Biomedical subjects

S Rudaz

Publications and source records attributed to S Rudaz.

At least 19 recordsLinked to original sources

PubMed2006

[Comparison of three approaches for uncertainty estimation].

Three different approaches for the estimation of uncertainty measurements using the same analytical method were compared, namely validation, robustness and inter-laboratory studies. The uncertainty obtained with the robustness study! predicted well the uncertainty of the inter-laboratory study. On the other hand, the uncertainty estimation obtained with the validation study is lower than those obtained with the two other approaches but is still acceptable as long as the analytical method will be used in a single laboratory.

Chromatography, Liquid↗

PubMed2005

Development and validation of a new reversed-phase ion pairing liquid chromatographic method with fluorescence detection for penciclovir analysis in plasma and aqueous humor.

A simple, sensitive and reliable HPLC ion-pairing method with fluorescence detection, was developed for penciclovir determination in plasma and aqueous humor, with a Zorbax SB-aq C18 (100 mmx2.1 mm) column. Plasma samples were treated by solid-phase extraction with Oasis MCX (30 mg) cartridges. Ganciclovir, an antiviral drug structurally related to penciclovir, was used as internal standard (I.S.). Aqueous humor samples were directly injected into the chromatographic system. Separation was performed by a gradient elution with a mobile phase consisting of a mixture of acetonitrile and phosphate buffer 50mM containing 5mM of sodium octanesulfonate, pH 2.0, at a flow rate of 0.3 ml/min. The method was validated and showed good performances in terms of linearity, sensitivity, precision and trueness. Quantification limit was obtained at 0.05 microg/ml for aqueous humor and at 0.1 microg/ml for plasma. Finally, the proposed analytical method was used to measure penciclovir in clinical samples for a pharmacokinetic study, after oral administration of famciclovir.

Acyclovir↗

PubMed2005

Conversion of cyclosporine A prodrugs in human tears vs rabbits tears.

The aim of this study was to evaluate the rate and mechanism of conversion of two water-soluble prodrugs of cyclosporine A (CsA) intended for topical delivery to the eye. The new molecules were designed according to the double prodrug concept: a solubilizing moiety was grafted onto CsA via an ester function, which could be hydrolysed via a two-step process (enzymatic and chemical). Prodrug solutions were prepared extemporaneously in an isotonic and neutral aqueous medium compatible with ophthalmic use. The rates of conversion into the parent molecule were determined by incubating the prodrugs in fresh rabbit or human tears or in a phosphate buffer solution (PBS) at pH 7.4. Both prodrugs were converted into CsA within the first minute in the presence of rabbit tears with rate constants of k=5.9x10(-3)min(-1) and k=3.8x10(-3)min(-1), respectively, for UNIL088 and UNIL089, whereas chemical conversion in PBS was negligible (k=0.5x10(-3)min(-1) for both molecules). Incubation of UNIL088 in human tears showed a significantly high conversion rate. It is concluded that the developed double prodrugs underwent a bioconversion in physiological media and thus represent promising candidates for topical delivery of CsA to the eye.

Animals↗

PubMed2004

Development of a bioreactor based on trypsin immobilized on monolithic support for the on-line digestion and identification of proteins.

The preparation and characterization of a new trypsin-based bioreactor is here described for on-line protein digestion and peptide analysis. Trypsin was immobilized on an epoxy-modified silica monolithic support with a single reaction step and the amount of immobilized enzyme was found to be 66.07 mg (+/-11.75 S.D.)/column (n = 6). The bioreactor was coupled through a switching valve to an analytical column for the on-line digestion, peptide separation and identification of test proteins by ESI-MS-MS. The influence of various parameters (flow rate, temperature, buffer pH and molarity, etc.) on enzymatic activity was investigated by an experimental design and the mostly significant factor was found to be the flow rate. The efficacy of the reported on-line bioreactor for tryptic mapping is reported for somatostatin and myoglobin, selected as model compounds. Tryptic peptide maps obtained by on-line digestion of myoglobin were compared to those obtained by traditional off-line digestion. Sequence coverage obtained with the on-line protocol (21 peptides, 75.16% coverage of myoglobin sequence) was found to be comparable to the one obtained with the off-line protocol (18 peptides, 76.47% coverage). Sensitivity for myoglobin digestion and identification was 0.1 mg/ml. The reproducibily of the peptide maps in terms of retention time was from 1.53 to 4.31%, R.S.D.

Amino Acid Sequence↗

PubMed2004

Protein precipitation for the analysis of a drug cocktail in plasma by LC-ESI-MS.

Three protein precipitation (PP) procedures with acetonitrile (ACN), perchloric acid (PA) and trichloroacetic acid (TCA) were investigated for the analysis of a drug cocktail from human plasma samples containing three pharmaceutical compounds and their primary metabolites. For this purpose, a capillary liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) method was developed for the simultaneous analysis of the six tested compounds in less than 6 min. Matrix effect was tested for each PP procedure by means of a post-column infusion system. The three PP techniques were found effective in removing proteins from human plasma and were fully compatible with capillary LC-ESI-MS analysis. However, with acid precipitations, low analyte recovery and a high variability, probably due to analyte coprecipitation, were obtained. Finally, ACN was found to be the most effective PP technique with a recovery higher than 80% and CV inferior to 6%.

Blood Proteins↗

PubMed2004

Restricted access materials and large particle supports for on-line sample preparation: an attractive approach for biological fluids analysis.

An analytical process generally involves four main steps: (1) sample preparation; (2) analytical separation; (3) detection; and (4) data handling. In the bioanalytical field, sample preparation is often considered as the time-limiting step. Indeed, the extraction techniques commonly used for biological matrices such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE) are achieved in the off-line mode. In order to perform a high throughput analysis, efforts have been engaged in developing a faster sample purification process. Among different strategies, the introduction of special extraction sorbents, such as the restricted access media (RAM) and large particle supports (LPS), allowing the direct and repetitive injection of complex biological matrices, represents a very attractive approach. Integrated in a liquid chromatography (LC) system, these extraction supports lead to the automation, simplification and speeding up of the sample preparation process. In this paper, RAM and LPS are reviewed and particular attention is given to commercially available supports. Applications of these extraction supports, are presented in single column and column-switching configurations, for the direct analysis of compounds in various biological fluids.

Animals↗

PubMed2003

Use of large particles support for fast analysis of methadone and its primary metabolite in human plasma by liquid chromatography-mass spectrometry.

A bioanalytical method was developed for the quantitation of methadone (MTD) and its primary metabolite, (EDDP) in plasma. The extraction step was performed within a capillary column packed with large particles (35x0.3 mm I.D.; d(p) 30 micrometer) at high flow-rate conditions (450 microliter/min). The separation was performed on a microbore analytical column (55x2 mm I.D.; d(p) 3 micrometer) coupled to a mass spectrometer (MS). This procedure was based on a column-switching unit. Analytes of interest were retained on the precolumn by hydrophobic interactions and backflushed from the precolumn to the analytical column. The detection was carried out with a MS single quadrupole equipped with an electrospray interface. The total analysis time was 6 min. The limits of quantification were evaluated at 10 and 25 ng/ml for MTD and EDDP, respectively. At this level, good accuracies were obtained for both analytes with repeatability values less than 18%.

Chromatography, Liquid↗

PubMed2001

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.

Antidepressive Agents, Second-Generation↗

PubMed2001

Nonaqueous capillary electrophoresis-mass spectrometry for separation of venlafaxine and its phase I metabolites.

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.

Antidepressive Agents, Second-Generation↗

PubMed2001

On-line capillary electrophoresis-electrospray mass spectrometry for the stereoselective analysis of drugs and metabolites.

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.

Amphetamines↗

PubMed2001

Experimental designs to investigate capillary electrophoresis-electrospray ionization-mass spectrometry enantioseparation with the partial-filling technique.

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.

Buffers↗

PubMed2001

A new poly(ortho ester)-based drug delivery system as an adjunct treatment in filtering surgery.

PURPOSE: Pharmacologic modulation of wound healing after glaucoma filtering surgery remains a major clinical challenge in ophthalmology. Poly(ortho ester) (POE) is a bioerodible and biocompatible viscous polymer potentially useful as a sustained drug delivery system that allows the frequency of intraocular injections to be reduced. The purpose of this study was to determine the efficacy of POE containing a precise amount of 5-fluorouracil (5-FU) in an experimental model of filtering surgery in the rabbit. METHODS: Trabeculectomy was performed in pigmented rabbit eyes. An ointmentlike formulation of POE containing 1% wt/wt 5-FU was injected subconjunctivally at the site of surgery, during the procedure. Intraocular pressure (IOP), bleb persistence, and ocular inflammatory reaction were monitored until postoperative day 30. Quantitative analysis of 5-FU was performed in the anterior chamber. Histologic analysis was used to assess the appearance of the filtering fistula and the polymer's biocompatibility. RESULTS: The decrease in IOP from baseline and the persistence of the filtering bleb were significantly more marked in the 5-FU-treated eyes during postoperative days 9 through 28. Corneal toxicity triggered by 5-FU was significantly lower in the group that received 5-FU in POE compared with a 5-FU tamponade. Histopathologic evaluation showed that POE was well tolerated, and no fibrosis occurred in eyes treated with POE containing 5-FU. CONCLUSIONS: In this rabbit model of trabeculectomy, the formulation based on POE and containing a precise amount of 5-FU reduced IOP and prolonged bleb persistence in a way similar to the conventional method of a 5-FU tamponade, while significantly reducing 5-FU toxicity.

Animals↗

PubMed2000

Simultaneous stereoselective analysis of venlafaxine and O-desmethylvenlafaxine enantiomers in clinical samples by capillary electrophoresis using charged cyclodextrins.

Capillary electrophoresis (CE) was used for the simultaneous chiral determination of venlafaxine (Vx), a new antidepressant drug and its main active metabolite. O-desmethyl venlafaxine (ODV). Among the charged cyclodextrins (CD) tested, phosphated gamma-CD was the most appropriate. Resolution of Vx and ODV was obtained with 50 mM phosphate buffer (pH 2.5) containing 20 mg/ml of phosphated gamma-CD. After optimisation of the method (including robustness), validation was carried out. Vx and ODV concentrations, as well as the enantiomeric ratio, were investigated in clinical samples. Chiral determination of Vx and ODV was performed after a simple liquid-liquid extraction (LLE). In the tested concentration range (25-500 ng/ml), coefficients of correlation were superior to 0.996. Within-day and between-day accuracy and precision were determined at three different concentrations for each enantiomer. Analyses of clinical samples (n = 16) exhibited non-racemic ratios for Vx and ODV, which suggests a stereoselective metabolism in humans.

Antidepressive Agents, Second-Generation↗

PubMed2000

Enantioselective analysis of methadone in saliva by liquid chromatography-mass spectrometry.

Saliva was tested and evaluated as a biological matrix for methadone (Mtd) monitoring. Conventional method using a narrow bore C18 column, and an enantioselective method using a narrow bore alpha1-acid glycoprotein column, were developed using liquid chromatography coupled with a mass spectromeric (MS) detector. After optimisation of MS conditions by flow injection analysis, selected ion monitoring detection was used to enhance sensitivity. The total Mtd concentration and the enantiomeric ratio in saliva were validated using an experimental design. The methods were applied to samples provided by heroin addicts undergoing a Mtd treatment. Results on total Mtd determination showed a very poor correlation between saliva and serum, whereas the enantiomeric ratios of Mtd gave a very good one.

Chromatography, Liquid↗

PubMed1999

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.

Analgesics, Opioid↗

PubMed1999

Chiral stationary phases in HPLC for the stereoselective determination of methadone.

An extensive study of the behavior of three chiral stationary phases (CSP) is presented for the stereoselective determination of methadone. The following chromatographic columns were selected: a cellulose, Chiralcel OJ; a modified cyclodextrin. Cyclobond I 2000 RSP, and a protein, Chiral-AGP. Retention factors, enantioselectivity, efficiency, and resolution were tested by modifying the composition of the mobile phase as well as the temperature. The mechanism for the chiral recognition of methadone on each support was discussed. Optimal chromatographic parameters were obtained for the three supports tested, and methadone enantiomers were separated in less than 20 minutes. The cellulose-based column gave the best resolution, but this CSP was not adapted to clinical analyses of methadone. Under optimized conditions, the cyclodextrin- and protein-based columns allowed an excellent separation of methadone enantiomers, but no interference with the primary metabolite was found only with Chiral-AGP.

Chromatography, High Pressure Liquid↗

PubMed1999

Development of validated stereoselective methods for methadone determination in clinical samples.

A stereoselective analysis of methadone (Mtd) in whole blood and serum was developed using liquid chromatography on a protein based chiral stationary phase. Liquid-liquid extraction (LLE) and solid phase extraction methods were applied before chromatographic analysis. The extraction procedure, as well as the choice of the biological matrix, showed significant differences in the extraction yield and in the precision of the assays. Serum was selected for this assay and LLE was chosen as the preparation step because of its simplicity and rapidity. The total procedure was validated and applied to clinical samples. Samples taken from 45 heroin-addicted patients were analyzed. A correlation was found between the dose administered and Mtd concentration (total and R-form), but interindividual variability of the total normalized Mtd was seen (concentration varied from 90 to 530 ng/ml). Furthermore, two populations were apparently observed with a mean Mtd concentration of 200 and 475 ng/ml, respectively. Stereoselective analyses showed that more than 50% of the patients presented a nonracemic ratio, and particularly about 25% showed a preferential metabolism of the active R-Mtd enantiomer. Therefore, the stereoselective determination of Mtd is necessary to improve the quality of the treatment of heroin addiction.

Chromatography, Liquid↗

PubMed1999

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-beta- and phosphated-gamma-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 microg/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 microg/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.

Antidepressive Agents, Second-Generation↗