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.     

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.     

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。     

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 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.     

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).     

Determination of alginate copolymer in pharmaceutical formulations by micellar electrokinetic chromatography

A micellar electrokinetic chromatography method was developed for the determination and quantification of sodium alginate. The alginate peak migrated in the very short time of 1.33 min and calibrated easily though the polydisperse properties of alginates. The minimum detection limit (LOD) of the method was calculated as 0.393 mg/ml. This analysis method was successfully applied to the alginate quantification in an antacid pharmaceutical formulation. Precise and reproducible analysis results were obtained, with liquid formulations injected directly without any pre-separation process.     

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.     

Separation of the glucuronides of entacapone and its (Z)-isomer in urine by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatography (MECC) method was developed for the separation of the 3-O-glucuronides of entacapone and its (Z)-isomer, the two main urinary metabolites of entacapone in humans. Entacapone is a novel, potent inhibitor of catechol-O-methyltransferase (COMT) intended for use as an adjunct in the treatment of Parkinson’s disease. Urine samples spiked with synthetic 3-O-glucuronides were used to study the effects of running buffer pH, composition and applied voltage on separation of the closely migrating glucuronides. The 3-O-glucuronide of nitecapone, was used as internal standard. The greatest improvement in separation was achieved by increasing the running buffer ionic concentration. Changes in pH had little effect on the separation, whereas increase in sodium dodecyl sulfate (SDS) concentration slightly improved resolution. Baseline separation and good selectivity relative to urine components were achieved by using a phosphate (25 mM)–borate (50 mM)–SDS (20 mM) running buffer, pH 7.0, in a 75 μm×60/67 cm fused-silica capillary at 15 kV and a 335 nm cut-off filter in the UV detector. The limits of detection (LOD) at a signal-to-noise ratio of 3 were about 0.25 μg/ml (5.2·10 ⁻⁷M) (injection 0.5 p.s.i./8 s). The linear detection range was 2–100 μg/ml (r²>0.999). Good repeatability of injection and relative migration times were obtained.     

Chiral separation of vinpocetine using cyclodextrin-modified micellar electrokinetic chromatography

A cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) technique has been developed for enantioseparation of vinpocetine using an inexpensive 2-hydroxypropyl-β-CD (HP-β-CD) as the chiral selector (CS). The best chiral separation was achieved using 40 mM HP-β-CD as the CS in 50 mM phosphate buffer (pH 7.0) consisting of 40 mM sodium dodecyl sulfate (SDS) at a separation temperature and separation voltage of 25°C and 25 kV, respectively. To the author's best knowledge, this is the first CD-MEKC study able to successfully separate the four stereoisomer of vinpocetine in separation time of 9.5 min and resolution of 1.04-3.87.     

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.     

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.     

Determination of aucubin and catalpol in Plantago species by micellar electrokinetic chromatography

Micellar electrokinetic chromatography (MEKC) was used for the separation and determination of two iridoid glycosides, aucubin and catalpol, in several Plantago species growing in Croatia: P. altissima L., P. argentea Chaix, P. coronopus L., P. holosteum Scop. (subsp. depauperata, subsp. holosteum and subsp. scopulorum), P. lagopus L., P. lanceolata L., and P. maritima L. Hot water extraction (HWE) was applied for the isolation of iridoid substances. Significant differences appeared between the iridoid contents in the examined species. The yield of aucubin and catalpol was up to 0.27% and 1.81% of the dry mass of the leaves, respectively. Besides aucubin and catalpol, two related compounds were determined in the plant samples.     

Separation of 1,4-benzodiazepines by micellar elektrokinetic capillary chromatography

In this work the applicability of micellar elektrokinetic capillary chromatography (MECC) for the determination of benzodiazepines (BZD) has been studied. The applied method was used for the simultaneous separation of 8 BZDs (alprazolam, bromazepam, chlordiazepoxide, diazepam, flunitrazepam, medazepam, oxazepam, nitrazepam), and also for the study of stability in acidic medium. A fast and reliable method has been developed; using a separation buffer composed of sodium tetraborate 25 mM (pH 9.5), SDS (50 mM) and methanol (at least 12%) as an organic modifier.     

Determination of 1-phenyl-3-methyl-5-pyrazolone-labeled carbohydrates by liquid chromatography and micellar electrokinetic chromatography

In this paper, the method for the derivatization of carbohydrates with 1-phenyl-3-methyl-5-pyrazolone (PMP) was simplified. One-third of the derivatization time was saved. Five monosaccharide derivatives have been well separated by MEKC and HPLC under optimized conditions. Good reproducibility could be obtained with relative standard deviation (RSD) values of the migration times within 5.0 and 2.3%, respectively. Furthermore, the developed methods have been successfully applied to the analysis of carbohydrates in Aloe powder and food. These methods are quite useful for routine analysis of monosaccharides and oligosaccharides in real samples.     

Assay for spermidine synthase activity by micellar electrokinetic chromatography with laser-induced fluorescence detection

An assay for spermidine synthase (SPDS) activity in rat liver has been developed using micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection to enable the discovery of SPDS inhibitors. The assay was established by estimating the amount of spermidine (SPD) produced from the putrescine (PUT) present by SPDS. The SPD in an enzyme reaction mixture of homogenized rat liver could directly react with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescence derivatization reagent. The NBD derivatives of SPD and PUT could be separated and detected by MEKC-LIF detection within 15 min. The IC(50) value measured for SPDS inhibitor, 4-methylcyclohexylamine, in rat liver by this assay was consistent with published data. Our SPDS assay using MEKC-LIF is simple and allows easy determination of SPDS activity in homogenized samples without troublesome procedures such as preparation of antibody or fluorescence-labeled substrate. The assay should be effective for discovering the SPDS inhibitors using biological samples.     

Micellar electrokinetic chromatography with polyelectrolyte complexes as micellar pseudo-stationary phases

The separation of dansyl (DNS-AAs) and carbobenzoxy (CBZ-AAs) amino acids using micellar electrokinetic chromatography employing polyelectrolyte-surfactant complexes (PSC) formed in the reaction between polyacrylic acid (PAA) and dodecyltrimethylammonium bromide (DTAB) as pseudo-stationary phases was described. The PSCs were stabilized by hydrophobic interactions of alkyl chains of the surfactant ions and converted to an intramolecular micellar-like phase. The running buffer was a 50mM solution of sodium phosphate (pH 6.0) containing 4.6-20.2mM PSC, in which a part of carboxyl groups of PAA was blocked by aliphatic amines. For the systems with 7.9mM of PAA/DTAB complex (phi=0.30, phi-composition of water-soluble polyelectrolyte complex) as a pseudo-stationary phase, the peaks of six dansyl amino acids (DNS-AAs) were baseline resolved. The separation in this case is based on a complex distribution mechanism of the dansyl derivatives between the free buffer and the intramolecular micellar-like phase of the water-soluble PSC. On the other hand, the additives of PAA/DTAB complex (phi=0.30) to the running buffer does not essentially affect on the electrophoretic behaviour of the CBZ-AAs, the variant MEKC is not realized. The influence of the concentration of the complex of PAA/DTAB on the electrophoretic behaviour of analytes was investigated. Relative retentions and relative selectivities were used for describing electrophoretic behaviour of the amino acid derivatives.     

Use of micellar electrokinetic chromatography to measure palmitoylation of a peptide

Palmitoylation is the thioester linkage of the fatty acid, palmitate (C16:0), to cysteine residues on a protein or peptide. This dynamic and reversible post-translational modification increases the hydrophobicity of proteins/peptides, facilitating protein-membrane interactions, protein-protein interactions and intracellular trafficking of proteins. Manipulation of palmitoylation provides a new mechanism for control over protein location and function, which may lead to better understanding of cell signaling disorders, such as cancer. Unfortunately, few methods exist to quantitatively monitor protein or peptide palmitoylation. In this study, a capillary electrophoresis-based assay was developed, using MEKC, to measure palmitoylation of a fluorescently-labeled peptide in vitro. A fluorescently-labeled peptide derived from the growth-associated protein, GAP-43, was palmitoylated in vitro using palmitoyl coenzyme A. Formation of a doubly palmitoylated GAP-peptide product was confirmed by mass spectrometry. The GAP-peptide substrate was separated from the palmitoylated peptide product in less than 7 min by MEKC. The rate of in vitro palmitoylation with respect to reaction time, GAP-peptide concentration, pH, and inhibitor concentration were also examined. This capillary electrophoresis-based assay for monitoring palmitoylation has applications in biochemical studies of acyltransferases and thioesterases as well as in the screening of acyltransferase and thioesterase inhibitors for drug development.