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See detailSimultaneous determination of insulin and its analogues in pharmaceutical formulations by micellar electrokinetic chromatography (MEKC)
Lamalle, Caroline ULg; Servais, Anne-Catherine ULg; Crommen, Jacques ULg et al

Poster (2013)

Insulin plays an important role in the homeostasis of blood glucose concentration. A deficiency of this hormone causes diabetes, which can be treated by subcutaneous injection of synthetic insulin ... [more ▼]

Insulin plays an important role in the homeostasis of blood glucose concentration. A deficiency of this hormone causes diabetes, which can be treated by subcutaneous injection of synthetic insulin. Besides human regular insulin, several modified analogues have been developed to accelerate (Lispro, Aspart, Glulisin) or delay (Glargin, Detemir) its absorption. Moreover, protamine is sometimes associated with human, Lispro or Aspart insulin to give a crystalline form, which delays the action of insulin, providing it with a prolonged absorption profile after injection. Diabetes is one of the most common metabolic diseases in the world; its prevalence increases continuously. A lot of patients are therefore concerned with the treatment, which is relatively expensive and requires a prescription. Some pharmaceutical formulations can sometimes be found without prescription on the parallel market but the risk of drug counterfeiting is then considerably increased. The poor quality of these drugs can lead to harmful consequences for the public health. It is therefore essential to develop a suitable method for the identification and quantification of human insulin and its analogues. Ortner et al. have already proposed micellar electrokinetic chromatography (MEKC) methods to analyse simultaneously human insulin and its five analogues but formulations containing protamine were not tested. Since the number of these formulations is significant, we included them in our study. The first optimisation step involved the sample preparation procedure. An acidic sample solution (0.01 M HCl) was finally selected to solubilise protamine and Glargin. Then the background electrolyte composition was investigated to separate the components present in the formulations. A basic buffer (50 mM ammonium acetate pH 9) was selected, providing an important and stable electroosmotic flow, a negative charge to insulin and related compounds and avoiding any adsorption to the capillary wall. The addition of sodium dodecyl sulfate and acetonitrile were also found crucial for selectivity. The six insulins and the two major excipients (phenol and m-cresol) were fully separated within 15 minutes The aforementioned method was then adapted to permit the separation of each insulin from its degradation products. [less ▲]

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See detailEnantioseparations in nonaqueous capillary electrophoresis using charged cyclodextrins.
Servais, Anne-Catherine ULg; Fillet, Marianne ULg

in Methods in Molecular Biology 970: Chiral Separations (2013)

The enantioseparation of acidic and basic compounds can be successfully achieved in nonaqueous capillary electrophoresis using single-isomer charged beta-cyclodextrin (beta-CD) derivatives of opposite ... [more ▼]

The enantioseparation of acidic and basic compounds can be successfully achieved in nonaqueous capillary electrophoresis using single-isomer charged beta-cyclodextrin (beta-CD) derivatives of opposite charge to that of the analytes. This chapter describes how to separate the enantiomers of three basic substances selected as model compounds, i.e., alprenolol, bupranolol, and terbutaline, using the negatively charged heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-CD. The enantiomers of three acidic drugs (tiaprofenic acid, suprofen, and flurbiprofen) are resolved using a monosubstituted amino beta-CD derivative, namely, 6-monodeoxy-6-mono(3-hydroxy)propylamino-beta-CD. [less ▲]

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See detailApplication of dual cyclodextrin systems in capillary electrophoresis enantioseparations.
Servais, Anne-Catherine ULg; Fillet, Marianne ULg

in Methods in Molecular biology 970: Chiral Separations (2013)

The enantioseparation of acidic and neutral compounds can be successfully achieved in capillary electrophoresis (CE) using dual cyclodextrin (CD) systems. This chapter describes how to separate the ... [more ▼]

The enantioseparation of acidic and neutral compounds can be successfully achieved in capillary electrophoresis (CE) using dual cyclodextrin (CD) systems. This chapter describes how to separate the enantiomers of acidic or neutral substances using dual CD systems made up of a negatively charged CD derivative, i.e., sulfobutyl-beta-CD (SB-beta-CD) or carboxymethyl-beta-CD (CM-beta-CD), in combination with a neutral one, namely, heptakis(2,3,6-tri-O-methyl)-beta-CD (TM-beta-CD). An acidic compound (carprofen) and a weakly acidic drug (pentobarbital) were selected as model compounds. [less ▲]

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See detailDetermination of inhibitory potency of argatroban toward thrombin by electrophoretically mediated microanalysis
Pochet, Lionel; Servais, Anne-Catherine ULg; Farcas, Elena ULg et al

in Talanta (2013), 116

Developing an EMMA method for enzymatic assay remains a challenge, particularly using UV detection. Indeed, it is necessary to optimize the separation conditions while allowing the enzymatic reaction to ... [more ▼]

Developing an EMMA method for enzymatic assay remains a challenge, particularly using UV detection. Indeed, it is necessary to optimize the separation conditions while allowing the enzymatic reaction to occur within the capillary respecting kinetic constraints and achieving enough sensitivity. In this work, such EMMA methodology was set up to evaluate the inhibitory potency of drugs toward thrombin, a serine protease implicated in the coagulation cascade. To achieve our goal, the separation buffer, the injection sequence, the internal standard and the chromogenic substrate were investigated. The newly developed system was then assessed determining the kinetic Km constant for the selected substrate and compared with the results obtained with a continuous spectrophotometer cell assay. Secondly, the Ki inhibitory constant of the thrombin inhibitor argatroban was determined and found in agreement with the published value. [less ▲]

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See detailHigh inorganic triphosphatase activities in bacteria and mammalian cells: Identification of the enzymes involved.
Kohn, Grégory ULg; Delvaux, David ULg; Lakaye, Bernard ULg et al

in PLoS ONE (2012), 7(9), 43879

Background: We recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this ... [more ▼]

Background: We recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. Methodology/Principal Findings: Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPPi) is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPPi but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. Conclusions and General Significance: We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPPi in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPPi, which could be cytotoxic because of its high affinity for Ca2+, thereby interfering with Ca2+ signaling. [less ▲]

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See detailDevelopment of a generic micellar electrokinetic chromatography method for the separation of 15 antimalarial drugs as a tool to detect medicine counterfeiting
Lamalle, Caroline ULg; Marini Djang'Eing'A, Roland ULg; Debrus, Benjamin ULg et al

in Electrophoresis (2012), 33

Since antimalarial drugs counterfeiting is dramatically present on the African market, the development of simple analytical methods for their quality control is of great importance. This work consists in ... [more ▼]

Since antimalarial drugs counterfeiting is dramatically present on the African market, the development of simple analytical methods for their quality control is of great importance. This work consists in the CE analysis of 15 antimalarials (artesunate, artemether, amodiaquine, chloroquine, piperaquine, primaquine, quinine, cinchonine, mefloquine, halofantrine, sulfadoxine, sulfalen, atovaquone, proguanil, and pyrimethamine). Since all these molecules cannot be ionized at the same pH, MEKC was preferred because it also allows separation of neutral compounds. Preliminary experiments were first carried out to select the most crucial factors affecting the antimalarials separation. Several conditions were tested and four parameters as well as their investigation domain were chosen: pH (5–10), SDS concentration (20–90 mM), ACN proportion (10–40%), and temperature (20–35°C). Then, the experimental design methodology was used and a central composite design was selected. Mathematical modeling of the migration times allowed the prediction of optimal conditions (29°C, pH 6.6, 29 mM SDS, 36% ACN) regarding analyte separation. The prediction at this optimum was verified experimentally and led to the separation of 13 compounds within 8 min. Finally, the method was successfully applied to the quality control of African antimalarial medicines for their qualitative and quantitative content. [less ▲]

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See detailMigration behaviour study of charged and uncharged compounds in micellar electrokinetic chromatography systems containing various proportions of SDS micelles and acetonitrile as organic modifier
Lamalle, Caroline ULg; Servais, Anne-Catherine ULg; Crommen, Jacques ULg et al

Poster (2012)

In 1984, Terabe and co-workers introduced a modified version of CZE, called micellar electrokinetic chromatography (MEKC), in which surfactant-formed micelles are included in the running buffer providing ... [more ▼]

In 1984, Terabe and co-workers introduced a modified version of CZE, called micellar electrokinetic chromatography (MEKC), in which surfactant-formed micelles are included in the running buffer providing a two phases chromatographic system for the separation of neutral compounds. Depending on their hydrophobicity, compounds can interact with the core of the micelles. Many MEKC methods have been described for the separation of neutral and basic compounds and for various applications. However, the migration behaviour of cationic, anionic and neutral analytes mixture is not well established regarding to the surfactant concentration and to the proportion of organic solvent in the background electrolyte (BGE). With such a mixture, it is important to remember that the separation of charged solutes in MEKC involves a combination of chromatographic and electrophoretic separation mechanisms. Moreover, it is worth noting that with charged analytes, two kinds of interactions with the micelles may occur; not only with the hydrophobic core but also with the head groups of the micelles through electrostatic interactions. In systems in which the surfactant has an opposite charge to that of the solute, ion pairing may occur. On the contrary, when a solute and the surfactant have similar charges, coulombic forces may cause the repulsion of these molecules. The goal of this study is to improve our understanding of the migration behaviour of charged and uncharged analytes in MEKC systems. With this aim in view, effective mobility (electrophoretic mobility under the influence of micelles) of neutrals, cations and anions were mesured at neutral, basic and acidic pH with BGE containing different SDS concentrations and ACN proportions. This study is also focused on the changes of migration order between CZE and MEKC systems using different BGE compositions. In the MEKC systems investigated in the study, SDS concentration and ACN proportion show a tremendous effect on the effective mobilities and migration order of the model compounds. While anions interact very weakly with SDS micelles, neutrals and cations interact with SDS through hydrophobic and ionic bonds. These interactions become stronger with the increase of SDS concentration and weaker with high ACN proportion. With 20 mM of SDS in the BGE, CZE behaviour is observed till 40% of ACN. But, when the SDS concentration is high and the ACN proportion is low, the migration order of analytes is reversed compared to CZE: EOF first, then the anions, followed by the neutrals and finally the cations. The migration order inside each group (cations, neutrals and anions) depends on the hydrophobicity of the analytes. Different organic solvents were also investigated to study the ion-pair formation. Those observations confirm the interest of using MEKC not only for the separation of neutral compounds but also variously charged analytes. [less ▲]

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See detailMicellar electrokinetic chromatography (MEKC) systems for the separation of mixtures of charged and uncharged compounds
Lamalle, Caroline ULg; Servais, Anne-Catherine ULg; Fradi, Inès et al

Poster (2012)

The migration behaviour of charged and uncharged analytes in MEKC was investigated under different conditions. Effective mobilities – electrophoretic mobilities under the influence of the negatively ... [more ▼]

The migration behaviour of charged and uncharged analytes in MEKC was investigated under different conditions. Effective mobilities – electrophoretic mobilities under the influence of the negatively charged micelles – of cations, anions and neutrals were measured at neutral, basic and acidic pH values (7.5, 11 and 2.2) using background electrolytes containing different sodium dodecyl sulfate (SDS) concentrations (0-90 mM) and acetonitrile proportions (0-75 %, v/v). The SDS concentration and acetonitrile proportion were found to have a tremendous effect on the effective mobilities and the migration order of the tested compounds. Although the SDS micelles interact more strongly with neutrals and cations, the migration of anionic compounds is also affected by the SDS concentration, indicating that hydrophobic interactions can occur between the micelles and these compounds. Since cationic, anionic and neutral solutes exhibit rather different migration behaviours, it is possible to considerably enhance the separation selectivity by properly adjusting the SDS concentration and the acetonitrile proportion in the background electrolyte. These observations confirm the interest of using MEKC not only for the separation of neutral substances but also for the analysis of mixtures of charged compounds. [less ▲]

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See detailMicellar electrokinetic chromatography systems for the separation of mixtures of charged and uncharged compounds
Lamalle, Caroline ULg; Servais, Anne-Catherine ULg; Fradi, Ines ULg et al

in Journal of Separation Science (2012), 35(15), 1933-1939

In this study, the migration behavior of charged and uncharged analytes was investigated under different conditions. Effective mobilities - electrophoretic mobilities under the influence of micelles - of ... [more ▼]

In this study, the migration behavior of charged and uncharged analytes was investigated under different conditions. Effective mobilities - electrophoretic mobilities under the influence of micelles - of cations, anions, and neutrals were measured at neutral, basic, and acidic pH (7.5, 11, and 2.2) using background electrolytes containing different sodium dodecyl sulfate (SDS) concentrations (0-90 mM) and acetonitrile (ACN) proportions (0-75%). SDS concentration and ACN proportion were found to have a tremendous effect on the effective mobilities and migration order of the model compounds. Although the SDS micelles preferably interact with neutrals and cations, hydrophobic bonds can also occur with anions. Cations, anions, and neutrals having rather different migration behaviors, it is possible to considerably enhance the selectivity of the method by adjusting properly the SDS concentration and the ACN proportion. These observations confirm the interest of using micellar electrokinetic chromatography not only for the separation of neutral substances but also to analyze charged compounds. [less ▲]

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See detailChemo- and enantio-selective method for the analysis of amino acids by capillary electrophoresis with in-capillary derivatization.
Fradi, Ines ULg; Servais, Anne-Catherine ULg; Lamalle, Caroline ULg et al

in Journal of Chromatography. A (2012), 1267

A novel dual chiral CE method was developed for the separation of l- and d-amino acids (AAs), using in-capillary derivatization with 9-fluoroenylmethyl chloroformate (FMOC). Firstly, using pre-column ... [more ▼]

A novel dual chiral CE method was developed for the separation of l- and d-amino acids (AAs), using in-capillary derivatization with 9-fluoroenylmethyl chloroformate (FMOC). Firstly, using pre-column derivatization, the enantioseparation of FMOC-AAs was optimized according to the nature of cyclodextrins (CD). A background electrolyte (BGE) composed of 30mM beta-CD, 30mM octakis(2,3-dihydroxy-6-O-sulfo)-gamma-CD (OS-gamma-CD), 40mM tetraborate and 15% isopropanol (IPA) was selected and led to 17 baseline resolved pairs (R(s)=1.7-5.8) and two partially resolved pairs (Lys, R(s)=0.5 and Arg, R(s)=1.2). Experimental conditions for in-capillary derivatization were then optimized. Several parameters, such as mixing voltage and time, concentration of labeling solution and the length of the spacer plug were studied. The optimal conditions for in-capillary derivatization procedure were obtained using successive hydrodynamic injections (30mbar) of AAs for 2s, borate buffer for 4s and 10mM FMOC solution for 6s, followed by a mixing at 3kV for 72s and wait time of 1min. Moreover, a particular attention was paid to improve separation chemoselectivity. The effect on stereoselectivity and chemoselectivity of different factors, such as decrease of pH and tetraborate concentration and the addition of sodium dodecyl sulfate (SDS), was investigated using the in-capillary derivatization procedure. The best separation of a standard mixture of ten AA racemates was observed using a BGE containing 30mM beta-CD, 30mM OS-gamma-CD, 25mM SDS, 40mM sodium tetraborate and 17% IPA. [less ▲]

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See detailComparative enantioseparation of talinolol in aqueous and non-aqueous capillary electrophoresis and study of related selector-selectand interactions by nuclear magnetic resonance spectroscopy.
Chankvetadze, Lali; Servais, Anne-Catherine ULg; Fillet, Marianne ULg et al

in Journal of Chromatography. A (2012), 1267

The enantiomers of the chiral beta-blocker drug talinolol were separated with two single component sulfated beta-cyclodextrin (CD) derivatives, namely heptakis (2,3-di-O-methyl-6-sulfo)-beta-CD) (HDMS ... [more ▼]

The enantiomers of the chiral beta-blocker drug talinolol were separated with two single component sulfated beta-cyclodextrin (CD) derivatives, namely heptakis (2,3-di-O-methyl-6-sulfo)-beta-CD) (HDMS-beta-CD) and heptakis (2,3-di-O-acetyl-6-sulfo)-beta-CD) (HDAS-beta-CD), in aqueous and non-aqueous capillary electrophoresis (CE). The enantiomer affinity pattern of talinolol toward these two CDs was opposite in both aqueous and non-aqueous CE. However, the enantiomer affinity pattern for a given CD derivative did not change when aqueous buffer was replaced with non-aqueous background electrolyte. The structures of the analyte-selector complexes in both, aqueous and non-aqueous electrolytes were studied using rotating frame nuclear Overhauser effect (ROESY) NMR spectroscopy. Inclusion complex formation between the enantiomers of talinolol and HDAS-beta-CD was confirmed in aqueous buffer, while the complex between the enantiomers of talinolol and HDMS-beta-CD was of the external type. The complex of the talinolol enantiomers with HDAS-beta-CD in non-aqueous electrolyte was also of the external type. In spite of external complex formation excellent separation of the enantiomers was observed in non-aqueous CE. [less ▲]

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See detailOptimization of the liquid chromatography enantioseparation of chiral acidic compounds using cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector and polar organic mobile phases.
Dossou, K. S. S.; Farcas, Elena ULg; Servais, Anne-Catherine ULg et al

in Journal of Chromatography. A (2012), 1234

The LC enantioseparation of chiral acidic and zwitterionic drugs selected as model compounds was optimized using chlorine containing cellulose based chiral stationary phases and polar organic mobile ... [more ▼]

The LC enantioseparation of chiral acidic and zwitterionic drugs selected as model compounds was optimized using chlorine containing cellulose based chiral stationary phases and polar organic mobile phases. The main solvent of the mobile phase was acetonitrile, the temperature was settled at 25 degrees C and a stationary phase with cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector (3-Cl-4-Me-PC) was selected. In the screening step, the nature and concentration of both acidic and basic additives were found to have a significant effect on retention, selectivity and resolution. Acetic acid (AcA) was selected as acidic additive for the optimization step since it could lead to the enantioseparation of more acidic compounds than trifluoroacetic acid (TFA) and formic acid (FA), while among the three basic additives tested, diethylamine (DEA) most often gave better results with respect to enantioresolution and selectivity than butylamine (BuA) and triethylamine (TEA). The optimization was performed using a central composite face-centered design with two factors, namely the concentration of acetic acid (0.1-0.3%) and the concentration of DEA (0.01-0.1%) in the mobile phase. On the basis of the results obtained in the screening and optimization steps, a strategy for the rapid development of methods for the enantioseparation of acidic or neutral compounds was proposed. [less ▲]

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See detailCombination of capillary electrophoresis, molecular modelling and nuclear magnetic resonance to study the interaction mechanisms between single-isomer anionic cyclodextrin derivatives and basic drug enantiomers in a methanolic background electrolyte.
Servais, Anne-Catherine ULg; Rousseau, Anne; Dive, Georges ULg et al

in Journal of Chromatography. A (2012), 1232

In order to improve our knowledge of the mechanisms of enantiomer recognition pattern in nonaqueous systems, an approach combining nonaqueous CE (NACE), molecular modelling and NMR was undertaken ... [more ▼]

In order to improve our knowledge of the mechanisms of enantiomer recognition pattern in nonaqueous systems, an approach combining nonaqueous CE (NACE), molecular modelling and NMR was undertaken. Bupivacaine and propranolol were selected as model compounds and their interactions with two single-isomer highly charged beta-CD derivatives, namely heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-CD (HDMS-beta-CD) and heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-CD (HDAS-beta-CD), were studied. The CD-bupivacaine complexes were evaluated by 2-D Rotating-frame Overhauser Effect SpectroscopY (ROESY) experiments. From these experiments, it can be assumed that inclusion complexes are not formed, whatever the CD derivative used. Molecular modelling was performed at the RHF/MINI-1 or B3LYP/6-31G(d) level. External as well as inclusion type complexes with the alkyl chain of propranolol into both CD cavities were located. Interaction energies calculated for bupivacaine and propranolol correlated with the enantiomer migration order observed in the NACE experiments using both anionic CD derivatives. The interaction of propranolol with HDMS-beta-CD or HDAS-beta-CD gives rise to a family of external and inclusion complexes in which some are more probably obtained. [less ▲]

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See detailOptimization of micro-HPLC peak focusing for the detection and quantification of low hepcidin concentrations.
Mansion, François ULg; Chiap, Patrice ULg; Houbart, Virginie ULg et al

in Journal of Separation Science (2011), 34(15), 1820-1827

Micro-high-performance liquid chromatography is a miniaturized, economic and ecological chromatographic system allowing the use of reduced size chromatographic columns. Coupled with electrospray ... [more ▼]

Micro-high-performance liquid chromatography is a miniaturized, economic and ecological chromatographic system allowing the use of reduced size chromatographic columns. Coupled with electrospray ionization tandem mass spectrometry, this technique can be used to detect and quantify low concentrations of peptides. In this study, hepcidin was used as the model compound and analysed using octadecylsilica stationary phase by means of a gradient elution mode at a flow rate of 4 muL/min. Several parameters were studied to optimize peak focusing. Using the methodology of experimental design, the mobile-phase gradient conditions and the sample composition were optimized in order to maximize the sensitivity and minimize retention time. Stability of the target peptide in solution was also demonstrated. [less ▲]

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