compromising separation efficiency. Parameters, such as type of surfactants, composition of background electrolyte (BGE), type of capillary, matrix effect, organic modifiers, etc., were evaluated in optimization of the method. The enrichment factors for targeted analytes were impressive, particularly; cationic surfactants were shown to be suitable for analysis of nitrosamines due to their ability to act as hydrogen bond donors. Ammonium perfluorooctanoate (APFO) also showed remarkable results in term of peak shapes and number of theoretical plates. It was shown that the separation results were best when a high conductivity sample was paired with a BGE of lower conductivity. Using higher surfactant concentrations (up to 200 mM SDS) than usual (50 mM SDS) for micellar electrokinetic chromatography (MEKC) improved the sweeping. A new method for micro-extraction and enrichment of highly polar neutral analytes (N-Nitrosamines in particular) based on three-phase drop micro-extraction was introduced and its performance studied. In this method, a new device using some easy-to-find components was fabricated and its operation and application demonstrated. Compared to conventional extraction methods (liquid-liquid extraction), consumption of organic solvents and operation times were significantly lower.

Determination of reserpine in urine by capillary electrophoresis with electrochemiluminescence detection

A fast and sensitive approach to detect reserpine in urine using micellar electrokinetic capillary chromatography with electrochemiluminescence (ECL) of Ru(bpy)(3)(2+) detection is described. Using a 25 mum i.d. capillary as separation column, the ECL detector was coupled to the capillary in the absence of an electric field decoupler. Field-amplified injection was used to minimize the effect of ionic strength in the sample and to achieve high sensitivity. In this way, the sample was analyzed directly without any pretreatment. The method was validated for reserpine in the urine over the range of 1 x 10(-6) - 1 x 10(-4) mol/L with a correlation coefficient of 0.996. The RSD for reserpine at a level of 5 mumol/L was 4.3%. The LOD (S/N = 3) was estimated to be 7.0 x 10(-8) mol/L. The average recoveries for 10 mumol/L reserpine spiked in human urine were 94%.