Claudia Desiderio

Use of negatively charged sulfobutyl ether-β-cyclodextrin for enantiomeric separation by capillary electrophoresis

Abstract A newly modified charged β-cyclodextrin (sulfobutyl ether-β-cyclodextrin) was investigated as a chiral selector in capillary electrophoresis in a study of the enantiomeric separation of a variety of underivatized anionic and cationic compounds of pharmaceutical interest and uncharged phenyl alcohols and dansyl-amino acids. Owing to the presence of four sulfonic groups, the chiral selector is negatively charged at all pH values used (2.5–9) and the complexation caused an increase in migration time for each compound studied. At a relatively low pH (2.5) the chiral selector could only be used at low concentration (0.1-0.5 mg/ml) for basic compounds, whereas at higher pH (6–9) the modified cyclodextrin in the concentration range 0–20 mg/ml was used. The concentration of the chiral selector, the distance from the aromatic group of the asymmetric centre of the analytes and the chemical composition and pH of the background electrolyte influenced the complexation, selectivity and resolution. Good enantiomeric separation was obtained for terbutaline at all pH values studied, whereas for other racemic compounds, warfarin, acenocoumarol, promethazine, bupivacaine and some dansyl-amino acids and phenyl alcohols, the pH range 6–9 was effective for optimizing the chiral resolution. Non-polar substituent groups on the asymmetric carbon of the analytes seem to enhance the complexation and the stereoselectivity.

Chiral analysis by capillary electrophoresis using antibiotics as chiral selector.

The separation of chiral compounds by capillary electrophoresis (CE) is a very interesting field of research in different areas such as pharmaceutical, environmental, agricultural analysis etc. The separation of two enantiomers can be achieved in CE using a chiral environment interacting with the two analytes on forming diastereoisomers with different stability constants and thus different mobilities. A wide number of chiral selectors have been employed in CE and among them glycopeptide antibiotics exhibited excellent enantioselective properties towards a wide number of racemic compounds. Vancomycin, ristocetin A, rifamycins, teicoplanin, kanamycin, streptomycin, fradiomycin, and two vancomycin analogues, added to the background electrolyte (BGE), are the antibiotics studied by CE running the separation in untreated and/or coated fused-silica capillary. Due to adsorption and absorption phenomena, some drawbacks can be expected when using bare fused-silica capillary, e.g., changes of electroosmotic flow (EOF), broaden peaks, reduced efficiency and low sensitivity. Coated capillary and counter current mode can be the solution to overcome the above mentioned problems. This review surveys the separation of enantiomers by CE when macrocyclic antibiotics are used as chiral selector. The enantioselectivity can be easily controlled modifying several parameters such as antibiotic type and concentration, pH, ionic strength and concentration of the background electrolyte, organic modifier etc. The paper also presents a list of the latest chiral separations achieved by CE where antibiotics were used as chiral selector.

Chiral capillary electrophoresis–electrospray mass spectrometry coupling using vancomycin as chiral selector

Abstract Capillary electrophoresis–electrospray mass spectrometry coupling (CE–ESI-MS) using vancomycin (VC) as a chiral selector is described in this study. The self electrophoretic mobility of VC, as a positively charged chiral selector at low pH, allows us to avoid the contamination of the ion source of the ESI-MS with the chiral selector in a simple way. The usefulness of this technique is illustrated for the analysis of chiral anionic compounds of pharmaceutical importance. Additionally, examples of a stereospecific determination of ibuprofen and its phase I metabolites as well as etodolac and its metabolites in urine samples are described. The advantages of MS for peak identification, peak purity testing and for selective monitoring of overlapping peaks are demonstrated.

Enantiomeric separation of acidic herbicides by capillary electrophoresis using vancomycin as chiral selector

Abstract In this study vancomycin has been used as chiral selector for the enantiomeric separtion of several free acid herbicides, namely aryloxypropionic (mecoprop, fenoprop and dichlorprop), N -benzoyl- N -(3-chloro-4-fluorophenyl)-2-aminopropionic acid (flamprop) and aryloxyphenoxypropionic (haloxyfop, fluazifop, diclofop and fenoxaprop). The partial filling method was used in order to increase the sensitivity of the electrophoretic method; vancomycin was not present at the detector path during the detection due to its positive charge and to the absence/reduction of the electroosmotic flow at the operating pH. The pH of the BGE, the capillary temperature and the vancomycin concentration influenced both effective mobility and resolution of the studied compounds. The increase of vancomycin concentration caused a general increase of migration time, resolution and selectivity, the best results have been achieved when a 6 m M concentration of chiral selector was used. Using such a concentration of vancomycin, baseline resolution was obtained for all the studied enantiomers in 4.5–8.5 min. The optimized CE method has been tested for teh analysis of haloxyfop free acid metabolite in a soil sample spiked with racemic haloxyfop ethoxyethyl ester solution. The metabolite resulted to be a mixture of R and S enantiomers where the R form was about 72% (peak areas ratio). The method showed good precision for both migration time and corrected peak areas with a detection limit of 5×10 −7 M .

Use of vancomycin silica stationary phase in packed capillary electrochromatography: II. Enantiomer separation of venlafaxine and O-desmethylvenlafaxine in human plasma

A capillary electrochromatography method, using vancomycin chiral stationary phase packed capillary, was optimized for the simultaneous chiral separation of the antidepressant drug venlafaxine and its main active metabolite O-desmethylvenlafaxine. Simultaneous baseline enantiomeric separation of the two compounds was obtained using a mobile phase composed of 100 mM ammonium acetate buffer pH 6/water/acetonitrile (5:5:90, v/v). The electrokinetic injection for sample introduction provided a limit of quantitation for both the compounds of 0.05 microg/ml racemate concentration suitable for the analysis of venlafaxine and metabolite in biological samples. The acetonitrile mobile phase concentration was found to modulate the analytes elution times, the enantiomeric resolution and the efficiency of the separation. The column was tested for repeatability and linearity showing RSD values (%) in the range of 0.13-0.24, 2.47-3.66 and 1.35-2.50 for migration time, sample/internal standard peak area ratio and enantiomeric resolution, respectively and correlation coefficients higher than 0.9990. The method was applied to the analysis of clinical samples of patients under depression therapy showing a stereoselective metabolism for venlafaxine.

Use of vancomycin silica stationary phase in packed capillary electrochromatography I. Enantiomer separation of basic compounds.

Chiral separation of basic compounds was achieved by using 75 or 100 μm ID fused‐silica capillaries packed with a vancomycin‐modified diol silica stationary phase. The capillary was firstly packed for about 12 cm with a slurry mixture composed of diol‐silica (3:1) then with the vancomycin modified diol‐silica (3:1) (23 cm), and finally with diol‐silica (3:1) for about 2 cm. Frits were prepared by a heating wire at the two ends of the capillary; the detector window was prepared at 8.5 cm from the end of the capillary where vancomycin was not present. The influence of the mobile phase composition (pH and concentration, organic modifier type and concentration) on the velocity of the electroosmotic flow, chiral resolution and enantioselectivity was studied. Good enantiomeric resolution was achieved for atenolol, oxprenolol, propranolol, and venlafaxine using a mobile phase composition of 100 mM ammonium acetate solution (pH 6)/water/acetonitrile (5:5:90 v/v/v) while for terbutaline a mixture of 5:15:80 v/v/v provided the best separations. The use of methanol instead of acetonitrile caused a general increase of enantiomer resolution of the studied compounds together with a reduction of efficiency and detector response. However, the combination of acetonitrile and methanol in the mobile phase (as, e.g., 10% methanol and 80% acetonitrile) allowed to improve the enantiomer resolution with satisfactory detector response.

Enantiomeric resolution study by capillary electrophoresis Selection of the appropriate chiral selector

Abstract The enantiomeric separation of several arylpropionic acids, namely carprofen, cicloprofen, flurbiprofen, ibuprofen, indoprofen, ketoprofen, naproxen and suprofen has been studied by capillary zone electrophoresis using different chiral selectors added to the background electrolyte with the aim to find the optimum experimental conditions for both qualitative and quantitative purposes. The chiral selectors used included two β-cyclodextrin derivatives and a glycosidic antibiotic, namely 2,3,6-tri-O-methyl-β-CD, heptamethylamino-β-CD and vancomycin. When the CDs were used the chiral selector was present in both capillary and electrode compartments while for vancomycin the partial filling method was used (the chiral selector was not present at the detector window in order to improve the sensitivity; vancomycin is strongly absorbing at low wavelengths). Enantiomeric resolution was recorded for all the compounds studied except for ibuprofen when heptamethylamino-β-CD was used; resolution generally increased by increasing the chiral selector concentration. Good sensitivity and good precision for both migration times and corrected peak areas (Asample/migration time) were achieved using the three chiral selectors. Among the three chiral additives employed vancomycin proved to be the most effective for the enantiomeric separation of the studied arylpropionic acids. The optimized method achieved analysis with the shortest time (

Enantiomeric separation of fluoxetine and norfluoxetine in plasma and serum samples with high detection sensitivity capillary electrophoresis.

A capillary electrophoresis method was optimized for the stereoselective analysis of the antidepressant drug fluoxetine and its main demethylated metabolite norfluoxetine using a cyclodextrin‐modified sodium phosphate buffer at pH 2.5. The combination of a neutral and a negatively charged cyclodextrin, dimethylated‐β‐ and phosphated‐γ‐respectively, provided the baseline enantiomeric separation of the two compounds. The very low concentrations of chiral selectors employed together with the use of a high sensitivity detection cell of special design (zeta‐shaped) in a diode array UV detector allowed us to reach a limit of detection of 0.005 and 0.01 μg/mL for fluoxetine and norfluoxetine, respectively. Analysis of fluoxetine and norfluoxetine standard mixtures showed a reproducibility of migration times and peak area and linearity in the concentration range of 0.1—2.0 μg/mL. The optimized method was applied to the analysis of clinical serum and plasma samples of patients under depression therapy. In all the analyzed samples the enantiomeric forms of fluoxetine and norfluoxetine were easily identified. The fluoxetine and metabolite enantiomeric ratio confirmed the stereoselectivity of the metabolic process of the fluoxetine drug in accordance with the literature data.

The Surprising Composition of the Salivary Proteome of Preterm Human Newborn

Saliva is a body fluid of a unique composition devoted to protect the mouth cavity and the digestive tract. Our high performance liquid chromatography (HPLC)-electrospray ionization-MS analysis of the acidic soluble fraction of saliva from preterm human newborn surprisingly revealed more than 40 protein masses often undetected in adult saliva. We were able to identify the following proteins: stefin A and stefin B, S100A7 (two isoforms), S100A8, S100A9 (four isoforms), S100A11, S100A12, small proline-rich protein 3 (two isoforms), lysozyme C, thymosins β4 and β10, antileukoproteinase, histone H1c, and α and γ globins. The average mass value reported in international data banks was often incongruent with our experimental results mostly because of post-translational modifications of the proteins, e.g. acetylation of the N-terminal residue. A quantitative label-free MS analysis showed protein levels altered in relation to the postconceptional age and suggested coordinate and hierarchical functions for these proteins during development. In summary, this study shows for the first time that analysis of these proteins in saliva of preterm newborns might represent a noninvasive way to obtain precious information of the molecular mechanisms of development of human fetal oral structures.

Simultaneous stereoselective analysis by capillary electrophoresis of tramadol enantiomers and their main phase I metabolites in urine

Capillary zone electrophoresis was successfully applied to the enantiomeric resolution of racemic tramadol and its six phase I metabolites using carboxymethylated beta-cyclodextrin (CMB) added to the background electrolyte (BGE). Baseline resolution of tramadol and its metabolites was obtained in less than 30 min using a 50 mM phosphate buffer (pH 2.5) containing 5 mM of CMB. Chiral determinations of tramadol and its main three metabolites, O-demethyltramadol (M1), N-demethyltramadol (M2) and O-demethyl-N-demethyltramadol (M5), were performed in urine after a simple double liquid-liquid extraction of 200 microliters of biological material. In the tested concentration range (0.5-20 micrograms/ml, except for M2: 0.5-10 micrograms/ml) coefficients of correlation superior than 0.994 were obtained. Within-day variation determined on three different concentrations for each enantiomers showed accuracies ranging from 95.4% to 103.2%. The relative standard deviation (RSD) of these assays was determined to be less than 10.0%. Day-to-day variation presented accuracies ranging from 96.3% to 106.5% with a RSD less than 9.0%. After oral administration of 100 mg of tramadol hydrochloride to an healthy volunteer, the urinary excretion was monitored during 30 h. About 15% of the dose was excreted as unchanged tramadol. The enantiomeric ratios of all the excreted analytes, T, M1, M2 and M5, were found to be very different to 1.0, showing that a stereoselective metabolism of tramadol clearly occurred.