Synthesis and study of a molecularly imprinted polymer for specific solid-phase extraction of vinflunine and its metabolite from biological fluids.

A molecularly imprinted polymer (MIP) was synthesized in order to specifically extract vinflunine, an anticancer agent, and its metabolite (4-O-deacetylvinflunine) from bovine plasma and artificial urine by solid-phase extraction (SPE). Vinorelbine, a non-fluorinated analogue of vinflunine, was selected as a template for MIP synthesis. The selectivity of MIP versus the template (vinorelbine) and other alkaloids (catharanthine, vinblastine, vincristine, vinflunine and 4-O-deacetylvinflunine) was shown by a SPE protocol carried out with non-aqueous samples. A second protocol was developed for aqueous samples with two consecutive washing steps (AcOH-NH2 OH buffer (pH 7, I=10 mM)-MeOH mixture 95:5 v/v and ACN-AcOH mixture 99:1 v/v) and an elution step (MeOH-AcOH mixture 90:10 v/v). Thus, MIP-SPE of bovine plasma brought high recoveries, 81 and 89% for vinflunine and its metabolite, respectively. This protocol was slightly modified for artificial urine samples in order to obtain a good MIP/NIP selectivity; furthermore, elution recoveries were 73 and 81% for vinflunine and its metabolite, respectively. Repeatability was assessed in both biological matrices and RSD (%) were inferior to 4%. The MIP also showed a suitable linearity (r(2) superior to 0.99), between 0.25 and 10 µg/mL for plasma, and between 1 and 5 µg/mL for artificial urine.

Chiral analysis of milnacipran by a nonchiral HPLC--circular dichroism: improvement of the linearity of dichroic response by temperature control.

The determination of the enantiomeric excess (e.e.) of a basic drug has been investigated in LC using a nonchiral stationary phase and a circular dichroism (CD) detector in order to avoid expensive chiral columns. The CD detector records both dichroic (Deltaepsilon) and UV (epsilon) signals at the same wavelength and calculates the anisotropy factor (g=Deltaepsilon/epsilon), which is linearly related to the e.e. The enantiomeric and chemical composition of a chiral drug can be simultaneously determined on a nonchiral HPLC support. However, the g factor from the CD signal is temperature dependent. Indeed, the temperature has an influence on the stability of the CD signal and the linear regression between g factor and the e.e. of 1R,2S-enantiomer. So, a decrease in temperature gives rise to an improvement of the above-mentioned linearity correlation. After optimization of chromatographic parameters (porous graphitic carbon-based column, methanol/ phosphate buffer as mobile phase) and selection of CD wavelength, a linear regression of g factor versus e.e. of 1R,2S-enantiomer was obtained at temperature-controlled CD detection and an LOQ of 94% was found. The enantiomeric composition of milnacipran was determined with good accuracy.

Validation of a method using an achiral liquid chromatography sorbent and a circular dichroism detector. Analysis of the efaroxan enantiomers.

The known HPLC method using an achiral C8 silica sorbent and a circular dichroism (CD) detector for the determination of efaroxan enantiomeric excess has been validated. After optimization of the mobile phase, the enantiomers were detected at 278 nm offering maximum ellipticity between two optically active forms. The calibration curve of the anisotropy factor (g) versus the enantiomeric excess was linear with a correlation coefficient (r2) of 0.9985. The accuracy of the method was assessed by comparing the enantiomeric excess obtained by measuring the g factor (C8 column, CD and UV detections) with those determined by enantioselective HPLC (Chiralpak AD-H column, UV detection). Statistical tests (level of confidence of 95%) were assessed to compare the two orthogonal methods. The straight line gave a correlation coefficient of 0.9995, an intercept not significantly different from zero (0.0549) and a slope of 1.026. The precision evaluated on retention time (RSD<0.6%), g factor (RSD<8.3%) and CD peak area (RSD<7.5%) was suitable both in term of intra- and inter-day precisions. The proposed method has the advantages of being fast and precise without using expensive chiral column. Non-enantioselective HPLC-CD was suitable for the simultaneous determination of the optical and chemical purity of efaroxan.

HPLC quantitation of the four stereoisomers of benzoxathiepin derivatives with cellulose phenyl type chiral stationary phase and circular dichroism detection.

The chiral separation of a new antianginal agent has been investigated on a chiral cellulose column with UV and circular dichroism (CD) detection. This benzoxathiepin derivative under development has two stereogenic centers whose (R,S) stereoisomer shows an interesting antianginal activity. After optimisation of the mobile phase composition, a baseline-resolved separation of the four stereoisomers was achieved on a Chiralcel OJ-H chiral column by using methanol-ethanol-diethylamine (25:75:0.1, v/v/v) as mobile phase. The CD detection system allowed quantitation and a linear response was observed within a 10-200 microg mL-1 concentration range (r2=0.9966) and limit of quantification down to 2 microg mL-1 was achieved.

Spectrofluorimetric study of eflucimibe-gamma-cyclodextrin inclusion complex.

Eflucimibe, a novel and highly potent acyl-coenzyme A cholesterol O-acyl-transferase (ACAT) inhibitor, is sparingly soluble in aqueous media and exhibits a very weak natural fluorescence. However, when increasing concentrations of gamma-cyclodextrin (gamma-CD) are added, an increase in the fluorescence signal is observed, attesting the formation of a non-covalent inclusion complex between eflucimibe and the gamma-CD. In this work, the stoichiometry of the complex and the corresponding association constant have been determined from fluorescence data by Benesi-Hildebrand's method (double reciprocal plots). As a result, a 1:1 stoichiometric ratio and a 20 M(-1) formation constant were obtained. This apparent formation constant was determined in water containing 10% methanol, which was needed to improve 'aqueous' solubility of the drug in a CD-free medium. Owing to the extreme hydrophobicity of eflucimibe, these results provide valuable information for pharmaceutical formulation studies.

Optimization of the separation of Vinca alkaloids by nonaqueous capillary electrophoresis.

A rapid method for the determination of Vinca alkaloids by nonaqueous capillary electrophoresis with diode array detection has been developed. A group of 11 alkaloids (catharanthine, vinorelbine, anhydrovinblastine, vinflunine, vindoline, 4-O-deacetylvinorelbine, 4-O-deacetylvinflunine, vindesine, vinblastine, 4'-deoxy-20',20'-difluorovinblastine, vincristine) could be readily separated within 10 min. The compounds were separated using a capillary of 38 cm effective length, a running buffer composed of 50 mM ammonium acetate and 0.6 M acetic acid in a methanol-acetonitrile (75:25, v/v) mixture. A constant voltage of 25 kV with a ramp time of 1 min and a 344.7 x 10(3) Pa pressure, applied simultaneously to inlet and outlet buffer vials, were used during sample analysis. Five of these alkaloids were selected for optimization of the separation and for validation studies with respect to specificity, linearity, range, limits of quantification and detection and then accuracy. The feasibility of the assay was demonstrated by analyzing a commercial sample of vinorelbine (Navelbine, ampoule at 10 mg/ml of vinorelbine base). The results were compared with a high-performance liquid chromatography method.

Quantification of very low enantiomeric impurity of efaroxan using a dual cyclodextrin system by capillary electrophoresis.

A rapid method for the enantiomeric purity determination of efaroxan has been developed by capillary electrophoresis (CE) using a dual cyclodextrin (CD) system. The influence of the nature and the concentration of CDs on separation parameters has been studied. High resolution (R(s)=7) and peak efficiency (104,000-121,000 theoretical plates) values were obtained for efaroxan enantiomers by adding two cyclodextrins, one neutral (7.5 mM DM-beta-CD) and the other negatively charged (3 mM CM-beta-CD) to the running buffer composed of 100 mM phosphoric acid-triethanolamine (pH 3). These resolution and peak efficiencies values allowed the quantitation of the (S)-enantiomer of efaroxan at very low enantiomeric excess even if the minor component migrates after the major one. This method was fully validated for the enantiomeric impurity determination of the (S)-form of efaroxan at the 0.05% level. Calibration curve, expressed by the peak areas ratio versus the enantiomeric purity was linear over the 0.05-1% enantiomeric impurity range (r2=0.9996). Limits of detection (LOD) and quantification (LOQ), expressed in term of (S)-enantiomer impurity were 0.02% and 0.05%, respectively. The accuracy of the method at 0.12%, 0.50% and 0.80% enantiomeric impurity levels for the (S)-form were determined. Recoveries were in 94-102% range for each quality control sample and were determined with good precision (intra-day R.S.D.=3.54%, inter-day R.S.D.=5.33%).

Sensitivity improvement of circular dichroism detection in HPLC by using a low-pass electronic noise filter: Application to the enantiomeric determination purity of a basic drug.

The quality control of chiral drugs requires the determination of their enantiomeric purity. Nowadays, circular dichroism (CD) spectroscopy is gaining increasing importance in pharmaceutical analysis because of the commercially available CD detector in liquid chromatography. The separation of the two enantiomers of a basic drug (efaroxan) was achieved by high performance liquid chromatography using an amylose-derivated column with both UV and CD detections. A baseline-resolved separation (resolution: 5) was obtained after optimization of the mobile phase composition with hexane-ethanol-diethylamine (90:10:0.05; v/v/v). The use of a commercial low-pass electronic noise filter of the CD signal has improved the signal-to-noise ratio by a factor twelve and allowed the quantitation of each enantiomer in the 1.25-300 microg ml(-1) concentration range. The CD linear calibration curve, expressed in terms of stereoisomer height ratio versus concentration ratio, was plotted over the 0.4-6% range. A correlation coefficient greater than 0.999 was obtained by least-squares regression and the limit of detection for the distomer/eutomer ratio was estimated at 0.14%. Although the method validation showed good repeatability on the retention times (RSD < 0.9%), on the peak height ratios (RSD < 8.7%) of each enantiomer only up to 99.2% enantiomeric purity was achieved.

Development of a method for simultaneous determination of eflucimibe and its three major metabolites in rat plasma by liquid chromatography/electrospray tandem mass spectrometry: a preliminary study.

A high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry (HPLC/ESI-MS/MS) method has been developed for the simultaneous determination of eflucimibe, a powerful acyl-coenzyme A cholesterol O-acyltransferase (ACAT) inhibitor, and its main metabolites, in plasma. The ESI and MS/MS parameters were investigated and optimised for each of the four compounds in the positive ion mode. Plasma samples were deproteinised by precipitation with acetonitrile and directly analysed by HPLC/ESI-MS/MS in less than 4 min. Quantitation was performed in the multiple reaction monitoring (MRM) mode for highest sensitivity, selecting the protonated molecules [M+H](+) as precursor ions. The method was demonstrated to be specific and sensitive, and a linear response was observed within a 1-25 ng/mL concentration range. Correlation coefficients (r(2)) greater than 0.9960 were obtained by least-squares regression, and limits of detection down to 0.2 ng/mL were calculated. Therefore, this HPLC/ESI-MS/MS method appears to be an efficient tool, able to provide valuable information for a pharmacological purpose.

Chiral separation of the four stereoisomers of a novel antianginal agent using a dual cyclodextrin system in capillary electrophoresis.

Reported here is an analytical method enabling the stereochemical resolution of a new antianginal compound possessing two stereogenic centers, leading to four stereoisomers. Only one of these isomers is currently under development as a novel antianginal agent and consequently, the other three isomers are considered as unwanted chiral impurities. Therefore, an enantioselective method is required in order to check its enantiomeric purity. This paper presents a method exploiting the high efficiency of capillary electrophoresis and the complexing properties of cyclodextrins to achieve the separation of the four stereoisomers of this weakly basic compound (pKa = 7.4). For this purpose, the combination of a neutral cyclodextrin, hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD), and an anionic cyclodextrin, carboxymethyl-beta-cyclodextrin (CM-beta-CD), was added to the separation buffer running in an uncoated silica capillary. After selection of the suitable cyclodextrin system, satisfactorily separation conditions were as follows: 30 mM phosphate buffer (pH 6.4) containing 10 mM of HP-gamma-CD and 10 mM of CM-beta-CD, running voltage +30 kV. The resulting run time and resolutions were respectively about 17 min and between 1.95 and 2.84. Linearity curves (0.993 < r2 < 0.998) are also shown.

Dual, hyperalgesic, and analgesic effects of the high-efficacy 5-hydroxytryptamine 1A (5-HT1A) agonist F 13640 [(3-chloro-4-fluoro-phenyl)-[4-fluoro-4-{[(5-methyl-pyridin-2-ylmethyl)-amino]-methyl}piperidin-1-yl]methanone, fumaric acid salt]: relationship with 5-HT1A receptor occupancy and kinetic parameters.

The aim of the present study was to establish the relationship between the plasma and brain concentration-time profiles of F 13640 [(3-chloro-4-fluoro-phenyl)-[4-fluoro-4-{[(5-methyl-pyridin-2-ylmethyl)-amino]-methyl}piperidin-1-yl]methanone, fumaric acid salt] after acute administration and both its hyper- and hypoanalgesic effects in rats. The maximal plasma concentration (C(max)) of F 13640 after i.p. administration of 0.63 mg/kg was obtained at 15 min and decreased to half its maximal value after about 1 h. The amount of F 13640 collected by means of in vivo microdialysis in hippocampal dialysates could be measured reliably after 0.63 and 2.5 mg/kg, reached its maximum at about 1 h, and fell to half of its maximal value at about 3 h. 5-Hydroxytryptamine 1A (5-HT(1A)) receptor occupancy was estimated by ex vivo binding in rat brain sections. F 13640 inhibited [(3)H]8-hydroxy-2-[di-n-propylamino] tetralin binding ex vivo in rat hippocampus, entorhinal cortex, and frontal cortex (ED(50), 0.34 mg/kg i.p.). Maximal inhibition was reached at approximately 30 min after 0.63 mg/kg F 13640 and fell to half of its value after about 4 to 8 h. After injection (15 min) in the paw pressure test, F 13640 (0.63 mg/kg i.p.) induced an initial hyperalgesia that was followed 4 h later by a paradoxical analgesia that lasted until 8 h. In contrast, in the formalin test, F 13640 inhibited pain behaviors until 4 h after drug administration. F 13640 also produced elements of the 5-HT syndrome that lasted up to 4 h after administration. These results demonstrate that F 13640 induces hyperalgesia and/or analgesia with a time course that parallels the occupancy of 5-HT(1A) receptors and the presence of the compound in blood and brain.

Application of capillary electrophoresis with field-amplified sample injection for the detection of new adrenoreceptor antagonist enantiomers in plasma in the low ng/mL concentration range.

Throughout the separation of chiral basic drugs by capillary electrophoresis (CE) with neutral hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as chiral selector, the sensitivity of detection has been improved by using field-amplified sample injection (FASI). In the present work, this on-line stacking method has been used to detect low ng/mL levels of cationic enantiomers of a new adrenoreceptor antagonist in plasma. A systematic study of the parameters affecting on-line concentration of these enantiomers (nature of the preinjection plug, composition of sample solvent, injection times of water and sample plugs) has been performed enabling the detection sensitivity of antagonist enantiomers to be improved by 180 times compared with usual hydrodynamic injection. The quantification of each adrenoreceptor antagonist enantiomer in plasma samples was then performed in the 2-100 ng/mL (or 8-400 nM) concentration range after a solid-phase extraction step. Using this FASI-CE-UV procedure, the limit of quantification (LOQ) for each enantiomer was in the low ng/mL concentration range (3 ng/mL or 10 nM).