Micellar electrokinetic capillary chromatography for the separation of cefalexin and its related substances

Micellar electrokinetic capillary chromatography (MEKC) was examined for analysis of cefalexin and its related substances. Good selectivity was obtained with two different buffer solutions: a sodium acetate buffer (50 mM, pH 5.25) containing sodium dodecyl sulfate (50 mM SDS) or sodium phosphate buffer (40 mM, pH 7.0) containing 100 mM SDS. Both methods permit cefalexin to be completely separated from its ten related substances within 20 min. The robustness of the method, using pH 5.25 acetate buffer, was examined by means of a full-fraction factorial design to test the influence of buffer pH, concentration of SDS and buffer concentration. The parameters for validation such as linearity, precision, limit of detection and limit of quantitation are also reported. The results show that method 1 is suitable for the analysis of cefalexin.     

Assay of the Related Compounds Thiamphenicol, Florphenicol, and Chloramphenicol by CE

The purpose of this study was to examine the migration behaviour of a mixture of thiamphenicol, its two analogues florphenicol and chloramphenicol, and four impurities found in thiamphenicol. Capillary zone electrophoresis was performed with phosphate–borate or borate running buffers, pH 8.5, with or without addition of sodium dodecylsulphate (SDS). Although the compounds could not be resolved in the absence of SDS, supplementation of both types of buffer, at concentrations of 100 mM and higher, with 50 mM SDS enabled separation. On the basis of migration time and resolution, 100 mM borate buffer containing 50 mM SDS was identified as the best for the study. The accuracy and precision of the method were good under these conditions.     

Separation and determination of closely related lantibiotics by micellar electrokinetic chromatography

A sensitive micellar electrokinetic chromatography (MEKC) method was developed for the separation and determination of four closely related lantibiotics: gallidermin, cinnamycin, duramycin and nisin. Factors affecting the separation of the lantibiotics such as pH, phosphate buffer concentration, SDS concentration and wavelength for UV detection were investigated. By optimizing these experimental conditions, successful separation was achieved between class 1A lantibiotics (nisin and gallidermin) and class 1B lantibiotics (duramycin and cinnamycin). The four lantibiotics were separated within 12 min in 50 mM phosphate buffer at pH 3.95 ± 0.1 containing 80 mM SDS with UV detection of 214 nm. The LOD (S/N = 3) were 61 ng/mL for gallidermin, 57 ng/mL for cinnamycin, 55 ng/mL for duramycin and 58 ng/mL for nisin. The method was successfully applied to real samples such as fermentation broth, bovine colostrum and predrop beer. This method yielded satisfactory results, with quantitative recoveries of spiked lantibiotics in the three samples ranging from 86.1 to 99.6%.     

Monomeric and polymeric anionic gemini surfactants and mixed surfactant systems in micellar electrokinetic chromatography. Part I: characterization and application as novel pseudostationary phases

Sodium di(undecenyl) tartarate monomer (SDUT), a vesicle-forming amphiphilic compound possessing two hydrophilic carboxylate head groups and two hydrophobic undecenyl chains gemini surfactant, was prepared and polymerized to form a polymeric gemini surfactant (i.e., poly-SDUT). These anionic surfactant systems with carboxylate (SDUT and poly-SDUT) and sulfate (sodium dodecyl sulfate, SDS) head groups as well as mixed surfactant systems (SDS/SDUT, SDS/poly-SDUT, and SDUT/poly-SDUT) were then applied as novel pseudostationary phases in micellar electrokinetic chromatography (MEKC). The SDUT and poly-SDUT were characterized using various analytical techniques. Retention factors of 36 benzene derivatives were calculated in 20 mM phosphate buffer of each surfactant system. The retention factor values of the test solutes show that there are distinctive selectivity differences between the surfactant systems. Solute-pseudostationary phase interactions in MEKC were also examined by determining the free energy of transfer of the substituted functional groups from the aqueous buffer phase into the pseudostationary phase.     

Cation-selective exhaustive injection and sweeping MEKC for direct analysis of methamphetamine and its metabolites in urine

Direct analysis of methamphetamine, amphetamine, and p-hydroxymethamphetamine in urine was achieved by cation-selective exhaustive injection and sweeping micellar EKC. A bare fused-silica capillary (40 cm, 50 microm id) was filled with phosphate buffer (80 mM, pH 3, containing 20% ACN). Then a high-conductivity buffer (100 mM phosphate, pH 3; 6.9 kPa for 2.5 min) was injected. Samples were loaded using electrokinetic injection (10 kV, 600 s) which created long zones of cationic analytes. To enhance sensitivity by sweeping, the stacking step was performed using a phosphate buffer (50 mM, pH 3, containing 20% ACN and 100 mM SDS) at -20 kV before separation by MEKC. This method was capable of detecting the analytes at ppb levels. The calibration plots were linear (r(2) >or= 0.9948) over a range of 100-5000 ng/mL for methamphetamine, and 100-2000 ng/mL for amphetamine and p-hydroxymethamphetamine. The LODs (S/N = 3) were 20 ng/mL for methamphetamine, and 15 ng/mL for amphetamine and p-hydroxymethamphetamine. The method was applied to analysis of 14 urine samples of addicts and is suitable for screening suspected samples for forensic purposes. The results showed good agreement with fluorescence polarization immunoassay and GC-MS.     

Optimization of separation and migration behavior of chloropyridines in micellar electrokinetic chromatography

The separation and migration behavior of pyridine and eight chloropyridines, including three monochloropyridines, four dichloropyridines, and 2,3,5-trichloropyridine were investigated by micellar electrokinetic chromatography using either sodium dodecyl sulfate (SDS) as an anionic surfactant or SDS-Brij 35 mixed micelles. Various parameters such as buffer pH, SDS concentration, Brij 35 concentration and methanol content that affect the separation were optimized. Complete separation of these chloropyridines was optimally achieved with a phosphate buffer containing SDS (30 mM) and methanol (10%, v/v) at pH 7.0. The resolution and selectivity of analytes could be considerably affected by the addition of methanol and/or Brij 35 to the background electrolyte. The migration order of these chloropyridines depends primarily on their hydrophobicity. However, electrostatic interactions may also play a significant role in the determination of the migration order of the positional isomers of chloropyridines.     

Quantitative determination of isoniazid in biological samples by cation-selective exhaustive injection-sweeping-micellar electrokinetic chromatography

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis, infects approximately one third of the current world population. Isoniazid is one of the most frequently used first-line anti-TB drugs. In this study, we developed a sensitive cation-selective exhaustive injection–sweeping–micellar electrokinetic chromatography method (CSEI-Sweep-MEKC) for analyzing isoniazid in human plasma. Parameters including acetonitrile (ACN) percentage in the separation buffer; the injection time, and concentration of the high-conductivity buffer; sodium dodecyl sulfate (SDS) concentration; phosphate concentration in the sample matrix; and the sample injection time were all optimized to obtain the best analytical performance. The optimal background electrolyte comprised 50 mM phosphate buffer, 100 mM SDS, and 15% ACN. Non-micelle background electrolyte, containing 75 mM phosphate buffer and 15% ACN, was first injected into the capillary, followed by a short plug of 200 mM phosphate (high-conductivity buffer). Run-to-run repeatability (n = 3) and intermediate precision (n = 3) of peak area ratios were found to be lower than 8.7% and 11.4% RSD, respectively. The accuracy of the method was within 98.1–106.9%. The limit of detection of isoniazod in human plasma was 9 ng mL⁻¹. Compared with conventional MEKC, the enhancement factor of the CSEI-Sweep-MEKC method was 85 in plasma samples. The developed method was successfully used to determine isoniazid concentration in patient plasma. The results demonstrated that CSEI-Sweep-MEKC has the potential to analyze isoniazid in human plasma for therapeutic drug monitoring and clinical research.     

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.     

Towards a general approach for the impurity profiling of drugs by micellar electrokinetic chromatography

A general micellar electrokinetic chromatographic (MEKC) strategy for the impurity profiling of drugs was developed involving a sodium dodecyl sulfate (SDS) and a cetyltrimethylammonium bromide (CTAB) MEKC system. With this combination, in principle, each sample component passes the detector in at least one of the two MEKC systems provided that separation buffers of the same pH are used in both systems. In order to select the proper MEKC systems, the electroosmotic flow (EOF) and micelle migration time (t(mc)) were determined for separation buffers of several pH values, containing various amounts of surfactant and organic modifier. The selectivity of the MEKC systems was studied using a mixture of compounds with a wide range of physico-chemical properties. The final selection of two adequate MEKC systems for this approach was based on the requirements that the t(mc) (i.e., analysis time) of both systems was below 20 min and that the t(mc)/t(eof) ratio was above 3 or 2 for the SDS and CTAB system, respectively. Furthermore, the systems should provide high efficiency, exhibit differences in selectivity and use moderate concentrations of modifier and surfactant, so that, if needed, further optimization is possible. The selected MEKC systems contained 60 mM SDS or 10 mM CTAB, respectively, in a phosphate buffer (pH 7.5) with 10% acetonitrile. Some test compounds with extreme mobilities were used to demonstrate the suitability of the MEKC approach to detect each component of a sample. The potential of the proposed MEKC combination for impurity profiling was demonstrated by the analysis of fluvoxamine with several impurities at the 0.1% level.     

Therapeutic deferoxamine and deferiprone monitoring in β‐thalassemia patients’ plasma by field‐amplified sample injection and sweeping in capillary electrophoresis

One CE method was established for detecting deferoxamine (DFO) and deferiprone (DFR) in plasma. For β‐thalassemia patients, DFO and DFR are major medicines to treat the iron overload caused by blood transfusion. Field‐amplified sample injection combined with sweeping was used for sensitivity enhancement in CE. This method was performed on an uncoated fused‐silica capillary. After liquid–liquid extraction, the plasma samples were electrokinetically injected into capillary at +10 kV for 180 s. The phosphate buffer (100 mM) containing 50 mM triethanolamine was used as the BGE (pH 6.6). Separation buffer was phosphate buffer (100 mM, pH 3.0) containing 150 mM SDS. This method showed good linearity (r ≥ 0.9960). Precision and accuracy were evaluated by the results of RSD and relative error of intrabatch and interbatch analyses, and all of the absolute values were less than 6.12%. The LODs (S/N = 3) were 200 ng/mL for DFO, and 25 ng/mL for DFR. The LOQ (S/N = 10) of DFO and DFR were 600 and 75 ng/mL, respectively. This method was applied for clinical applications of five β‐thalassemia patients.     

Simultaneous determination of inorganic anions, carboxylic and aromatic carboxylic acids by capillary zone electrophoresis with direct UV detection

Co-electroosmotic capillary zone electrophoresis (CZE) with direct UV detection was developed for simultaneous determination of inorganic anions, carboxylic and aromatic carboxylic acids. These solutes were separated using a 30 mM phosphate buffer containing 1.0 mM tetradecyltrimethylammonium bromide (TTAB) and 20% (v/v) acetonitrile at pH of 6.5 and directly detected by UV at 190 nm. Calibration curves were linear in the range 0.01-2.0 mM, depending of the solutes. The detection limits ranged from 1.0 to 8.0 microM and the relative standards deviations (n=5) in range from 1.9 to 3.6% for the peak area. The proposed method was used to determine inorganic anions and carboxylic and aromatic acids in soil and plant tissue extracts.     

Studies on bovine serum albumin-sodium dodecyl sulfate complexes using pyrene fluorescence probe and 5-doxylstearic acid spin probe

Interactions and characteristics of 0.1% bovine serum albumin (BSA)-sodium dodecyl sulfate (SDS) in 20 mM phosphate buffer solution were investigated by means of fluorescence spectroscopy and electron spin resonance (ESR) spectroscopy. In BSA-SDS system, the intensity ratio, Im3/Im1, of the third vibronic band of the pyrene monomer to the first vibronic band showed a small peak at about 0.1 mM SDS in the phosphate buffer below cmc. In accordance with this Im3/Im1 ratio, the intensity ratio, Ie/Im1, of fluorescence from the pyrene excimer to that from the monomer showed a pseudo-plateau (0.08-0.8 mM) and suggested the existence of micelle-like aggregates below the cmc. Temperature dependence of ln(Ie/Im1) in pyrene fluorescence in the SDS-BSA system was examined as a function of SDS concentration. The activation energy of pyrene diffusion for excimer formation in a micelle was estimated to be 19.2 kJ mol(-1) for the BSA-SDS system. ESR spectra of 5-doxylstearic acid (5-DSA) showed that the probe location is restricted at SDS concentrations above the cmc, and that the probe also is highly restricted in motion for BSA-bound SDS micelles.     

Separation of twenty-one naphthalene sulfonates by means of capillary electrophoresis

The production and use of naphthalene sulfonates can easily cause pollution of surface and other types of waters. In the present study, capillary electrophoresis in combination with UV absorption detection was used to separate 21 amino- and hydroxy-substituted naphthalene sulfonates which included multiple isomeric compounds. The influence of various parameters such as pH (which turned out to be extremely important), temperature of the surrounding air flow, and the use of buffer additives (micelles, cyclodextrins, organic modifiers) was studied. Complete separation of all analytes including the isomers, was achieved in two runs with a 50 mM boric acid/borate buffer, containing either 100 mM sodium dodecylsulfonate or 15% acetonitrile. The limits of detection obtained for the individual compounds typically were 20μgI⁻¹. River water samples spiked at this concentration level could be analysed using a simple three-step sample clean-up procedure.     

Cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography for analysis of morphine and its four metabolites in human urine

A cation-selective exhaustive injection and sweeping micellar EKC (CSEI-Sweep-MEKC) was established to analyze morphine and its four metabolites, including codeine, normorphine (NM), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G). After SPE, the urine samples were analyzed by this CE method. The phosphate buffer (75 mM, pH 2.5) containing 30% methanol was first filled into an uncoated fused-silica capillary (40 cm, 50 microm id), then a high-conductivity buffer (120 mM phosphate, 10.3 kPa for 99.9 s) followed. The pretreated urine sample was loaded by electrokinetic injection (10 kV, 600 s). The stacking and separation were performed by using phosphate buffer (25 mM, pH 2.5) containing 22% methanol and 100 mM SDS at -20 kV, and detected at 200 nm. During method validation, calibration plots were linear (r > or = 0.998) over a range of 30-3000 ng/mL for morphine, NM, and codeine, 100-2000 ng/mL for M6G, and 80-3200 ng/mL for M3G. The LODs (S/N = 5, sampling 600 s at 10 kV) were 10 ng/mL for morphine, NM, and codeine, 35 ng/mL for M6G, and 25 ng/mL for M3G. This stacking CE method could increase 2500-fold sensitivity of codeine, when comparing with CZE. Five addicts' urine specimens were analyzed. Their results were compared with those of LC-MS-MS, and showed good coincidence. This method could be feasible for monitoring morphine and its metabolites in forensic interest and pharmacokinetic investigations.     

On-chip chiral and achiral separation of amphetamine and related compounds labeled with 4-fluoro-7-nitrobenzofurazane

Amphetamine and analogous compounds have been labeled with 4-fluoro-7-nitrobenzofurazane and analyzed on a microfabricated chip. Separation of norephedrine, ephedrine, cathinone, pseudoephedrine, methcathinone, amphetamine and methamphetamine is demonstrated using micellar electrokinetic capillary chromatography (MEKC) and laser-induced fluorescence (LIF) detection. Chiral separations of individual drugs were studied using neutral and negatively charged cyclodextrins (CDs) with and without the addition of an organic modifier and/or sodium dodecyl sulfate (SDS). The best results were obtained using a highly sulfated gamma-CD (HS-gamm-CD) in combination with a low concentration of SDS. To obtain complete separation of a mixture of (+/-)-norephedrine, (+/-)ephedrine, (+/-)-pseudoephedrine, (+/-)-methcathinone, (+/-)-amphetamine and (+/-)-methamphetamine it was necessary to add a small amount (1.5 mM) of SDS to the separation buffer. Optimized chiral separation was achieved within 7 min using an S-folded separation channel, a separation voltage of 8 kV and a buffer consisting of 50 mM phosphate (pH 7.35), 10 mM HS-gamma-CD and 1.5 mM SDS.     

Selective determination of gamma-aminobutyric acid, glutamate and alanine by mixed micellar electrokinetic chromatography and fluorescence detection

A mixed micellar electrokinetic chromatography method with fluorescence detection was developed to simultaneously monitor gamma-aminobutyric acid (GABA), glutamate (Glu) and alanine (Ala) in biological samples. Amino acids were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA). The separation of three NDA-labeled isomers (GABA, alpha-ABA, beta-ABA) was studied in detail with different micelles solutions such as sodium dodecyl sulfate (SDS), beta-cyclodextrin (beta-CD) and sodium cholate (SC). Simultaneous resolution of GABA, Glu and Ala from 21 amino acids was achieved within 5 min using 20 mM phosphate buffer at pH 8.7 containing 24 mM SC and 26 mM SDS. The detection limits were 4.0 x 10(-8), 1.1 x 10(-8) and 1.3 x 10(-8) M, for GABA, Glu and Ala, respectively, with S/N = 2. The method was applied to monitor the changes of amount of GABA, Glu and Ala in tobacco leaf in response to cold and dark stress.     

Field-amplified sample stacking and nonaqueous capillary electrophoresis determination of complex mixtures of polar aromatic sulfonates

Nonaqueous CE and field-amplified sample stacking have been used in the determination of complex mixtures of polar aromatic sulfonates (AS; mainly benzene- and naphthalenesulfonates) of environmental concern. The analytical procedure consists of an on-column aqueous sample enrichment, followed by the nonaqueous electrophoretic determination of stacked aromatic sulfonates. Various organic solvents were used as separation medium, acetonitrile and N-methylformamide gave the best results. Optimum capillary electrophoresis separation is obtained with ammonium acetate (25 mM) dissolved in N-methylformamide-methanol (90:10) as background electrolyte. This combined method was applied to the analysis of surface water samples spiked with selected aromatic sulfonates derivatives.     

Solvation dynamics of DCM in a polypeptide-surfactant aggregate: gelatin-sodium dodecyl sulfate

Solvation dynamics of 4-(dicyanomethylidene)-2-[p-(dimethylamino)styryl]-6-methyl-4H-pyran (DCM) is studied in a polypeptide-surfactant aggregate consisting of gelatin and sodium dodecyl sulfate (SDS) in potassium dihydrogen phosphate (KP) buffer. The average solvation time (tauS) in gelatin-SDS aggregate at 45 degrees C is found to be 1780 ps, which is about 13 times slower than that in 15 mM SDS in KP buffer at the same temperature. The fluorescence anisotropy decay in gelatin-SDS aggregate is also different from that in SDS micelles in KP buffer. DCM displays negligible emission in the presence of gelatin in aqueous solution. Thus the solvation dynamics in the presence of gelatin and SDS is exclusively due to the probe (DCM) molecules at the gelatin-micelle interface. The slow solvation dynamics is ascribed to the restrictions imposed on the water molecules trapped between the polypeptide chain and micellar aggregates. The critical association concentration (cac) of SDS for gelatin is determined to be 0.5 +/- 0.1 mM.     

Micellar electrokinetic and high-performance liquid chromatographic determination of potential manufacturing impurities in pholcodine

Quantitative high-performance liquid chromatographic (HPLC) and micellar electrokinetic chromatographic (MEKC) methods have been developed for the determination of four structurally related potential manufacturing impurities, including morphine, of the opiate derivative pholcodine. Pholcodine and the four impurities were separated by MEKC in less than 14 min using a 70 cm x 75 microm I.D. uncoated fused-silica capillary (25 kV at 30 degrees C) and a running buffer consisting of 10% acetonitrile (v/v) in 20 mM borate-phosphate buffer pH 8.0 containing 40 mM sodium dodecyl sulphate (SDS). The MEKC method was compared to a HPLC method using a 5 microm Luna phenyl-hexyl column (150 x 4.6 mm I.D.) eluted with a mobile phase consisting of a mixture of 10% (v/v) acetonitrile, 7% (v/v) tetrahydrofuran in 20 mM phosphate buffer pH 8.0. Both methods were fully validated and a comparison was made regarding selectivity, linearity, precision, robustness and limits of detection and quantitation. The presence of the impurities in different samples of pholcodine drug substance was investigated using both methods.     

Fast CE for combinatorial catalysis

Two solvent-modified MEKC methods were developed for the quantitative analysis of heterocyclic amines synthesised using intramolecular ring closure via catalysed hydroamination. The first method was capable of resolving six of the amines (precursors and products) with a sample-to-sample injection time of 2 min employing a 20 mM borate buffer, pH 9.2 with 20 mM SDS and 5% v/v n-butanol (n-BuOH). A second low-pH method using 20 mM phosphate buffer, 100 mm SDS, 5% v/v n-BuOH and 20% v/v iso-propanol (i-PrOH) was able to resolve an additional pair of compounds with a sample-to-sample time of 3.5 min. Application of the first method to the analysis of a sample containing catalyst as well as amines placed directly in a 96-well plate showed excellent performance, with migration time and peak height and area reproducibility being less than 0.9 and 9.6%, respectively. The quantity of conversion by catalyst was calculated to be 68 +/- 7%, which was in excellent agreement with the 67 +/- 5% obtained by more conventional (1)H NMR experiments, with the added advantage that this method is also cheaper, quicker and more amendable to high-throughput screening of combinatorial libraries.