Pharmaceutical analysis using micellar electrokinetic capillary chromatography

The potential utility in pharmaceutical analysis of a capillary electrokinetic separation technique that employs a micellar "pseudo-stationary phase" is discussed and illustrated. Chromatograms of separations of vitamin metabolites and derivatized amino acids are presented to illustrate the high efficiency of the technique and the ability to simultaneously separate the charged and neutral components of pharmaceutical samples. The analytical characteristics of the technique and the importance of optimizing experimental parameters, such as surfactant concentration and capillary column diameter, are discussed and demonstrated with the aid of chromatograms.     

Determination of plasma heparin by micellar electrokinetic capillary chromatography

The micellar electrokinetic capillary chromatography (MECC) method is reported for the separation of heparin, and for the possibility of direct determination of free heparin in plasma. The conditions for MECC were: pH 8.5, 25 mM sodium dodecyl sulfate (SDS), 25 mM borate buffer, with a 30 cmx50 mum ID fused-silica capillary. The sample was detected with a UV-detector at 270 nm with heparin as external standard. The recovery rate was 95.6-98.7%. This method was linear in the range 80-7000 U l(-1). The within-run and between-run relative standard deviations were lower than 3.1 and 4.5%, respectively. It is suggested that this MECC method may be used to determine blood samples containing high levels of heparin.     

Analysis of lamotrigine and its metabolites in human plasma and urine by micellar electrokinetic capillary chromatography

A reliable micellar electrokinetic capillary chromatographic method was developed and validated for the determination of lamotrigine and its metabolites in human plasma and urine. The variation of different parameters, such as pH of the background electrolyte (BGE) and Sodium dodecyl sulfate (SDS) concentration, were evaluated in order to find optimal conditions. Best separation of the analytes was achieved using a BGE composed of 10 mM borate and 50 mM SDS, pH 9.5; melatonin was selected as the internal standard. Isolation of lamotrigine and its metabolites from plasma and urine was accomplished with an original solid-phase extraction procedure using hydrophilic-lypophilic balance cartridges. Good absolute recovery data and satisfactory precision values were obtained. The calibration plots for lamotrigine and its metabolites were linear over the 1-20 microg/mL concentration range. Sensitivity was satisfactory; the limits of detection and quantitation of lamotrigine were 500 ng/mL and 1 microg/mL, respectively. The application of the method to real plasma samples from epileptic patients under therapy with lamotrigine gave good results in terms of accuracy and selectivity, and in agreement with those obtained with an high-performance liquid chromatography (HPLC) method.     

Analysis of water-soluble vitamins by micellar electrokinetic capillary chromatography

Seven water-soluble vitamins were determined simultaneously by micellar electrokinetic capillary chromatography with UV detection. All these compounds were separated from each other within ca. 22 min by using a carrier containing sodium dodecyl sulphate as the surfactant. On-column detection at 254 nm with ethyl p-aminobenzoate as the internal standard allowed sensitive, accurate and reproducible determination of these compounds. Five principal constituents of a vitamin injection were determined with relative standard deviations of less than 2.1%.     

Optimization strategies in micellar electrokinetic capillary chromatography

Summary Computer-assisted procedures for the one-parameter optimization of the surfactant concentration and the concentration of urea or D-glucose as modifiers in micellar electrokinetic capillary chromatography have been developed. These procedures permit a rapid optimization of one parameter on the basis of only two experiments. Predicted values are compared to empirically obtained optimum values. The influence of the modifier concentration on the critical micelle concentration of sodium dodecyl sulfate was experimentally determined in buffers commonly employed in micellar electrokinetic chromatography. The alteration of retention factors of solutes caused by the influence of urea addition on the critical micelle concentration of sodium dodecyl sulfate was calculated under the assumption of constant distribution coefficients and compared to experimentally obtained values. It was demonstrated that the addition of urea or of D-glucose does not alter the phase ratio substantially.     

Analysis of lignans using micellar electrokinetic chromatography

Micellar electrokinetic chromatography (MEKC) was used to separate twelve lignan compounds originating from Phyllanthus plants. To increase the reliability of peak identification, two micellar systems, the sodium dodecyl sulfate (SDS) and sodium deoxycholate (SDC) systems, were investigated. Because of the high lipophilicity of the lignan analytes, tetrahydrofuran was added to the SDS micellar system to increase its separating ability. In contrast to SDS system, no organic solvent was needed with SDC micelles. Both micellar systems gave a satisfactory separation within a reasonable analysis time. On considering accuracy for quantitation, the SDS method was validated and then used to determine the content of the lignans in two Phyllanthus plants. The selectivity (elution order of the lignans) was significantly different between the SDS and SDC micellar systems. Retention in SDC-MEKC seemed to be dominated by the hydrophobicity of the lignan solutes, while in SDS-MEKC, retention was greatly influenced by hydrogen bonding interactions.     

Determination of lignans in Schisandra chinensis using micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatography (MEKC) has been developed as a promising method for the determination of lignans in plant samples. The separation conditions have been optimized with respect to the different parameters including sodium dodecyl sulfate (SDS) and acetonitrile concentration, pH of the background electrolyte, separation voltage, and capillary temperature. The background electrolyte consisting of 40 mM SDS and 35% acetonitrile in 10 mM tetraborate buffer (pH 9.3) was found to be the most suitable electrolyte for this analysis. The applied voltage of 28 kV (positive polarity) and the capillary temperature 25 degrees C gave the best separation of lignans. The interday reproducibility of the peak areas and the migration times was below 2.0%. The results of MEKC analyses were compared with those obtained by capillary electrochromatography (CEC) and reversed-phase high-performance liquid chromatography (RP-HPLC). The possibilities of using this method for the determination of lignans in drug and in serum samples were also tested.     

Determination of nonsteroidal anti-inflammatory drugs in human specimens by capillary zone electrophoresis and micellar electrokinetic chromatography

Therapeutic drug monitoring of anti-inflammatory drugs is necessary for the identification of the agents that cause toxic events and for the decision on the treatment for intoxication. Recently, capillary electrophoresis (CE) has been developed for the simple and rapid analyses of a variety of chemical agents. Micellar electrokinetic chromatography (MEKC) can separate acidic, neutral and basic anti-inflammatory drugs in serum. Furthermore, serum samples are directly applied to the CE system without any pretreatments, and some anti-inflammatory drugs can be separated from serum albumin in the MEKC analysis. On the other hand, capillary zone electrophoresis (CZE) enables us to determine a few microg/mL levels of acidic anti-inflammatory drugs with simple running buffer and stacking technique. A rapid and simultaneous determination of several analgesic anti-inflammatory agents, including ibuprofen, acetaminophen, indomethacin, and salicylic acid in human serum has been developed by using CZE. Therefore, the CZE and MEKC analysis may become a potentially useful alternative to high-performance liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA) for therapeutic drug monitoring, particularly in serum of patients suffering from intoxication by overdosage of nonsteroidal anti-inflammatory agents.     

Determination of antitubercular drugs by micellar electrokinetic capillary chromatography (MEKC)

A method for the determination of isoniazid (ISO), pyrazinamide (PYR) and rifampicin (RIF) in pharmaceutical products, by micellar electrokinetic capillary chromatography (MEKC) with ultraviolet detection is described. The influence of pH, concentration of surfactants, buffer and organic solvents, over the separation were studied as experimental variables. The optimal separation was carried out at 30 degrees C and 20 kV, using a 40 mM borate buffer and 100 mM sodium dodecylsulphate (SDS) adjusted to pH 8.5. Under these conditions, the analysis is accomplished in about 8 min. The method was applied to the determination of these compounds in different pharmaceuticals with good results when compared with a reference liquid chromatographic (LC) method.     

Pesticide analysis by micellar electrokinetic capillary chromatography

On-capillary sample concentration using sample stacking for the improvement of detection limits for various pesticides separated by micellar electrokinetic capillary chromatography was examined. The dependence of the stacking on different parameters was investigated. An approximately 30-fold preconcentration was achieved by applying sample stacking. Employing a two-step enrichment process (solid-phase extraction combined with sample stacking), detection limits were improved and the sample volume for SPE was reduced. In addition, the total time for the analysis was considerably reduced. Detection limits were between 0.01 and 0.1 ng/ml under these enrichment conditions.     

Analysis of nucleic acid derivatives by micellar electrokinetic capillary chromatography

The use of micellar electrokinetic capillary chromatography (MECC) for the analysis of the major nucleobases, nucleosides, and nucleotides, and their chemically modified derivatives, has been developed and refined. The dimensions of the separating capillaries, the composition of the buffering systems, and the conditions used for electrophoresis were investigated in order to obtain the best performance. Particular emphasis was placed on the identification of the physiological constituents of nucleic acids and their chemically modified analogs: in vitro studies on calf thymus DNA exposed to genotoxic agents have demonstrated that adducted bases and nucleolides can be identified by MECC.     

Separation of D-lysergic acid diethylamide derivatives using micellar electrokinetic capillary chromatography

By adjusting column temperature and applied electric field, a fast separation in micellar electrokinetic capillary chromatography was developed for the separation of D-lysergic acid diethylamide derivatives. A baseline separation of nine derivatives was accomplished with a run time of less than 12 min by utilizing elevated column temperature (60 degrees C) and an applied electric field of 387 V/cm. The number of plates generated per unit time for the separations completed at elevated temperatures was significantly higher when compared to separations at the same applied electric field but at lower temperatures (20 degrees C).     

Specific determination of cysteine in human urine by capillary micellar electrokinetic chromatography

This paper describes a method for the analysis of cysteine in human urine using capillary micellar electrokinetic chromatography and on‐column reaction with 2,2′‐dipyridyl disulfide. In this reaction cysteine is quantitatively transformed into a mixed disulfide concomitantly with formation of an equimolar amount of 2‐thiopyridone that is further separated by capillary micellar electrokinetic chromatography and determined spectrophotometrically at 343 nm. The concentration of cysteine is thus estimated indirectly from the result of 2‐thiopyridone determination. The linear detection range for concentration versus peak area for the assay is from 0.05 to 5 mM (correlation coefficient 0.989) with a detection limit of 2.5 μM and a limit of quantitation of 8.5 μM. The inter‐day reproducibility of the peak area was 2.18% and the inter‐day reproducibility of the migration time 0.51%. The method is relatively rapid, simple, and can be easily automated. Moreover, its detection limit covers the concentration range at which cysteine is present in biological samples such as human urine.     

Separation of coumarins by micellar electrokinetic capillary chromatography

Nine coumarins were successfully separated simultaneously using micellar electrokinetic capillary chromatography with 4-hydroxybenzoic acid as an internal standard. A carrier composed of buffer solution (20 mM sodium dodecyl sulfate-15 mM sodium borate-15 mM sodium dihydrogenphosphate)-acetonitrile (24:1) was found to be the most suitable electrolyte for this separation. The analysis time (22 min) was shorter than that using high-performance liquid chromatography (47 min). Contents of coumarins in the crude drug of Angelicae Tuhou Radix could be easily determined by the proposed method.     

Enantiomer separation of drugs by micellar electrokinetic chromatography using chiral surfactants

A review surveying enantiomer separations by micellar electrokinetic chromatography (MEKC) using chiral surfactants is described. MEKC is one of the most popular techniques in capillary electrophoresis, where neutral compounds can be analyzed as well as charged ones, and the use of chiral micelles enable one to achieve the enantioseparation. The chiral MEKC systems are briefly reviewed according to the types of chiral surfactants along with typical applications. As chiral micelles or pseudostationary phases in MEKC, various natural and synthetic chiral surfactants are used, including several low-molecular-mass surfactants and polymerized surfactants or high-molecular-mass surfactants. Cyclodextrin modified MEKC using chiral micelles is also considered.     

Use of a fluorosurfactant in micellar electrokinetic capillary chromatography

Beads prepared from a thermosensitive polymer, hydroxypropylcellulose, exhibit temperature-dependent porosity. At temperatures below 40°C the beads are swollen having large pores, while at temperatures above 45°C the beads are in a shrunken state having smaller pores. In the presence of 1 M NaCl the transition temperature decreased to about 30°C. In a swollen state the size of pore is large enough to accommodate lysozyme (mol. mass 14 400) and -chymotrypsin (mol. mass 21 600) but not bovine serum albumin (mol. mass 67 000). When the beads are shrunken, all the proteins are eluted from the column packed with hydroxypropylcellulose beads in the volume close to the void volume of the column.     

Serum iohexol analysis by micellar electrokinetic capillary chromatography

A simple and rapid ( approximately 4 min) method for the measurement of iohexol in serum for assessing the glomerular filtration rate is described. It is based on direct serum injection on the capillary by MEKC. The method is linear between 8 and 260 mg/L, with an RSD of peak height of 2.9%. Several simple steps have contributed to an improved daily precision, such as choosing a high pH buffer, increasing the SDS concentration, frequent standardization, and eliminating any sample pretreatment.