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A rapid and sensitive method was developed for the simultaneous determination of fluoxetine and its primary metabolite, norfluoxetine, in plasma. It was based on a column-switching approach with a precolumn packed with large size particles coupled with a liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS). After a simple centrifugation, plasma samples were directly injected onto the precolumn. The endogenous material was excluded thanks to a high flow rate while analytes were retained by hydrophobic interactions. Afterwards, the target compounds were eluted in back flush mode to an octadecyl analytical column and detected by ESI-MS. The overall analysis time per sample, from plasma sample preparation to data acquisition, was achieved in less than 4 min. Method performances were evaluated. The method showed good linearity in the range of 25-1000 ng mL(-1) with a determination coefficient higher than 0.99. Limits of quantification were estimated at 25 ng mL(-1) for fluoxetine and norfluoxetine. Moreover, method precision was better than 6% in the studied concentration range. These results demonstrated that the method could be used to quantify target compounds. Finally, the developed assay proved to be suitable for the simultaneous analysis of fluoxetine and its metabolite in real plasma samples.

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.

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.