Determination of urinary hippuric acid by micellar electrokinetic capillary chromatography

We propose a method for the simultaneous determination of hippuric acid (HA) and creatinine based on capillary micellar electrokinetic chromatography. Experimental conditions were 20 mM sodium phosphate, pH 7.20, 25 mM sodium dodecyl sulfate, 5% (v/v) acetonitrile. Electropherograms evidenced HA and creatinine peaks in less than 12 min. The method showed good linearity for both analytes and satisfactory within-day precision. The present method, which is accurate, sensitive, rapid and simple, may be applied to single-spot urine samples.     

Simultaneous determination of paracetamol, caffeine and propyphenazone in ternary mixtures by micellar electrokinetic capillary chromatography

A new micellar electrokinetic capillary chromatographic method has been developed to analyze the pharmaceutical preparations containing ternary combination of paracetamol (PAR), caffeine (CAF) and propyphenazone (PRO). Best results were obtained by using 20mM pH 9.0 borate buffer containing 30mM sodiumdodecylsulphate as the background electrolyte. Diflunisal (DIF) was used as internal standard (IS). The separation was performed through a fused silica capillary (50microm internal diameter, 44cm total length, 35.5cm effective length) at 25 degrees C with the application of 3s of hydrodynamic injection at 50mbar pressure and a potential of 29kV. Detection wavelength was 200nm. Under these conditions, the migration times were found to be 5.174min for PAR, 5.513min for CAF, 7.195min for DIF, and 9.366min for PRO. Linearity ranges for the method were determined as 2-200microgmL(-1) for PAR and CAF and 3-200microgmL(-1) for PRO. Limit of detections were found as 0.6microgmL(-1) for PAR and CAF and 0.8microgmL(-1) for PRO. According to the validation study, the developed method was proved to be accurate, precise, sensitive, specific, rugged and robust. Three pharmaceutical preparations, which are produced by different drug companies in Turkey, were analyzed by the developed method. One of the same preparations was also analyzed by the derivative ratio spectro zero-crossing spectrophotometric method reported in literature. No significant differences were found statistically.     

Determination of thiamphenicol in human plasma by micellar electrokinetic capillary chromatography

A micellar electrokinetic chromatographic method is described for the determination of thiamphenicol in human plasma. The plasma sample was basified by adding K₂HPO₄ and was then extracted with ethyl acetate. After the solvent was evaporated, the residue was reconstituted in water. Approximately 40 nl of the solution were injected hydrodynamically. The running buffer was 20 mM borate (pH 9.2) containing 40 mM sodium dodecyl sulfate and 10% acetonitrile. The applied voltage was 18 kV and the detector wavelength was set at 195 nm. On-column sample stacking was achieved during the analysis to enhance the sensitivity; the limit of quantitation was 0.1 μg/ml. Linearity was over the range of 0.2 to 10 μg/ml. Recovery was 93.7±3.3%, the intra-day precision and accuracy was 99.6±2.8%; the inter-day precision and accuracy was 98.4±3.4%. The concentration of thiamphenicol in human plasma from eight volunteers was measured after administering thiamphenicol capsules orally.     

Determination of diterpenoid triepoxides in Tripterygium wilfordii by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatographic (MEKC) method has been established for the identification and determination of diterpenoid triepoxides in the Chinese herb Tripterygium wilfordii Hook F. and its preparations. Studies of the influence of boric acid and borax buffer concentration and pH, and of sodium dodecylsulphate (SDS) concentration have been carried out, and the optimum separation for the triepoxides was achieved using 20 mM boric acid and 10 mM borax with 20 mM SDS as the running buffer. MEKC was found to exhibit good accuracy, precision and repeatability. The sensitivity of the assay was sufficient to monitor the three active components in T. wilfordii and its preparations.     

Separation of new antidepressants and their metabolites by micellar electrokinetic capillary chromatography

Selective serotonin reuptake inhibitors (SSRIs), serotonin noradrenergic reuptake inhibitors (SNaRIs) and noradrenergic and specific serotoninergic antidepressant (NaSSA) are widely used in the treatment of depression. An increase in antidepressant intoxications led to the development of reliable analytical methods for their analysis. A new determination procedure for these compounds (milnacipran, venlafaxine, desmethylvenlafaxine, mirtazapine, desmethylmirtazapine, citalopram, desmethylcitalopram, fluvoxamine, paroxetine, sertraline and fluoxetine) was developed by micellar electrokinetic capillary chromatography (MEKC) with diode array detection (DAD). Separation and determination were optimised on an uncoated fused-silica capillary (600 mm, 75 microm I.D.). The migration buffer consisted of 20 mM sodium borate, pH 8.55, with 20 mM SDS and 15% isopropanol, at an operating voltage of 25 kV. The column temperature was maintained at 40 degrees C. Injection in the capillary was performed in the hydrodynamic mode (0.5 p.s.i., 15 s). In these conditions, the migration time of the antidepressants was less than 11 min. In most cases, calibration curves were established for 30 - 2000 ng/ml (r > 0.995). The limit of detection and the limit of quantification were ranged between 10 and 20 and between 20 and 30 ng/ml, respectively, for all the molecules. This method allowed the determination of some of these compounds in biological fluids (blood, urine) in post-mortem cases. Samples (1 ml) were extracted with diethyl ether (5 ml) at pH 9.6 and reconstituted in diluted migration buffer. Similar results were obtained by a HPLC-DAD determination, performed as a reference method. These results suggest that this MEKC method can be useful for the determination of new antidepressants in post-mortem cases.     

Simultaneous determination of nitrazepam and its metabolites in urine by micellar electrokinetic capillary chromatography

We applied micellar electrokinetic capillary chromatography to simultaneous separation and determination of nitrazepam and its major metabolites, 7-aminonitrazepam and 7-acetamidonitrazepam, in spiked urine. Prior to electrophoresis, the three compounds were successfully extracted from the spiked urine with commercial disposable solid-phase cartridges. The optimum running buffer for the separation was prepared by combining 85 parts of 60 mM sodium dodecyl sulphate—6 mM phosphate—borate, adjusted to pH 8.5, with 15 parts of methanol. The separation order, completed within 25 min, was 7-aminonitrazepam > 7-acetamidonitrazepam > nitrazepam, at an applied potential of 20 kV. We obtained reproducible electropherograms in successive repetitions, and few other peaks or interferences appeared in the electropherogram. The detection limits of the three compounds were 50–100 pg (0.1–0.2 μg/ml of analyte in spiked urine), and the recoveries were 78.9–100.8% for 1 μg/ml and 84.1–100.3% for 5 μg/ml. The application of this method to forensic or clinical samples is demonstrated.     

Separation of bisbenzylisoquinoline alkaloids by micellar electrokinetic chromatography

The micellar electrokinetic chromatographic (MEKC) separation of seven bisbenzylisoquinoline alkaloids has been developed. The effects of various separating factors were studied. Optimum separation was achieved using a buffer (pH 9.2) of 20 mM sodium borate and 20 mM sodium dihydrogen phosphate buffer containing 55 mM sodium cholate; the optimum voltage and injection time were 21 kV and 0.05 min, respectively. Highest peak efficiency was obtained when the analytes were dissolved in 10 mM sodium dodecyl sulphate as sample matrix for injection. The elution order of the bisbenzylisoquinoline alkaloids was related to their lipophilicity. The resolution, run time and detection limits of the MEKC method were compared with those of an HPLC method developed previously.     

Determination of dialysate creatinine by micellar electrokinetic chromatography

Micellar electrokinetic chromatography with UV absorbance detection has been applied for fast and selective determination of creatinine in samples of postdialysate fluid. Optimization of the method was performed, with the best results being obtained using a 30 mM borate-100 mM sodium dodecyl sulphate background electrolyte, pH 9, with the detector set at 235 nm and an applied voltage of 17 kV across a fused-silica capillary of 67 cm/75 micro m I.D. The linear range of the technique was over 2 orders of magnitude (5-1000 micro M). The developed analytical procedure is useful for the monitoring of clinical hemodialysis treatment, because creatinine levels in real undiluted samples of postdialysate range from 80 to 350 micro M. The separation system allows the analysis of about six to seven samples of spent dialysate per hour in almost real time. The determinations are not influenced by other components of dialysate fluid nor by other surrogates extracted from patient blood. The results of analysis using the developed procedure and the kinetic spectrophotometric Jaffe method conventionally used in clinical settings for creatinine determination are fully comparable. Successful clinical evaluation of the analytical system was performed. The developed system is useful for bloodless estimation of bioanalytical parameters of hemodialysis sessions such as creatinine-time profiles and total creatinine removal. Both these parameters are important in clinical models of hemodialysis therapy.     

Determination of biogenic amines in fish implicated in food poisoning by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatography (MECC) method for the simultaneous determination of seven biogenic amines in fish was developed. The peaks of all components were successfully separated within 11.5 min. MECC was performed with 0.06 M sodium deoxycholate in 0.02 M borate buffer (pH 9.2)–methanol (95:5, v/v) solvent. The average recoveries for all components ranged from 84.4 to 100.3%. The application of this method to detect amines in fried marlin fillet implicated in a food poisoning incident indicated that a high level (56.24 mg/100 g) of histamine was present in the sample. Another 10 fish samples collected from markets were also analyzed and did not contain detectable levels of histamine (<2.5 mg/100 g).     

Determination of temozolomide in serum and brain tumor with micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatographic (MEKC) with photodiode-array detection was applied to determine temozolomide (TMZ) in human serum and brain tumor. The limit of quantitation (LOQ) was 0.096 μg/mL using 325 nm as detection wavelength. The method made possible that the TMZ could be detected in in vivo serum samples without sample pretreatment. In order to detect TMZ at lower concentration, an extraction with ethyl acetate was applied to preconcentrate the analyte. Small amount of brain tumor tissues (less than 1g) were lyophilized and pretreated using extraction as a clean up and concentrating step. After removing the organic solvent a final sample volume of only 10 μL was analyzed. The obtained peak concentrations (8.2-10.1 μg/mL) and T(max) (44-65 min) of TMZ in serum were similar to the data reported by others, the in vivo TMZ concentrations found in brain tumor ranged between 0.061 and 0.117 μg/g.     

N-乙酰氨基苯磺酰氟用于柱前衍生氨基酸的毛细管胶束电动色谱分离

采用一种新型标记试剂,N-乙酰氨基苯磺酰氟（PAABS—F）作为柱前衍生试剂,建立了用毛细管胶束电动色谱分离16种常见氨基酸的方法。以硼砂-三（羟甲基）氨基甲烷（Tris）二元缓冲体系为背景电解液,考察了缓冲液的pH和离子强度、表面活性剂十二烷基硫酸钠（SDS）添加量、有机改性剂种类及分离电压等条件对分离效果的影响,实验结果表明：采用40mmol／L硼砂-Tris（摩尔比1：1）缓冲液（pH9．3）、添加140mmol／L的SDS、体积分数5％甲醇及10mmol／L三乙胺作为分离体系,可对16种PAABS—F氨基酸衍生物进行分离。     

Non-competitive immunoassay for alpha-fetoprotein using micellar electrokinetic capillary chromatography and laser-induced fluorescence detection

A non-competitive immunoassay based on micellar electrokinetic capillary chromatography (MECC) with laser-induced fluorescence (LIF) detection has been developed for the determination of alpha-fetoprotein (AFP). The anti-AFP antibody was labeled with fluorescein isothiocyanate (FITC) and the product was used as a fluorescent tracer, then AFP was mixed with the labeled antibody. After incubation, the immune AFP-antibody complex was separated from labeled free antibody by MECC. The parameters affecting separation such as the concentration of sodium dodecyl sulfate (SDS), the buffer pH and separation voltage were investigated and the following conditions were selected: 20 mM tetraborate containing 100 mM SDS at pH 9.50, and 20 kV separation voltage. The detection limit of this assay was 0.1 ng/ml with a linear range spanning two orders of magnitude. This method was applied to determine AFP in human serum.     

Separation of carbamazepine and five metabolites,and analysis in human plasma by micellar electrokinetic capillary chromatography

A rapid and feasible method was developed for the analysis of carbamazepine and its five metabolites (10,11-dihydro-10,11-epoxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine, 10,11-dihydro-10-hydroxycarbamazepine, 2-hydroxycarbamazepine and 3-hydroxycarbamazepine) in human plasma. Separation of the analytes is based on micellar electrokinetic chromatography, in untreated fused-silica capillary (48.5/40.0 cm length, 50 microm I.D.) with phosphate buffer (30 mM, pH 8.00) as background electrolyte, containing 50 mM sodium dodecylsulfate, and methanol (15%, v/v) as organic modifier. Clean up of human plasma samples was carried out by means of a solid-phase extraction procedure, which gave a high extraction yield for all six carbamazepines (>88%). The overall precision of the method gives a mean RSD of about 1.8%. The limit of quantitation for all analytes is < or = 0.30 microg ml(-1), the limit of detection < or = 0.12 microg ml(-1).     

Monitoring of dithiothreitol clearance by means of micellar electrokinetic chromatography

A new method for specific determination of dithiothreitol (DTT) using micellar electrokinetic chromatography and on-column reaction with reactive disulfide-2,2'-dipyridyldisulfide is described. DTT in this reaction is quantitatively transformed into a mixed disulfide concomitantly with formation of equimolar amount of the 2-thiopyridone that is further separated by micellar electrokinetic chromatography and determined spectrophotometrically at 343 nm. The concentration of DTT 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 2.5 mM (correlation coefficient 0.993) with a detection limit of 2.5 microM. The inter-day reproducibility of the peak area was 1.35% and the inter-day reproducibility of the migration time 0.56%. The method can be applied for DTT monitoring both in chemical and biological systems.     

Microemulsion and micellar electrokinetic chromatography of steroids

A mixture of ten steroids was separated by microemulsion and micellar (SDS and glycodeoxyholate) electrokinetic chromatography systems. Separations were done on a 50 cm (to the detector) × 50 μm I.D. fused-silica capillary. Complete separation of all the test compounds in the micellar mode was obtained with glycodeoxycholate (50 mM) in 25 mM borate buffer, pH 6.5, as the micelle-forming agent. The best results, however, were obtained using microemulsion electrokinetic chromatography in which higher aliphatic alcohols were used as the microemulsion-forming modifiers. The system consisted of n-hexanol (0.81%), SDS (3.31%) and n-butanol (6.61%) in 20 mM phosphate buffer, pH 10.0 (89.28%, w/w). In the microemulsion mode, linear calibration for steroid standards was obtained in the concentration range 3 × 10⁻⁴ − 3 × 10⁻⁵ mol l⁻¹ with a detection limit of 1 pmol. The method was validated and applied to an 11β-hydroxysteroid dehydrogenase assay in tissues.     

Determination of caffeine and its metabolites by micellar electrokinetic capillary electrophoresis

The determination of caffeine and its analogues is important for a wide variety of analyses and is performed in an assortment of matrices ranging from food to clinical samples. While reversed-phase HPLC has become the standard analysis protocol in most laboratories, capillary electrophoresis has the advantages of higher separation efficiency and shorter separation time. The micellar capillary electrophoresis (MECC) separation of caffeine and its metabolites, theobromine, paraxanthine, theophylline and 1,3,7-trimethyluric acid was investigated using sodium dodecyl sulphate (SDS) as the micellar phase. The effects of pH, micelle concentration, buffer concentration, ionic strength, buffer salts, applied voltage and injection time were studied to select the optimum conditions for the determination of caffeine and its four analogues in drugs, foods and body fluids. Caffeine and its three analogues were resolved within 120 s with detection limits less than 1 μg/ml. Samples could be analyzed utilizing direct injection with satisfactory resolution and reproducibility.     

Heart-cut two-dimensional separation method via hyphenation of micellar electrokinetic capillary chromatography and capillary zone electrophoresis using analyte focusing by micelle collapse

A novel two-dimensional (2D) separation method, which hyphenated micellar electrokinetic capillary chromatography (MEKC) and capillary zone electrophoresis (CZE), was developed for analysis of flavonoids in Leonurus cardiaca. The Leonurus cardiaca sample was separated and purified in first dimension by MEKC. Then only a selected portion of the first dimension separation was transferred into the second dimension by pressure. Finally, the zone of flavonoids was separated by CZE. As the key to successful hyphenation of MEKC and CZE, an analyte focusing by micelle collapse (AFMC) concentration method was employed between the two dimensions to release analytes from the micelle interior to a liquid zone and to overcome the sample zone diffusion caused by mobilization pressure. The whole heart-cut 2D separation process can be performed in a conventional CE analyzer. The relative standard deviation of peak height, peak area and migration time were in the range of 2.3-4.2%, 1.5-3.8% and 3.6-5.5%, respectively, and detection limits (S/N=3) were 15-55 ng/mL. The new methodology was applied with success for the flavonoids separation of Leonurus cardiaca.     

A simple peptide mapping method by partial filling micellar electrokinetic capillary chromatography with a zwitterionic-nonionic mixed micelle

A partial filling micellar electrokinetic capillary chromatography (PF-MEKC) method with a mixed micelle system composed of a zwitterionic surfactant named 3-(N,N-dimethylhexadecylammonium)propanesulfonate (PAPS) and a nonionic surfactant polyethylene glycol dodecyl ether (Brij 35) for peptide mapping is described. The method was demonstrated by the separation of tryptic digestion of bovine serum albumin (BSA). The optimal mixed micelle solution was 50 mM NH(4)OH-HCOOH buffer (pH 2.0) containing 32 mM PAPS and 0.6% (m/v) Brij 35. It was found that the mixed micelle system permitted a highly selective separation of the tryptic digestion. The high separation selectivity was probably due to the ion-pairing interaction between the zwitterionic surfactant molecules and the peptides.