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.     

Analysis of synthetic derivatives of peptide hormones by capillary zone electrophoresis and micellar electrokinetic chromatography with ultraviolet-absorption and laser-induced fluorescence detection

Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were used for the analysis of new synthetic derivatives of hypophysis neurohormones--vasopressin and oxytocin, and pancreatic hormone--human insulin (HI) and its octapeptide fragment, derivatized by fluorescent probe, 4-chloro-7-nitrobenzo[1,2,5]oxadiazol (NBD). The suitable composition of background electrolytes (BGEs) was selected on the basis of calculated pH dependence of effective charge of analyzed peptides. Basic ionogenic peptides were analyzed by CZE in the acidic BGE composed of 100 mM H3PO4, 50 mM Tris, pH 2.25. The ionogenic peptides with fluorescent label, NBD, were analyzed in 0.5 M acetic acid, pH 2.5. The best MEKC separation of non-ionogenic peptides was achieved in alkaline BGE, 20 mM Tris, 5 mM H3PO4, with micellar pseudophase formed by 50 mM sodium dodecylsulfate (SDS), pH 8.8. Selected characteristics (noise, detectability of substance, sensitivity of detector) of the UV-absorption detectors (single wavelength detector, multiple-wavelength photodiode array detector (PDA), both of them operating at constant wavelength 206 nm) and laser-induced fluorescence (LIF) detector (excitation/emission wavelength 488/520 nm) were determined. The detectability of peptides in the single wavelength detector was 1.3-6.0 micromol dm(-3) and in the PDA detector 1.6-3.1 micromol dm(-3). The LIF detection was more sensitive, the applied concentration of NBD derivative of insulin fragment in CZE analysis with LIF detection was three orders lower than in CZE with UV-absorption detector, and the detectability of this peptide was improved to 15.8 nmol dm(-3).     

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.     

Quantification of 4-aminopyridine in plasma by capillary electrophoresis with electrokinetic injection

A rapid and sensitive CE method for the determination of 4-aminopyridine in human plasma using 3,4-diaminopyridine as an internal standard was developed and validated. The analytes were extracted from 0.5-mL aliquots of human plasma by liquid–liquid extraction, using 8 mL of ethyl ether, and injected electrokinetically into capillary electrophoresis equipment. The instrumental conditions were obtained and optimized by Design of Experiments (DOE – factorial and response surface model), having as factors: separation voltage, ionic strength (buffer concentration), pH and temperature. The response variables were migration time, resolution, tailing factor and drug peak area. After obtaining mathematically predicted values for the response variables with best factors combinations, these were reproduced experimentally in good agreement with predicted values. In addition to optimal separation conditions obtained by Design of Experiments, sensitivity was improved using electrokinetic injection at 10 kV for 10 s, and a capillary with 50 cm effective length and 100 μm I.D. The final instrumental conditions were voltage at 19 kV, capillary temperature at 15 °C, wavelength at 254 nm, and phosphate buffer 100 mM, pH 2.5 as the background electrolyte. This assay was linear over a concentration range of 2.5–80 ng/mL with a lower limit of quantification of 2.5 ng/mL. The relative standard deviation for the assay precision was <7% and the accuracy was >95%. This method was successfully applied to the quantification of 4-aminopyridine (4-AP) in plasma samples from patients with spinal cord injury.     

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.     

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.     

Pherogram normalization in capillary electrophoresis and micellar electrokinetic chromatography analyses in cases of sample matrix-induced migration time shifts

When analyzing bio-matrix samples using capillary electrophoresis (CE) or micellar electrokinetic chromatography (MEKC), unwanted shifts in the time axis are often observed, both between samples and standards and between samples, thus hampering identification. These shifts are caused by either or both of two sample matrix-induced effects: variations in stacking conditions (effective field strength or migration length) and variations in electroosmotic flow. Based on elementary CE principles and provided that any two peaks in the pherograms can be linked, these variations can be separately accounted and quantitatively corrected for, so that perfectly overlapping pherograms of standards and samples can be obtained after normalization. The method was validated using samples of a DNA ladder, separated in a sieving polymer. In addition, a number of data files from CE and MEKC analyses (steroids, opioids, beta-blockers, amines, and inorganic anions) previously published by other authors were successfully normalized. A freeware computer programme, CEqualizer, for normalizing ASCII files of detector signals using the method described, is available to the CE community from http: //www.ceyork.f2s.com.     

Determination of carbohydrates as their p-sulfophenylhydrazones by capillary zone electrophoresis.

p-Hydrazinobenzenesulfonic acid was explored as an ultraviolet labeling reagent for capillary electrophoresis of mono-, di- and trisaccharides. The labeling reaction that produces p-sulfophenylhydrazines took less than 8 min, and introduced both chromphore and charged groups into the carbohydrate molecules. The derivatives of nine mono- and disaccharides were completely separated in 9 min using a 100 mM borate buffer at pH 10.24. On-column UV detection at 200 nm allowed the detection of glucose with a mass detection limit of 17.6 fmol or a concentration limit of 3.6 microM. Reproducible quantification of carbohydrates was achieved in the concentration range of 0.1-9.1 mM in reaction solution. The method was applied successfully to determine the monosaccharide composition of laminaran.     

Analysis of ellagic acid in pomegranate rinds by capillary electrophoresis and high-performance liquid chromatography

Two novel methods for the analysis of ellagic acid in pomegranate (Punica granatum) rinds are proposed. Capillary electrophoresis (CE) was performed in a bare fused-silica capillary using a buffer solution of tri(hydroxymethyl)aminomethane:potassium dihydrogen phosphate (pH 8.4) with an applied voltage of 20 kV and UV detection at 254 nm. HPLC analysis was performed with a Zobax SB C(18) column and a mobile phase consisting of methanol:ethyl acetate:potassium dihydrogen phosphate: phosphoric acid at a flow rate of 1.0 mL/min. Under optimised conditions, the HPLC retention and the CE migration times for ellagic acid were 10.32 and 12.23 min, respectively. Calibration curves of peak area vs. concentration gave correlation coefficients of 0.9999 for HPLC and 0.9990 for CE. The detection limits for HPLC and CE were 2.8 and 2.2 microg/mL, respectively. Average recoveries were 98.32 +/- 1.2% for HPLC and 96.52 +/- 2.8% for CE. Both methods were shown to be suitable for the determination of ellagic acid in pomegranate rinds extraction; however, the CE method required less solvent and gave better column efficiency, whilst the HPLC provided superior precision.     

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.     

High-performance capillary electrophoresis analysis of mate infusions prepared from stems and leaves of Ilex paraguariensis using automated micellar electrokinetic capillary chromatography

An automated micellar electrokinetic capillary chromatographic method has been developed in order to determine xanthines, e.g. caffeine, theobromine and theophylline, and chlorogenic acid in samples of yerba mate (Ilex paraguariensis). The target constituents were detected by photodiode array, and quantified by an external standard method. In addition, each constituent was collected separately and identified by EIMS. The method has been used to analyse 30 samples of mate infusions prepared at 30 and 75 degrees C with milled leaves and stems of 14 commercial brands which had been subjected to different elaboration processes. Suspended powdered material of each infusion was also analysed after three sieving steps. There was a remarkable difference in the relative xanthine composition of the finely suspended material, the amount of which varied according to the yerba mate brand, the elaboration process and the temperature of the infusion. The importance of these results with respect to gastrointestinal disorders which have been observed by habitual consumers of mate are discussed.     

Determination of free radical reaction products and metabolites of salicylic acid using capillary electrophoresis and micellar electrokinetic chromatography

Hydroxylated radical products of salicylic acid are often used as a relative measurement in free radical research. Several analytical methods exist to determine the amount of 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid. In this study we use capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) in order to determine these free radical products. The CZE experiment was optimized with a CZE simulation program in order to achieve an optimal pH. Calibration curves were recorded in the range 10⁻⁶–10⁻⁴ M and the detection limit was determined. For both CZE and MECC it was 2·10⁻⁷ M. Both methods resulted in a reproducible analysis of salicylate and its hydroxylated free radical products in 6 min.     

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。     

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.     

High resolution of oligosaccharide mixtures by ultrahigh voltage micellar electrokinetic capillary chromatography

Oligosaccharide mixtures released from ribonuclease B and human IgG have been separated using micellar electrokinetic capillary chromatography operated at 100 kV. The resolution of these closely related analytes at this high voltage was found to be superior to that obtained at 20 kV, a voltage which is ordinarily used in most capillary electrophoresis separations.     

Sample stacking for the analysis of penicillins by microemulsion electrokinetic capillary chromatography

We present a method for determining eight penicillin antibiotics using microemulsion electrokinetic chromatography (MEEKC). We studied how the composition of the microemulsion affected separation by modifying such parameters as the surfactant or the addition of organic solvents. The best microemulsion system consisted of 0.5% ethyl acetate, 1.2% 1-butanol, 2% Brij 35, 10% 2-butanol and 86.3% 10 mM borate buffer at pH 10. We studied the suitability of this microemulsion composition for analyzing a commercial drug. To improve the sensitivity of the method, we used the stacking technique reversed electrode polarity stacking mode (REPSM), which increased the detection limits by about 40-fold.     

Quantitative separation of 4-hydroxyproline from skeletal muscle collagen by micellar electrokinetic capillary electrophoresis

The phenylthiohydantoin (PTH) derivatives of 3- and 4-hydroxyproline (Hyp) were separated using micellar electrokinetic capillary electrophoresis (MEKC). The separation protocol was also used to determine Hyp content of bovine skeletal perimysial collagen preparations and whole muscle samples. Amino acids from hydrolyzed tissues were labeled using a two step procedure that involved initial reaction with o-phthalaldehyde (OPA) to modify primary amines followed by their precipitation under acidic conditions. In the second step, imino acids were reacted with phenyl isothiocyanate (PITC). This labeling method was rapid and the Hyp values determined in these biological samples were found to be in close agreement with conventional methods and other published reports.     

Analysis of ibuprofen in serum by capillary electrophoresis

A rapid method for analysis of the analgesic drug ibuprofen in serum by capillary zone electrophoresis in a borate buffer 160 mmol/l pH 8.5 is described. The method involves deproteinization with acetonitrile to remove serum proteins followed by direct injection on the capillary. The recoveries of standards added to the serum were 84–92%. The method is suited for analysis of samples with concentrations >10 mg/l. Many other analgesics such as ketoprofen, daypro and salicylates can also be determined by this method.