Characterisation of retention in micellar high-performance liquid chromatography, in micellar electrokinetic chromatography and in micellar electrokinetic chromatography with reduced flow

The retention (migration) behaviour of various barbiturates, phenylurea and triazine herbicides in micellar electrokinetic chromatography (MEKC) with uncoated fused-silica capillaries was compared with the behaviour in micellar electrokinetic chromatography with reduced electroosmotic flow (RF-MEKC) using capillaries modified with linear polyacrylamide. The error in the values of the retention factors caused by the neglection of the contribution of the electroosmotic flow in RF-MEKC was investigated and a method for correcting this error was suggested. The retention was characterised using the lipophilic and polar indices to characterise and to predict the retention as a function of the concentration of the surfactant (sodium dodecylsulphate) in the running buffer in MEKC and in RF-MEKC. Homologous series of n-alkylbenzenes and of n-alkan-2-ones were compared as the standard sets for the calibration of the retention (migration) index scale. The values of the lipophilic indices of a given solute measured in reversed-phase HPLC, MEKC and RF-MEKC are close to each other. Under ideal MEKC conditions, the values of the polarity indices are close to one for various sample solutes. However, for partially ionised compounds such as weakly acidic barbiturates, where the contribution of the electrophoretic migration is significant, the values of the polarity indices are significantly lower than one. Optimum conditions for separations of mixtures of triazine and phenylurea herbicides and of barbiturates using various techniques tested were compared.     

Theoretical models of separation selectivity for charged compounds in micellar electrokinetic chromatography

Equations and theoretical models for MEKC separation selectivity (α(MEKC) ) were established to explain a change in separation and electrophoretic mobility order of fully charged analytes, in which α(MEKC) is related to the dimensionless values of mobility selectivity in CZE (α(CZE)) and retention selectivity (α(k)) in MEKC, and where α(CZE) and α(k) are defined as the ratio of electrophoretic mobility in CZE and the ratio of retention factor (k) in MEKC for two charged analytes, respectively. Using four alkylparabens as test analytes, excellent agreement was found between the observed α(MEKC) and the proposed α(MEKC) models of test analytes in MEKC over a wide range of SDS concentrations and values of k. For example, in comparison with CZE separation of charged analytes, MEKC separation can enhance separation selectivity up to the maximum value when the selectivity ratio (ρ) is greater than 1.0 (ρ=α(k)/α(CZE)), while lower separation selectivity is obtained with ρ<1.0 (α(CZE) >α(k) >1).     

Comparison of micellar electrokinetic chromatography, liquid chromatography, and microbiologic assay for analysis of cephalexin in oral suspensions

Two well-accepted methodologies, based on a microbiologic assay (MA) and liquid chromatography (LC), and a novel methodology using micellar electrokinetic chromatography (MEKC), were compared for the determination of cephalexin in commercially available and simulated samples of oral suspensions. The MA, described in the Brazilian Pharmacopeia, was performed with a strain of Staphylococcus aureus ATCC 6538 as the test organism, following the cylinder-plate method. The LC analysis followed the European Pharmacopoeia, 3rd Ed., and was used with minor modifications. The MEKC analysis was based on a previous work of the group. Estimates of the repeatability relative standard deviations of the MA, LC, and MEKC methods in the analysis of a commercial sample were 0.34, 0.42, and 0.37%, respectively. The recovery obtained with LC was 99.90 +/- 1.11%; for MEKC, it was 100.09 +/- 0.56%. Although the 3 methodologies were statistically equivalent for the determination of cephalexin in oral suspensions, MA gave suitable repeatability despite being nonspecific and time-consuming. MEKC provided faster analysis and higher column efficiency, whereas LC presented superior sensitivity. The results indicated that MEKC can be used as an alternative method to MA and LC in routine quality control laboratories.     

Statistical evaluation of linear solvation energy relationship models used to characterize chemical selectivity in micellar electrokinetic chromatography

Characterization of retention and selectivity differences between surfactants in micellar electrokinetic chromatography (MEKC) using linear solvation energy relationships (LSERs) has been given a significant amount of attention in the last four years. This report evaluates the validity of using the two LSER models that have been used to fit retention in MEKC in the literature. The results and the fit of the revised model and parameters developed by Abraham and coworkers are compared to the original model developed by Kamlet, Taft, and coworkers. LSERs can generally only be used as a comparative tool to describe the selectivity differences between surfactant systems used in MEKC. With this in mind, it was determined that the results of both models essentially provide the same information about these differences. However, the revised model and parameters have been found to yield a statistically better fit of the MEKC retention data as well as providing more chemically sound LSER coefficients.     

MEKC Determination of Nilutamide in Human Serum Using Sweeping in High Salt Sample Matrix

The determination of the antiandrogen drug nilutamide in human serum by MEKC using flutamide as an internal standard is described. Several parameters influencing the separation, such as the running electrolyte composition concerning the micelle concentration and pH, are discussed. MEKC separation was achieved within 7 min using 50 mM sodium borate pH 9.0 with the addition of 50 mM sodium dodecylsulfate at +20 kV. The proposed method was applied to determination of nilutamide in spiked human serum samples after protein precipitation with acetonitrile. The increasing of sensitivity for determination of nilutamide in serum was done by sweeping in a high salt concentration sample matrix when the injection of a larger volume of sample diluted in 150 mM NaCl was applied. The limit of detection after the preconcentration step for nilutamide was 26 ??g L^?1.     

Separation of a group of N-phenylpyrazole derivatives by micellar electrokinetic chromatography: application to the determination of solute-micelle association constants and estimation of the hydrophobicity

Micellar electrokinetic chromatography (MEKC) was applied to the separation of a group of N-phenylpyrazole derivatives. Sodium dodecyl sulfate (SDS) as micellar system and 2-(N-cyclohexylamino)ethanesulfonic acid (CHES) as separation buffer (pH 10) were employed in the absence and presence of different percentages of medium chain alcohols (n-propanol or n-butanol). The separation of multicomponent mixtures of the solutes studied enabled the rapid determination of their retention factors which, in turn, allowed the study of the separation selectivity of compounds and the determination of their solute-micelle association constants (from the linear variation of the retention factors as a function of the total surfactant concentration in the separation buffer). Separation selectivity was studied according to the elution range and number of solutes separated in all the electrolyte solutions employed (45 micellar phases). The effect of the buffer concentration (0.05, 0.08 and 0.10 M), the alcohol nature (n-propanol or n-butanol) and the alcohol percentage (1, 3 or 5%) of the values obtained for the solute-micelle association constants was also studied. The best separation (12 solutes) was performed when a 0.08 M CHES buffer, pH 10, 0.02 M SDS modified by 5% n-butanol was used. The possibilities of using MEKC for evaluating the hydrophobicity of compounds was investigated through the study of the correlation between the logarithm of the retention factors of N-phenylpyrazole derivatives and their logarithm of the octanol-water distribution coefficients estimated by high performance liquid chromatography (HPLC).     

Comparability of retention indices of polar substances at elevated temperatures

Summary The non-linear dependency of the retention indices of drugs on temperature is demonstrated hence an indication of the actual working temperature is essential. Fixed temperatures for the determination of retention indices of drugs are proposed. Interlaboratory comparisons of the retention indices of 14 drugs frequently encountered in analytical practice are made for the first time on two stationary phases in three laboratories and on four GC apparatus. The data show that, in comparison with literature values, the interlaboratory deviation is reduced by 1/3 to 1/2 if same temperatures are used. Standardization of the temperature of determination of the retention indices of drugs and unknown substances in biological specimens on OV-1 and OV-17 is proposed.     

Simultaneous micellar electrokinetic chromatography and liquid chromatography of adriblastina and tarabine PFS Their application to some biological fluids

The current work presents analytical procedures for simultaneous determination of tarabine PFS and adriblastina by micellar electrokinetic chromatography (MEKC) and liquid chromatography (LC). For MEKC analysis, separations and identifications were accomplished using uncoated fused-silica capillary with hydrodynamic injections in the presence of 50mM borate/phophate pH 8.7 and 100mM SDS. The migration times of tarabine PFS and adriblastina were found to be 2.70 and 6.40min, respectively. Calibration curves were established for 10-300ng/mL (r=0.998) tarabine PFS and for 8-120microg/mL (r=0.999) adriblastina. For LC analysis, separations were performed on teicoplanin stationary phase with reversed mobile phase containing methanol:buffer pH 4.05 (20:80%, v/v) at 285nm. The retention times of tarabine PFS and adriblastina were 5.18 and 7.20min, respectively. Calibration curves were established for 3-90microg/mL (r=0.998) tarabine PFS and for 10-120microg/mL (r=0.999) adriblastina. Both MEKC and LC methods were applied for the simultaneous determination of analytes in urine samples.     

Quantification of testosterone and epitestosterone in biological samples by capillary electrophoresis with immunoaffinity extraction

Testosterone is one of the most common doping drugs abused by athletes. Therefore, it is necessary to develop a sensitive and simple method to monitor testosterone and its epimer epitestosterone. An off-line immunoaffinity extraction followed by capillary electrophoresis for simultaneous determination of testosterone and epitestosterone has been described in this paper. Anti-epitestosterone monoclonal antibody which is specific to both testosterone and epitestosterone had been prepared and immobilized on a Sepharose 4B stationary phase. The immunoaffinity column was used for sample cleanup, extraction and preconcentration. After elution and reconstitution, testosterone and epitestosterone in the sample were separated and quantified by micellar electrokinetic chromatography(MEKC) using the borate buffer (200 mM borate, pH 8.7) containing 40 mM sodium cholate as a chiral selector. The immunoaffinity column was evaluated in different parameters such as the retention mechanism, selectivity, binding capacity, elution protocol, and reusability. The separation of these two compounds by MEKC was also optimized. Limit of detection for testosterone and epitestosterone were 5 and 23 ng mL⁻¹, respectively. It was satisfactory to apply this method to analyze testosterone and epitestosterone in spiked urine sample with the recoveries from 78% to 109%.     

Rapid and accurate determination of deoxyribonucleoside monophosphates from DNA using micellar electrokinetic chromatography with a cationic surfactant additive

A micellar electrokinetic chromatography (MEKC) method for rapid and accurate determination of 2′-deoxyribonucleoside 5′-monophosphates (dNMPs), four structural elements of DNA, is described. MEKC separation at an optimized pH enabled complete separation of four dNMPs. The use of a cationic surfactant additive for MEKC led to the reversal of EOF, which enhanced the migration velocities of the negatively charged dNMPs. Under the optimized condition, full-baseline separation of the four dNMPs assuring accurate peak integration was obtained within 5 min. For the given separation condition, pH-mediated on-column sample stacking was optimized and applied to enhance sensitivity up to 6-fold. Analytical precision was improved by spiking iothalamate as an internal standard. The accuracy of dNMP quantitation was ensured with dNMP standard solutions determined by inductively coupled plasma-optical emission spectroscopy that measured phosphorous quantity. Performance of the proposed method was ultimately proven by accurate quantitation of a DNA oligonucleotide that was enzymatically hydrolyzed prior to dNMP analysis. The proposed MEKC method turned out to be a reliable analytical method for dNMPs that features high speed, high sensitivity, and high precision, and could be utilized for high-accuracy determination of the amount of DNA as well as the base composition of DNA.     

Matrix effects and selectivity issues in LC-MS-MS

Summary Chlorobenzenes, triazine and phenylurea herbicides were separated by normal micellar electrokinetic chromatography (MEKC) and by micellar electrokinetic chromatography with reversed flow (RF-MEKC) in running buffers containing organic solvents. The relationship between the two techniques is similar to that between reversed-phase and normal-phase HPLC. Using RF-MEKC, the separation of lipophilic compounds is often improved compared to normal MEKC. The migration in MEKC and in RF-MEKC was characterised by lipophilic and polar indices. The experimental values of the lipophilic indices of the compounds tested in the two techniques were close to the indices in reversed-phase HPLC (RP-HPLC). This enables the use of the indices determined in RP-HPLC for predicting the effects of changing composition of the running buffers on migration times in MEKC and in RF-MEKC. Presented at Balaton Symposium '01 on High-Performance Separation Methods, siófok, Hungary, September 2–4, 2001     

Compositional studies of high-temperature coal tar by GC/FTIR analysis of light oil fractions

Summary A method for determination of the composition of the light oil fractions in high-temperature coal tar by means of distillation, followed by gas chromatography on a crosslinked fused-silica, capillary column coated with optimum amount of stationary phase and identification by capillary gas chromatography/Fourier transform infrared spectrometry combined with GC retention indices (GC/FTIR-RI) is described. This method was effectively used to identify complex mixture such as coal tar without any standard samples, especially, adapted for isomeric compounds. More than 60 and 50 compounds were also separated and identified respectively in light oil fractions. This shows the capability of the capillary GC/FTIR combined with GC retention indices to identify isomers not accomplished by GC/MS.     

Temperature programmed retention indices: Calculation from isothermal data. Part 1: Theory

Direct conversion of isothermal to temperature programmed indices is not possible. In this work it is shown that linear temperature programmed retention indices can only be calculated from isothermal retention data if the temperature dependence of both the distribution coefficients and the column dead time are taken into account. Procedures are described which allow calculation of retention temperatures and from these, accurate programmed retention indices. Within certain limits the initial oven temperature and programming rate can be chosen freely. The prerequisite for this calculation is the availability of reliable isothermal retention data (retention times, retention factors, relative retention times, or retention indices) at two different temperatures for one column. The use of compiled isothermal retention indices at two different temperatures for the calculation of retention temperatures and thus temperature programmed indices is demonstrated. For the column for which programmed retention indices have to be determined, the isothermal retention times of the n-alkanes and the column dead time as a function of temperature have to be known in addition to the compiled data for a given stationary phase. Once the programmed retention indices have been calculated for a given column the concept allows the calculation of temperature programmed indices for columns with different specifications. The characteristics which can be varied are: column length, column inner diameter, phase-ratio, initial oven temperature, and programming rate.     

Fundamental studies on electrokinetic chromatography with PEGylated phospholipid micelles.

In this study, micelles prepared from distearoylphosphatidylethanolamine with covalently attached poly(ethylene) glycol) (PEG) of molecular weight 2000 (DSPE-PEG-2000) were employed in micellar electrokinetic chromatography (MEKC) as pseudostationary phases. Since DSPE-PEG-2000 contains long hydrophobic alkyl chains, an anionic phosphate group, and hydrophilic PEG chains, the prepared micelles are expected to provide a characteristic retention behavior for both neutral and ionic compounds. As a typical example, a baseline separation of phenol and 2-naphthol was successfully achieved by using the DSPE-PEG-2000 micelles as a background electrolyte for MEKC; such success clearly shows that the micelles can retain electrically neutral compounds. The MEKC separations of anionic and cationic compounds with a DSPE-PEG-2000 micellar solution and the enantioseparation of binaphthyl compounds with mixed micelles containing bile salt are also discussed.     

Simultaneous determination of cefepime and L-arginine by micellar electrokinetic chromatography and applications to commercial injections

A simple micellar electrokinetic chromatography (MEKC) with UV detection is described for simultaneous analysis of cefepime and L-arginine. The determination of cefepime and L-arginine in pharmaceutical preparations was performed at 25degreesC using a background electrolyte consisting of Tris buffer with sodium dodecyl sulfate (SDS) as the electrolyte solution. Several parameters affecting the separation of the drugs were studied, including the pH and concentrations of the Tris buffer and SDS. Under optimal MEKC conditions, good separation with high efficiency and short analysis times is achieved. Using cefazolin as an internal standard, the linear ranges of the method for the determination of cefepime and L-arginine were over 5-100 microg/mL; the detection limits of cefepime (signal to noise ratio = 3; injection 3.45 kPa, 3 s) and L-arginine (signal to noise ratio = 3; injection 3.45 kPa, 3 s) were 2 microg/mL and 4 microg/ mL, respectively. Applicability of the proposed method for the determination of cefepime and L-arginine in commercial injections was demonstrated.     

Simultaneous Determination of Various Classes of Pesticides Using Micellar Electrokinetic Capillary Chromatographyin Combination with Solid-Phase Extraction

The potential of micellar electrokinetic capillary chromatography (MEKC) with UV-detection for the simultaneous determination of various selected pesticides in water samples was investigated. The developed method using solid-phase extraction showed high efficiency and good resolution with detection limits in the 0.2 to 0.5 ppm range. Comparison of several SPE cartridges demonstrates their suitability for the extraction of pesticides with different hydrophobicity achieving 5000-fold enrichment. The described method involving SPE procedure and MEKC separation enables the successful determination of a wide spectrum of pesticides in water in the range of maximum residue limits (MRLs).     

Further applications for capillary gas chromatography in routine quantitative toxicological analyses

The use of capillary column gas chromatography for drug screening in forensic toxicology has become increasingly widespread. Screening procedures however are often lengthy and unsuitable for rapid confirmatory or quantitative applications. In order to develop a practical scheme for confirmatory/quantitative analysis, we have optimized a series of temperature profiles to allow the rapid quantitative determination of a wide range of acid/neutral and basic drugs in extracts from post mortem fluids and tissue. The appropriate profile is selected based on the retention index on a standard crosslinked methyl silicone column used to screen extracts. The use of a 5% phenyl methyl silicone phase allows complementary identification, and allows the separation of some pairs of compounds with identical retention indices in the screning procedure.     

Copolymerized polymeric surfactants: characterization and application in micellar electrokinetic chromatography

An achiral monomeric surfactant (sodium 10-undecenyl sulfate, SUS) and a chiral surfactant (sodium 10-undecenoyl L-leucinate, SUL) were synthesized and polymerized individually to form poly-SUS and poly-SUL. These surfactants were then copolymerized at various molar ratios to produce a variety of copolymerized surfactants (CoPSs), possessing both achiral (sulfate) and chiral (leucinate) head groups. The CoPSs, poly-SUS, poly-SUL, and sodium dodecyl sulfate were characterized using several analytical techniques. The aggregation numbers of the polymeric surfactants and the partial specific volumes were determined by the use of fluorescence quenching and density measurements, respectively. These polymeric surfactants were investigated as novel pseudostationary phases in micellar electrokinetic chromatography (MEKC) for the separation of chiral and achiral solutes. Solute hydrophobicity was found to have major influence on the MEKC retention of alkyl phenyl ketones. In contrast, hydrogen-bonding ability of benzodiazepines is the major factor that governs their retention, but hydrophobicity has an insignificant effect on MEKC retention of benzodiazepines.