Cationic gemini surfactant as dynamic coating in CE for the control of EOF and wall adsorption

The cationic gemini surfactant ethylene bis(1-dodecyldimethylammonium) dibromide was used as a dynamic coating to control EOF and prevent wall adsorption of basic proteins in CE for the first time. This gemini surfactant shows a more powerful capability in EOF reversal than traditional single-chained surfactant. The gemini surfactant reverses the EOF at a concentration level even less than 0.01 mM, and the EOF magnitude is affected by surfactant concentration, pH, ionic strength, and ions added in buffer. Highly efficient and rapid protein separation (N > 300,000) was obtained with buffer containing 2 mM gemini surfactant under pH ranging from 3 to 6. The effects of surfactant and buffer concentration on protein separation were investigated in detail. Under the optimal conditions, good repeatability (RSD of migration time <0.6% for run-to-run and <2.5% for day-to-day assays) and recovery (>90%) of tested proteins were obtained. This new dynamic coating is also suitable for biosample analysis.     

Routine thermometric titration of tricyclic antidepressant drugs and their formulations

A rapid procedure is described for the routine assay of amitryptyline, imipramine and clomipramine hydrochlorides and trimipramine maleate. The weakly basic drugs are titrated with perchloric acid in anhydrous acetic acid; the heat output of the proton-catalysed acetylation of 4-hydroxy-4-methylpentan-2-one with acetic anhydride indicates the equivalence point. For milligram amounts of the drugs, accuracy is good and relative standard deviations are usually better than 1%; sub-milligram amounts can also be assayed. Four commercial formulations were found to be within the British Pharmacopoeia 1980 specifications.     

Chiral analysis of pollutants and their metabolites by capillary electromigration methods

Chiral separation of enantiomers is one of the most challenging tasks for any analytical technique including CE. Since the first report in 1985 showing the great possibilities of CE for the separation of chiral compounds, the amount of publications concerning this topic has quickly increased. Although chiral electromigration methods have mainly been used for enantioseparation of drugs and pharmaceuticals, they have also been applied to analyze chiral pollutants. This article intends to provide an updated overview, including works published till January 2005, on the principal applications of CE to the chiral analysis of pollutants and their metabolites, with special emphasis on articles published in the last 10 years. The main advantages and drawbacks regarding the use of CE for chiral separation of pollutants are addressed including some discussion on the foreseen trends of electromigration procedures applied to chiral analysis of contaminants.     

Comparative chemical ionization mass spectra of tricyclic antidepressant amines and related compounds

Positive-ion methane chemical ionization (CI) mass spectra were obtained for seven underivatized tricyclic amines: amitriptyline, nortriptyline, protriptyline, imipramine, desipramine, cyclobenzaprine and cyproheptadine. Some discrepancies in previously reported spectra were noted. Spectra of protriptyline, cyclobenzaprine and cyproheptadine are reported for the first time. Comparisons are made among the CI spectra and with electron ionization spectra, and relative abundances of major CI ions among the seven compounds are rationalized in terms of substituent and geometric effects. In most cases low-mass iminium ions from anion abstraction were more abundant than [MH]⁺. Hydride abstraction and adduct formation with reagent-gas ions were important. The three heterocyclic amines gave abundant [M]⁺˙ by electron transfer. Protonation at the nitrogen atom on the side chain followed by amine elimination gave tricyclic aromatic fragment ions.     

Effect of detergent on electromigration of proteins: CE of very low density lipoprotein receptor modules and viral proteins

The different electrophoretic behavior of the members of two groups of proteins with respect to the absence or presence of detergent additives in the BGE was explored. Recombinant soluble concatemers of repeat 3 of the very low density lipoprotein (VLDL)-receptor fused at their N-terminus to maltose-binding protein (MBP) exhibited different electrophoretic mobilities in borate buffer (pH 8.3) in the absence and in the presence of dodecyl-PEG ether (D-PEG). This enabled the separation of the receptor fragments from MBP after enzymatic cleavage. In the presence of SDS, the mobilities of all proteins approached the same values with increase in detergent concentrations. In contrast, viral capsid proteins of a human rhinovirus (HRV) exhibited different migration in the presence of the additive. For the receptor proteins, extreme apparent high plate numbers were observed when the SDS concentration in the sample and the separation buffer differed. This effect might be erroneously interpreted as a high efficiency. However, it is due to the conductivity boundaries caused by the sample and leads to a total loss of separation.     

Simultaneous HPLC determination of 14 tricyclic antidepressants and metabolites in human plasma

A HPLC method has been developed for the simultaneous determination of seven tricyclic antidepressants (TCAs) and seven metabolites in human plasma. The analyte separation was obtained using a C8 reversed phase column and a mobile phase composed of 68% aqueous phosphate buffer at pH 3.0 and 32% ACN. The UV detector was set at 220 nm and loxapine was used as the internal standard. A careful pre‐treatment procedure for plasma samples was developed, using SPE on C2 cartridges, which gives satisfactory extraction yields (>80%) and good sample purification. The LOQs were always lower than 9.1 ng/mL and the LODs always lower than 3.1 ng/mL for all analytes. The method was successfully applied to plasma samples from depressed patients undergoing therapy with one or more TCA drugs. Precision data (RSD <8.1%), as well as accuracy results (recovery >80%), were satisfactory and no interference from other drugs was found. Hence the method seems to be suitable for the therapeutic drug monitoring of patients treated with TCAs under monotherapy or polypharmacy regimens.     

Analysis of tricyclic antidepressant drugs in plasma by means of solid-phase microextraction-liquid chromatography-mass spectrometry

Solid-phase microextraction coupled to liquid chromatography and mass spectrometry (SPME-LC-MS) was used to analyze tricyclic antidepressant drugs desipramine, imipramine, nortriptyline, amitriptyline, and clomipramine (internal standard) in plasma samples. SPME was performed by direct extraction on a PDMS/DVB (60 microm) coated fiber, employing a stirring rate of 1200 rpm for 30 min, pH 11.0, and temperature of 30 degrees C. Drug desorption was carried out by exposing the fiber to the liquid chromatography mobile phase for 20 min, using a labmade SPME-LC interface at 50 degrees C. The main variables experimentally influencing LC-MS response were evaluated and mathematically modeled. A rational optimization with fewer experiments was achieved using a factorial design approach. The constructed empirical models were adjusted with 96-98% of explained deviation allowing an adequate data set comprehension. The chromatographic separation was realized using an RP-18 column (150 mm x 2.1 mm, 5 microm particles) and ammonium acetate buffer (0.01 mol/l, pH 5.50) : acetonitrile (50 : 50 v/v) as mobile phase. Low detection levels were achieved with electrospray interface (0.1 ng/ml). The developed method showed specificity, linearity, precision, and limit of quantification adequate to assay tricyclic antidepressant drugs in plasma.     

A rapid and reliable method for extraction and assay of tricyclic antidepressant drugs in serum or plasma

Because of their narrow therapeutic ranges and differing patient tolerances, the monitoring of tricyclic antidepressants (TADs) commonly prescribed for control of endogenous depression is highly desirable for the satisfactory treatment of patients. The majority of methods for analysis of TADs require multiple extractions from plasma with organic solvents, accompanied by obligatory centrifugation steps. We describe a simple procedure for the rapid extraction of serum TADs, with high recovery of the analyte drugs. It involves sample preparation using Analytichem 'BOND-ELUT' C-2 reverse phase columns, and analysis by gas chromatography using a nitrogen-phosphorus detector. The method offers a quick, inexpensive and low-maintenance assay which is applicable to all the common tricyclic class drugs.     

Separation of tricyclic psychosedative drugs by high-speed ion-pair partition and liquid-solid adsorption chromatography.

The application of high-speed ion-pair partition and liquid-solid adsorption chromatography to the separation of twenty common tricyclic tranquilizers and antidepressant drugs is described. In the ion-pair system, amine-perchlorate ion-pairs were extracted from an aqueous stationary phase supported on 10-mum silica gel by organic eluents containing a chloromethane and a higher aliphatic alcohol, and chromatographic parameters for elution by eight eluent mixtures are presented. Using 5 mm times 120 mm columns good separations, according to chemical class, were achieved. For adsorption chromatography, the components were eluted from 20-mum spherical alumina using eluents containing methylene chloride, n-hexane or n-pentane, and acetic acid. Chromatographic parameters are given for eight eluent compositions. Components differing little in structure are well separated by liquid-solid adsorption chromatography. Compared with ion-pair partition chromatography, adsorption chromatography is much more selective for compounds of the same chemical type. The two methods are therefore complementary. Both methods gave plate heights in the range of 0.1 to 0.3 mm.     

Enantioselective separation of the novel antidepressant mirtazapine and its main metabolites by CEC

In this work, the simultaneous enantioseparation of the second-generation antidepressant drug mirtazapine and its main metabolites 8-hydroxymirtazapine and N-desmethylmirtazapine by chiral CEC is reported. The separation of all enantiomers under study was achieved employing a capillary column packed with a vancomycin-modified diol stationary phase. With the aim to optimize the separation of the three pairs of enantiomers in the same run, different experimental parameters were studied including the mobile phase composition (buffer concentration and pH, organic modifier type and ratio, and water content), stationary phase composition, and capillary temperature. A capillary column packed with vancomycin mixed with silica particles in the ratio (3:1) and a mobile phase composed of 100 mM ammonium acetate buffer (pH 6)/H(2)O/MeOH/ACN (5:15:30:50, by vol.) allowed the complete enantioresolution of each pair of enantiomers but not the simultaneous separation of all the studied compounds. For this purpose, a packing bed composed of vancomycin-CSP only was tested and the baseline resolution of the three couples of enantiomers was achieved in a single run in less than 30 min, setting the applied voltage and temperature at 25 kV and 20 degrees C, respectively. In order to show the potential applicability of the developed CEC method to biomedical analysis, a study concerning precision, sensitivity, and linearity was performed. The method was then applied to the separation of the enantiomers in a human urine sample spiked with the studied compounds after suitable SPE procedure with strong cation-exchange (SCX) cartridges.     

Stereoselective determination of hydroxychloroquine and its metabolites in human urine by liquid-phase microextraction and CE

Liquid-phase microextraction based on polypropylene hollow fibers and CE were applied for the chiral determination of hydroxychloroquine (HCQ) and its metabolites (desethylchloroquine, DCQ; desethylhydroxychloroquine, DHCQ; bisdesethylchloroquine, BDCQ) in human urine. The analytes were extracted from 3 mL of urine spiked with the internal standard (metoprolol) and alkalinized with 250 muL of 2 M NaOH. The analytes were extracted into 1-octanol impregnated in the pores of the hollow fiber, and into an acid acceptor solution inside the hollow fiber. The electrophoretic separations were carried out in 100 mmol/L Tris buffer (pH adjusted to 9.0 with phosphoric acid) containing 1% w/v S-beta-CD and 30 mg/mL HP-beta-CD with a constant voltage of +18 kV. The method was linear over the concentration range of 10-1000 ng/mL for each HCQ stereoisomer and 21-333 ng/mL for each metabolite stereoisomer. Within-day and between-day assay precision and accuracy for the analytes were studied at three concentration levels for each stereoisomer and were lower than 15%. The developed method was applied for the determination of the cumulative urinary excretion of HCQ, DCQ, and DHCQ after oral administration of rac-HCQ to a health volunteer. The results obtained are in agreement with previous literature data.     

Determination of clomipramine and its hydroxylated and demethylated metabolites in plasma and urine by liquid chromatography with electrochemical detection

A procedure for the determination of clomipramine and its 8-hydroxy, demethyl, 8-hydroxydemethyl and didemethyl metabolites in plasma and urine by high-performance liquid chromatography with electrochemical detection is described. A 1-ml plasma or urine sample is made alkaline with a carbonate buffer (pH 9.8) and extracted with 20% ethyl acetate in n-heptane. After back-extraction into an acid phosphate buffer (pH 2.4), an aliquot is injected into a 5-microns ion-paired reversed-phase column and eluted with a mobile phase containing a phosphate buffer with tetramethylammonium chloride-acetonitrile (57:43). The detection is coulometric with a first cell at +0.40 V, a second at +0.73 V and a guard cell set at 0.75 V for oxidation of the mobile phase. The method provides recoveries in the general range of 80-110% and a day-to-day precision of 3.7-8.8%, depending on the compound. The minimum quantifiable level for all compounds was 0.2 ng/ml with a 20-microliters injection. Steady-state plasma concentration data and urinary levels are reported for 24 depressed patients receiving daily either 75-150 mg orally or 50-75 mg by infusion.     

Localized adsorption at solid-liquid interfaces: the sticky site-hard wall model

The interface between a structured solid and a liquid is modelled by a flat surface with sticky sites placed on a regular lattice. It is shown that this three-dimensional adsorption model can be mapped into a two-dimensional lattice-gas model with the interaction energies of the particles on the sites related to the potential of mean force in the inhomogeneous fluid. In the simple case of a liquid of hard spheres, the first layer of liquid at contact with the solid exhibits an order-disorder phase transition and the total surface excess shows a maximum as the bulk density increases.     

Separation and detection of amino acid metabolites of Escherichia coli in microbial fuel cell with CE

In this work, CE‐LIF was employed to investigate the amino acid metabolites produced by Escherichia coli (E. coli) in microbial fuel cell (MFC). Two peptides, l ‐carnosine and l ‐alanyl‐glycine, together with six amino acids, cystine, alanine, lysine, methionine, tyrosine, arginine were separated and detected in advance by a CE‐LIF system coupled with a homemade spontaneous injection device. The injection device was devised to alleviate the effect of electrical discrimination for analytes during sample injection. All analytes could be completely separated within 8 min with detection limits of 20–300 nmol/L. Then this method was applied to analyze the substrate solution containing amino acid metabolites produced by E. coli. l ‐carnosine, l ‐alanyl‐glycine, and cystine were used as the carbon, nitrogen, and sulfur source for the E. coli culture in the MFC to investigate the amino acid metabolites during metabolism. Two MFCs were used to compare the activity of metabolism of the bacteria. In the sample collected at the running time 200 h of MFC, the amino acid methionine was discovered as the metabolite with the concentrations 23.3 μg/L.     

Conductometric and spectrofluorimetric characterization of the mixed micelles constituted by dodecyltrimethylammonium bromide and a tricyclic antidepressant drug in aqueous solution

Conductivity and static fluorescence measurements have been carried out at 25 degrees C to study the monomeric and micellar phases of aqueous solutions of mixed micelles constituted by a conventional cationic surfactant, dodecyltrimethylammonium bromide (D(12)TAB), and a tricyclic antidepressant drug, amitriptyline hydrochloride (AMYTP), with aggregation properties. From conductivity data, the total mixed critical micelle concentration and the dissociation degree of the mixed micelle have been obtained, while fluorescence experiments allow for the determination of the total aggregation number, and the micropolarity of micellar inside. Furthermore, the partial contribution of each surfactant to the mixed micellization process, through their critical micelle concentrations and their aggregation numbers have been determined, as well. The solubilization of the drug in the mixed micelles has been also studied through the mass action model, by determining the association constant between the micelles and the drug. From these results, the use of the micelles studied in this work as potential models for vectors of antidepressant drugs of the amitriptyline family has been discussed. The theoretical aspects of the mixed micellization process have been also analyzed.     

Optimizing the extraction, separation and quantification of tricyclic antidepressant drugs in human plasma with CE-ESI-TOF-MS using cationic-coated capillaries

In this study, the extraction and CE-ESI-TOF-MS analysis of tricyclic antidepressant (TCA) drugs imipramine, desipramine, clomipramine and norclomipramine in human plasma has been optimized. The CE capillaries were modified with ω-iodo-alkyl ammonium salt (M7C4I coating) to reduce analyte adsorption to the silica wall. The use of a strong cation exchange (SCX) solid-phase extraction (SPE) column specifically designed for the extraction of basic drug species from biofluids gave very clean extracts with high and reproducible recoveries. The extraction recoveries were ranging between 87 and 91% with % RSD values of 0.5-1.7% (n=3). The obtained strong cation exchange-SPE extracts of the TCA in human plasma only contained the analytes of interest. The optimized CE separation conditions were obtained by adding ACN and acetic acid to the sample while using an aqueous BGE. The CE-ESI-TOF-MS analysis was performed within 6 min for all TCA analytes under the optimized condition with peak efficiencies up to 1.4 x 10? plates/m and an average % RSD of the migration times of the analytes of 0.3% (n=5). The presented method can readily be used for the extraction and quantification of basic drug species in human biological fluids and in pharmaceutical formulations.     

Sample preparation with fiber-in-tube solid-phase microextraction for capillary electrophoretic separation of tricyclic antidepressant drugs in human urine.

Solid-phase microextraction (SPME) is a solvent-free sample preparation technique using a thin coating attached to the surface of a fused silica-fiber as the extraction medium, which has been successfully applied to the analysis of a wide variety of compounds by coupling to gas chromatography (GC). In recent years, in-tube SPME using GC capillary column as the extraction medium has also been developed and coupled with liquid chromatography (LC) for the preconcentration of nonvolatile compounds. In this study, an on-line interface between the fiber-in-tube SPME and capillary electrophoresis (CE) has been developed, and the preconcentration and separation of four tricyclic antidepressant (TCA) drugs, amitriptyline, imipramine, nortriptyline, and desipramine, were performed with the hyphenated system. Under the optimized condition, a better extraction performance than conventional in-tube SPME was obtained, even the length of the extraction medium was much shorter. The results clearly indicated that the fiber was working effectively as an extraction medium. For the separation of these four TCAs, capillary electrophoretic separation with beta-cyclodextrin as the buffer additive has been employed and the application of the developed system to the analysis of complex sample mixtures in a biological matrix is also demonstrated.