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In the frame of our molecular imaging activities, a PEGylated lipopeptide has been developed as a specific ligand for the human vascular endothelial growth factor receptor 2, which is considered as one of the important molecular marker of angiogenesis. In this study, the potential of affinity capillary electrophoresis (ACE) is evaluated to measure the interactions of an active PEGylated lipopeptide, its hydrolysis product and its precursor consisting of a peptide structure with different micelles including Brij-35, Tween-20, and pegylated phospholipids. Given the amphiphilic structure of the PEGylated lipopeptide, a MEKC method allowing the simultaneous separation of the compounds of interest was set up, using low percentages of acetonitrile. Analytes were resolved using a BGE consisting of 100 mM borate buffer pH 9.0, 1 mM Brij, and 25% acetonitrile. Optimized conditions were then used to perform ACE experiments. The affinity constants of the analytes with the micelles were calculated on the basis of their mobility decrease when surfactant concentration increased in the electrolyte. The use of different linearization models to estimate affinity constants was discussed and comparison of different surfactants was reported. PEGylated lipopeptide interacted more strongly with pegylated phospholipid micelles than with Brij-35 or Tween-20. Moreover, it is likely that the chemical structure of the compounds, and particularly the lipidic part of the molecules, significantly affects the interaction with micelles. In conclusion, the ACE method can be readily applied to investigate interactions of our targeting lipopeptides with various micelles currently used for the preparation of pharmaceutical vehicles., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Due to their high specificity and efficiency, antibodies are ideal ligands for target-specific ultrasound contrast agents. The present study focuses on the chemical stability of antibodies during functionalisation with sulfosuccinimidyl-pyridyldithiopropionamidohexanoate (SPDP), a heterobifunctional linker, which exposes free thiol groups upon treatment with a reducing agent. Thiolated antibodies can then react with thiol-reactive group, such as maleimide present on the microbubble surface to form stable covalent complexes. The immunoglobulin structure relies on several intra- and inter-chain disulfide bridges which might be affected by reducing agents. A capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) method with UV detection was applied to address the effect of the functionalisation process on the structural integrity of the antibodies and revealed that antibody disulfide bonds are prone to reduction as function of the reducing agents. Depending on the coupling conditions, various IgG fragments were identified reflecting different combinations between the light and heavy chains. Furthermore, two commonly used reducing agents, namely triscarboxyethylphosphine (TCEP) and 1,4-dithiothreitol (DTT) were compared under various preparation conditions. Results showed that reduction conditions based on DTT as a reducing agent under acidic pH were more appropriate to preserve intra- and inter-disulfide bridges of SPDP-modified antibodies.

Four chiral basic analytes, namely methadone, fluoxetine, venlafaxine, and tramadol, were selected as model compounds for investigating their stereoselective separation with highly sulfated gamma-cyclodextrin (HS gamma-CD) by capillary electrophoresis (CE)-UV and CE-mass spectrometry (MS). At high concentration of chiral selector, the preferentially bonded enantiomer migrated faster in the anodic mode to the detector and high resolutions were obtained for all analytes. In the cathodic mode, at lower highly sulphated cyclodextrin (HS-CD) concentration, basic compounds could be detected, with the weakly bonded enantiomer migrating first (enantiomeric migration order inversion). It was also then possible, at intermediate HS-CD concentration, that only one enantiomer migrated to the detector as cation while the other enantiomer complexed with the CD was negatively charged and presented an opposite mobility. The latter never reached the detector achieving a perfect enantiomeric selectivity. Infinite chiral resolutions were thus achieved by CE-UV as well as by CE-electrospray ionisation (ESI)-MS where concentrations of HS-CD were adapted according to the negative contribution of the nebulization gas pressure of the interface.

The simultaneous analysis of Ecstasy and its derivatives in an acetonitrile-methanol (80:20 v/v) mixture was previously shown to be strongly dependent on the nature of the electrolyte (acetate versus formate). To elucidate the phenomena involved, systematic experiments were conducted in this solvent medium. Conductivity measurements allowed to evaluate the ion-pairing rate in the background electrolyte (BGE) and thereby distinguish between electrolyte concentration and ionic strength. The influence of electrolyte concentration on analyte effective mobilities micro(eff)) was studied by capillary electrophoresis (CE). As micro(eff) extrapolated to infinite dilution proved to be independent of the nature of the electrolyte, selectivity changes could not be attributed to a modification in the protonation degree of amphetamines. Experimental mobility data were then confronted to existing theoretical mobility models to discriminate between ion pairing or simple ionic strength effect. Ion-pair formation in a BGE containing acetate was highlighted with an ion-pairing model and ion-pair formation constants between each amphetamine and acetate ion were calculated.

A nonaqueous capillary electrophoresis (NACE) method, coupled with either UV or electrospray mass spectrometry (ESI-MS), is described for the simultaneous analysis of seven beta-blockers. The same electrolyte, namely 25 mM ammonium formate and 1 M formic acid, was used with different investigated organic solvents. In addition to frequently used organic solvents such as methanol (MeOH) and acetonitrile (MeCN), formamide and its derivatives were investigated. Formamide (FA) and N-methylformamide (NMF) present several interesting physico-chemical properties, one of them being a high dielectric constant (e). Since FA and NMF possess a high UV cutoff, beta-blockers with an absorbance above 250 nm were selected as model compounds in order to compare NACE-UV and NACE-MS performances. FA and NMF showed different selectivity compared to water, MeOH or MeCN, and also demonstrated a higher efficiency in terms of the number of theoretical plates (especially NMF). To overcome their unfavorable optical properties, hyphenation with MS detection appears as a promising technique, thanks to its benefits in terms of selectivity, sensitivity and universality. The practical compatibility of FA and NMF with ESI-MS detection in combination with a sheath liquid configuration was demonstrated. In comparison to UV detection, sensitivity was increased, while a high efficiency was maintained. In addition, the low and stable generated currents observed were evidences for the successful hyphenation with ESI-MS. Hence, FA and NMF seemed to be promising alternatives in NACE-ESI-MS, either used as pure organic solvent or as a mixture with MeOH or MeCN.

Benzodiazepines, namely flunitrazepam and its three major metabolites, were successfully separated by microemulsion electrokinetic chromatography. Separation was achieved using an untreated fused-silica capillary (48 cm (effective length 40 cm) x 50 num) at 25 kV; detection was performed by UV at 220 nm. The microemulsion system consisted of 70 mM octane, 800 mM 1-butanol, 80 mM sodium dodecyl sulfate (SDS) and 10 mM borate buffer, pH 9. Very high efficiencies (up to 400 000 plates) and resolution better than 3 were achieved. Since this technique is not compatible with mass spectrometry (MS) detection, a capillary electrochromatographic (CEC) method was developed to separate flunitrazepam and its metabolites. The effects of mobile phase composition and pH as well as voltage and temperature were systematically investigated. The optimized CEC method allowed the baseline separation of the investigated compounds. For the on-line coupling of CEC with electrospray ionization-mass spectrometry, the column was connected to a void fused-silica capillary using a Teflon connection. This configuration was found efficient and suitable for hyphenation of commercial CEC and MS instrumentation using commercially available CEC columns.

The potential of nonaqueous capillary electrophoresis was investigated for the simultaneous separation of fluoxetine hydrochloride, its meta-isomer, and other related compounds. The resolution of these compounds was compared in aqueous and nonaqueous media. Baseline separation of the studied solutes required a buffer electrolyte solution composed of 25 mM ammonium acetate and 1 M acetic acid in acetonitrile, an applied voltage of 30 kV and a temperature of 20 degrees C. Selectivity was considerably affected by the nature of the solvent (water, methanol, and acetonitrile). Moreover, substituting acetate by formate in the background electrolyte resulted in migration time changes, which were attributed to an ion-pairing phenomenon. Finally, the method was successfully coupled on-line with electrospray ionization-mass spectrometry (ESI-MS) and allowed significant selectivity and sensitivity enhancement. The effect of ESI-MS parameters, such as nebulizing gas pressure, sheath liquid composition and flow rate, on resolution and method sensitivity was also discussed.

The on-line combination of partial-filling capillary electrophoresis and electrospray ionization mass spectrometry was demonstrated for the enantioseparation of pharmaceutical drugs and metabolites, namely amphetamines, methadone, venlafaxine and selected tropane alkaloids. The partial-filling technique proved to be a suitable and efficient approach to avoid mass spectrometry (MS) source contamination, as well as signal suppression due to nonvolatile additives. To achieve chiral separation, various chiral selectors were applied, including neutral and particularly negatively charged cyclodextrins. Because of the countercurrent contribution, charged cyclodextrins were found more suitable for the on-line MS detection of separated enantiomers. Hyphenation of capillary electrophoresis (CE) with mass spectrometry was found appropriate for the stereoselective analysis of methadone in real serum samples. Moreover, the use of MS in the selected ion monitoring mode resulted in a very high selectivity, as well as improved sensitivity compared to UV detection. Finally, with atropine as a model compound, the quantitative performances of the method were evaluated and showed high sensitivity, as well as good repeatability in terms of migration time and peak area ratio.

An experimental design approach is described to evaluate the main electrophoretic parameters involved in the enantioseparation of pharmaceuticals by capillary electrophoresis (CE) coupled to electrospray ionization-mass spectrometry (ESI-MS). For all experiments, the partial-filling technique was applied to avoid the chiral selector entering in the mass spectrometer ion source with a negative effect on the electrospray performance. To carry out enantioseparation, a volatile buffer constituted of 20 mM ammonium acetate at pH 4.0, and a polyvinyl alcohol-coated capillary were used. Methadone was employed as the model compound and three different cyclodextrins (CDs), namely sulfobutyl ether-beta-CD, carboxymethylated-beta-CD and hydroxypropyl-beta-CD, were selected in order to study the countercurrent process. Two different experimental designs were chosen: (i) a full-factorial design to examine the effects and significance of the investigated factors, and (ii) a central composite face-centered design to establish the mathematical model of the selected responses in function of experimental factors. The chiral selector concentration, percentage of the capillary filled with the chiral selector, and drying gas nebulization pressure were three relevant factors taken into consideration. For each CD, the methadone enantiomeric resolution, apparent selectivity, and migration time of the second enantiomer were established as responses. The latter were systematically related to experimental parameters with the help of multiple linear regression. It is noteworthy that the behaviour was different in function of the chiral selector charge. Results revealed that the nebulization pressure involved in the electrospray process and the CD concentration had a significant effect on the enantiomeric resolution, while the effect of the separation zone length was less pronounced. Finally, response surfaces were drawn from the mathematical model and experimental conditions were selected to allow a robust determination of methadone enantiomers by CE-MS.

Nonaqueous capillary electrophoresis coupled to UV detection is described for the separation and determination of steroidal alkaloids. After optimization of electrophoretic parameters, including the electrolyte nature and the organic solvent composition, a reliable separation of solasodine and solanidine was achieved in a methanol-acetonitrile (20:80, v/v) mixture containing 25 mM ammonium acetate and 1 M acetic acid. For quantitative purposes, a fused-silica capillary with a bubble cell was used and detection was performed at low wavelength (195 nm). Method performances, including migration time and peak area reproducibility, linearity, sensitivity and accuracy, were also evaluated. The method was applied to determine solasodine in Solanum elaeagnifolium berries and Solanum sodomaeum leaves and seeds. To further improve sensitivity in the analysis of solasodine-related compounds, solanidine, demissidine and tomatidine, the developed method was interfaced with electrospray ionization mass spectrometry. In the case of solasodine, the detection limit was estimated at 3 microg/ml for NACE-UV and at 0.05 microg/ml for NACE-MS, in the selected ion-monitoring mode.

Capillary zone electrophoresis was applied to separate caseinoglycomacropeptide glycoforms and characterize microheterogeneity of the glycopeptide. Particular attention was paid to the sialic acid content in caseinoglycomacropeptide obtained through different manufacturing processes. A chemometric approach was used to simultaneously study effects of acid concentration, hydrolysis time and temperature on sialic acid release from caseinoglycomacropeptide. Hydrolysis conditions that maximize sialic acid release were chosen. Sialic acid was determined using high performance anion exchange chromatography coupled with pulsed amperometric detection. Results were compared to those obtained by alternative techniques, such as colorimetric and enzymatic methods.

Aqueous and nonaqueous capillary electrophoresis (NACE) were investigated for separation of venlafaxine, a new second-generation antidepressant, and its three phase I metabolites. Working at basic pH, around the venlafaxine pKa value, was effective in resolving the investigated drugs, but created considerable peak tailing. To overcome electrostatic interactions between analytes and silanol groups, investigations were also carried out at acidic pH. However, despite the addition of up to 50% v/v of organic solvents (e.g., methanol or acetonitrile), complete separation of the studied compounds was not possible. NACE was found to be an appropriate alternative to resolve venlafaxine and its metabolites simultaneously. Using a conventional capillary (fused-silica, 64.5 cm length, 50 microm inner diameter), and a methanol-acetonitrile mixture (20/80 v/v) containing 25 mM ammonium formate and 1 M formic acid, complete resolution of these closely related compounds was performed in less than 3.5 min. Selectivity, efficiency and separation time were greatly affected by the organic solvent composition. As the electric current generated in nonaqueous medium was very low, the electric field was further increased by reducing the capillary length. This allowed a baseline resolution of venlafaxine and its three metabolities in 0.7 min. Selectivity was compared in aqueous and nonaqueous media in relation to the acid-base properties of the analytes as well as to the solvation degree. Finally, the method successfully coupled on-line to mass spectrometry with electrospray ionization interface allowed significant sensitivity enhancement.

A nonaqueous capillary electrophoresis method, coupled to UV and electrospray mass spectrometry (ESI-MS), is described for the simultaneous analysis of Ecstasy and other related derivatives. Several electrophoretic and ESI-MS parameters were systematically investigated, such as electrolyte nature and concentration, organic solvent and sheath liquid compositions, nebulization gas pressure and drying gas flow-rate. The best results were achieved with an acetonitrile-methanol (80:20, v/v) mixture containing 25 mM ammonium formate and 1 M formic acid, an applied voltage of 30 kV and a separation temperature of 15 degrees C. Under optimized CE-ESI-MS conditions, separation of the investigated drugs was performed in less than 6 min, with a high efficiency. Method precision based on migration time and peak area was determined and the limits of detection, which depend on the tested compound, were established between 20 and 70 ng ml(-1) in the selected ion monitoring mode. Finally, the described method was successfully applied to the analysis of amphetamines in urine after a liquid-liquid extraction.

Nine non steroidal anti-inflammatory drugs were simultaneously separated by nonaqueous capillary electrophoresis with a methanol-acetonitrile (40:60, v/v) mixture containing 20 mM ammonium acetate. The effect of solvent composition, electrolyte nature and concentration on the electrophoretic behavior of the selected drugs was systematically studied. Investigated electrolytes were ammonium, lithium and sodium acetate. Modification of the solvent and/or the electrolyte composition was found to alter the migration order of the pharmaceutical drugs. Finally, to assess method robustness, three sensitive electrophoretic parameters as well as their interactions were evaluated using a full factorial design at two levels.

On-line combination of partial filling capillary electrophoresis and electrospray ionization mass spectrometry was demonstrated for the simultaneous enantioseparation of tramadol and its main phase I metabolites. The partial filling technique was efficient at avoiding MS contamination by the chiral selector. Different experimental factors were investigated, including the chiral selector nature and concentration, plug length as well as the separation temperature. The best enantioseparation of the investigated compounds was achieved with a coated polyvinyl alcohol capillary and a 40 mM ammonium acetate buffer, pH 4.0, adding sulfobutyl ether beta-cyclodextrin (2.5 mg/ml) as the chiral selector. The charged cyclodextrin not only allowed enantioseparation of tramadol and its metabolites, but also improved the selectivity of compounds with the same molecular mass. Finally, CE-electrospray ionisation-MS was successfully applied to the stereoselective analysis of tramadol and its main metabolites in plasma after a simple liquid-liquid extraction.

A capillary zone electrophoresis (CZE) method, with sulfated beta-CD as chiral selector, was optimized by means of an experimental design for the enantioseparation of atropine. In this study, a central composite design was used and the following factors were varied simultaneously: buffer concentration, buffer pH and sulfated beta-CD concentration. The resolutions between littorine and its positional isomer ((-)-hyoscyamine) and between atropine enantiomers, as well as the separation time and generated current were established as responses. A model was obtained for each response by linear multiple regression of a second-degree mathematical expression. The most favorable conditions were determined by maximizing the resolution between atropine enantiomers and by setting the other responses at threshold values. Successful results were obtained with a 55 mM phosphate buffer at pH 7 in the presence of 2.9 mM sulfated-beta-CD at 20 degrees C and 20 kV. Under these optimized conditions, a baseline separation of littorine and atropine enantiomers was achieved in less than 5 min. Finally, the method allowed the enantiomeric separation of atropine in a pharmaceutical formulation and was also found to be suitable for the enantiomeric purity evaluation of (-)-hyoscyamine in plant extracts, in relation with the extraction procedure. It was demonstrated that supercritical fluid extraction induced less racemization than classical liquid-solid extraction procedures.

A capillary zone electrophoresis method was developed for the enantioseparation of celiprolol enantiomers, using a sulfated beta-cyclodextrin (beta-CD) as a chiral selector. The use of a coated capillary was necessary to achieve stable and reproducible enantioseparations. A central composite design was applied to optimize the method and four parameters were selected for this study: the buffer pH, the buffer concentration, the sulfated beta-CD concentration and the temperature. Resolution between celiprolol enantiomers as well as analysis time and generated current were established as responses. For each response, a model was obtained by a second-degree mathematical expression. From the models, the most favorable conditions were determined by optimizing the resolution between celiprolol enantiomers and by setting the two other responses at threshold values. Response surfaces were also used to assess the robustness of the analytical method around the optimal region. Successful results were obtained with a 52 mM acetate buffer at pH 4.0 in the presence of 3.0 mM sulfated beta-CD at a temperature of 19.5 degrees C. Under these optimized conditions, baseline separation of the celiprolol enantiomers was achieved in less than 10 min. The method showed good validation data in terms of precision, accuracy and linearity, and was found to be suitable in determining celiprolol enantiomers in pharmaceutical preparations and in biological fluids.

Capillary zone electrophoresis, coupled to UV and interfaced with electrospray ionization mass spectrometry (ESI-MS), is described for the simultaneous analysis of hyoscyamine and scopolamine. On-line UV detection occurred at 22 cm from the inlet of the capillary and ESI-MS monitoring was performed along the entire length of the capillary (85 cm). An alkaline solution of 40 mM ammonium acetate at pH 8.5 was suitable for the analysis of the alkaloids under consideration. Under the optimized conditions, including CE and ESI-MS parameters, the two alkaloids were resolved within a short time and with very high sensitivity. The differentiation of hyoscyamine and its positional isomer littorine, commonly encountered in plant material, is also presented using up-front collision-induced dissociation. Finally, the developed method was applied to the analysis of these alkaloids in Belladonna leaf extract and in Datura candida x D. aurea hairy root extract.

A simple nonaqueous capillary electrophoresis method is described for the separation of several atropine and scopolamine related drugs. The analysis of these pharmaceutical compounds was performed in a methanol-acetonitrile (25/5, v/v) mixture containing 25 mM ammonium acetate and 1 M acetic acid. The robustness was proved using a full factorial design at two levels. The method was validated and successfully applied for the determination of N-butylscopolamine in different pharmaceutical preparations. Results were compared to those obtained by a capillary electrophoresis method based on aqueous media.

Three capillary electrophoresis methods, using UV detection, were developed for the simultaneous determination of several tropane alkaloids, including atropine, scopolamine and synthetic derivatives. After optimization, the validated capillary zone electrophoresis methods were applied to the determination of these compounds in various pharmaceutical forms, such as ophthalmic and injection solutions, tablets, suppositories and aerosols. Capillary electrophoresis in the micellar mode was found to be more appropriate for the analysis of hyoscyamine and scopolamine in plant material. These two compounds are generally found together with other tropane alkaloids which present similar structures and charge to mass ratio. Furthermore, the separation of positional isomers, such as hyoscyamine and littorine generally encountered in plant extracts, was also considered. The developed method was applied to the analysis of hairy root extracts of Datura candida x Datura aurea, Datura quercifolia and Hyoscyamus albus.

Liquid chromatography and capillary zone electrophoresis, respectively coupled to an evaporative light scattering detector and a UV detector have been developed for the analysis of acarbose without any derivatization procedure. The electrophoretic separation of acarbose anomers was achieved through the manipulation of the working temperature. Both methods were validated and showed good validation data in terms of precision, accuracy and linearity. The validated methods were successfully applied to the dosage of acarbose in commercially available Glucobay tablets.

Non-aqueous capillary electrophoresis was investigated for its potential in the analysis of pharmaceutical compounds, namely tropane alkaloids and amphetamine derivatives. The separation of these drugs was compared in aqueous and organic media such as methanol and/or acetonitrile. Selectivity, migration times and efficiency were critically affected by the composition of the methanol/acetonitrile mixture, as well as by the nature and the concentration of the electrolyte. In particular, the migration orders of two positional isomers, littorine and hyoscyamine, were inverted in the presence of trifluoroacetic acid in the nonaqueous medium. The same behavior was observed for amphetamine-methamphetamine and for two methylenedioxyamphetamine derivatives.

The simultaneous determination of atropine and scopolamine derivatives, which have similar structures, was investigated by using capillary zone electrophoresis. The effects of buffer pH, buffer concentration and hydroxypropyl-beta-cyclodextrin concentration on migration time and resolution of the investigated compounds were systematically studied. The selected electrophoretic buffer consisted of a 80 mM sodium citrate pH 2.5, containing 2.5 mM hydroxypropyl-beta-cyclodextrin as the complexing agent. Quantitative analysis was validated by testing the reproducibility of the method, giving a relative standard deviation less than 1 and 2% for the intermediate precision of migration times and peak area ratios, respectively. The linearity of the method was assessed between 50 and 150% of the theoretical content (coefficient of correlation greater than 0.99). The proposed method was found to be suitable and accurate for the determination of these basic drugs in pharmaceutical preparations.

A capillary zone electrophoresis method is described for the simultaneous determination of atropine, homatropine and scopolamine. Successful results were obtained after optimization of the electrophoretic parameters such as buffer composition and pH. The best separation was achieved using a 100 mM Tris-phosphate running buffer at pH 7. The validation data proved that the method had the requisite selectivity, sensitivity, reproducibility and linearity to be used for the assay of these compounds in pharmaceutical formulations. Dosage of the separate drugs in ophthalmic preparations is also presented.