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P Chiap

Publications and source records attributed to P Chiap.

36 records · Page 2Linked to original sources
PubMed2002

Automated determination of pirlindole enantiomers in plasma by on-line coupling of a pre-column packed with restricted access material to a chiral liquid chromatographic column.

A fully automated liquid chromatographic method has been developed for the determination of the enantiomers of pirlindole, an antidepressant drug, in human plasma. The method is based on the use of a pre-column packed with restricted access material (RAM) (LiChrospher ADS RP-4) for sample clean-up coupled to a column containing a cellulose tris-(3,5-dimethylphenylcarbamate) based chiral stationary phase (Chiralcel OD-R) for the separation and quantitative analysis of pirlindole enantiomers. A 50-microl plasma volume was injected directly onto the pre-column using a mixture of phosphate buffer (pH 5.0) and methanol (97:3; v/v) as washing liquid. By rotation of a switching valve, the analytes were then eluted in the back-flush mode with the LC mobile phase. A complete separation of pirlindole enantiomers was obtained in 22 min on the Chiralcel OD-R column, using a mobile phase made of a mixture of phosphate buffer (pH 5.0) containing 50 mM sodium perchlorate and acetonitrile (65:35; v/v). The flow-rate was 0.6 ml/min and the analytes were detected fluorometrically using 295 and 340 nm as excitation and emission wavelengths, respectively. The method was then validated and was found to be linear in the 2.5-200 ng/ml range. The limit of detection was lower than 1 ng/ml. Repeatability and intermediate precision at a concentration of 50 ng/ml were about 1.5 and 3.5%, respectively.

Antidepressive Agents↗
PubMed2001

On-line coupling of partial filling-capillary zone electrophoresis with mass spectrometry for the separation of clenbuterol enantiomers.

The on-line coupling of capillary zone electrophoresis with mass spectrometry (CZE-MS) for the separation of enantiomers is hampered by the presence of nonvolatile chiral selectors such as cyclodextrins in the separation buffer. This problem can be overcome by use of the partial filling technique where only a part of the capillary is filled with the separation buffer containing chiral selectors. Since the electroosmotic flow is almost completely suppressed at acidic pH, that dimethyl-beta-cyclodextrin is neutral, no free cyclodextrin would reach the MS detector when using a partially filled capillary. By this method, clenbuterol enantiomers were successfully resolved and separated from salbutamol (internal standard) in aqueous solution and in plasma samples. A solid-phase extraction (SPE) was used for the preparation of plasma samples before analysis.

Clenbuterol↗
PubMed2001

Development and validation of an automated method for the liquid chromatographic determination of sotalol in plasma using dialysis and trace enrichment on a cation-exchange pre-column as on-line sample preparation.

A fully automated method for the determination of sotalol in human plasma was developed, involving dialysis through a cellulose acetate membrane, clean-up and enrichment of the dialysate on a strong cation-exchange pre-column and subsequent liquid chromatographic (LC) analysis with UV detection. All sample handling operations were carried out by means of an ASTED system. Before starting dialysis, the trace enrichment column (TEC) was conditioned. The plasma sample, to which the internal standard (atenolol) was automatically added, was then loaded in the donor channel and was kept static while the dialysis liquid, consisting of 0.017 M acetic acid, was passed through the acceptor channel in successive pulses. After each pulse, the dialysate was dispensed onto the TEC. When dialysis was discontinued, the analytes were eluted from the TEC by the LC mobile phase by rotation of a switching valve and transferred to the analytical column packed with octyl silica. The LC mobile phase was a mixture of methanol and pH 7.0 phosphate buffer containing 1-octanesulfonate at a concentration of 7.5 x 10(-4) M (19:81; v/v). The UV detection was performed at 230 nm. The influence of several parameters of the dialysis and trace enrichment processes on analyte recovery and method selectivity was investigated. The method was then validated. The mean absolute recovery for sotalol was about 60%. The limit of quantitation was 25 ng/ml and R.S.D. for repeatability and intermediate precision obtained at a concentration level of 50 ng/ml were 4.3 and 5.8%, respectively.

Automation↗
PubMed2000

Automated liquid chromatographic determination of atenolol in plasma using dialysis and trace enrichment on a cation-exchange precolumn for sample handling.

A fully automated method involving dialysis combined with trace enrichment was developed for the liquid chromatographic (LC) determination of atenolol, a hydrophilic beta-blocking agent, in human plasma. The plasma samples were dialysed on a cellulose acetate membrane and the dialysate was reconcentrated on a short trace enrichment column (TEC) packed with a strong cation-exchange material. All sample handling operations can be executed automatically by a sample processor (ASTED system). After TEC conditioning, the plasma sample, to which the internal standard (sotalol, another hydrophilic beta-blocker) was automatically added, was introduced in the donor channel and dialysed in the static/pulsed mode. The dialysis liquid consisted of 4.3 mM phosphoric acid. When the dialysis process was discontinued, the analytes were eluted from the TEC in the back-flush mode by the LC mobile phase and transferred to the analytical column, packed with octyl silica. The LC mobile phase consisted of phosphate buffer, pH 7.0-methanol (81:19; v/v) with 1-octanesulfonate. Atenolol and the internal standard were monitored photometrically at 225 nm. The different parameters influencing the dialysis and trace enrichment processes were optimised with respect to analyte recovery. The influence of two different kinds of cation-exchange material on analyte recovery and peak efficiency was also studied. The method was then validated in the concentration range 25-1000 ng/ml. The mean recovery for atenolol was 65% and the limit of quantitation was 25 ng/ml.

Adrenergic beta-Antagonists↗
PubMed2000

Determination of albendazole and its main metabolites in ovine plasma by liquid chromatography with dialysis as an integrated sample preparation technique.

Albendazole is a benzimidazole derivative with a broad-spectrum activity against human and animal helminth parasites. In order to determine the main pharmacokinetic parameters in sheep after oral and intravenous administration of a new formulation of albendazole (an aqueous solution), a fully automated method was developed for the determination of this drug and its main metabolites, albendazole sulfoxide (active metabolite) and sulfone in ovine plasma. This method involves dialysis as purification step, followed by enrichment of the dialysate on a precolumn and liquid chromatography (LC). All sample handling operations were executed automatically by means of an ASTED XL system. After conditioning of the trace enrichment column (TEC) packed with octadecyl silica with pH 6.0 phosphate buffer containing sodium azide, the plasma sample, in which a protein releasing reagent (1 M HCl) containing Triton X-100 was automatically added, was loaded in the donor channel and dialysed on a cellulose acetate membrane in the static-pulsed mode. The dialysis liquid consisted of pH 2.5 phosphate buffer. By rotation of a switching valve, the analytes were eluted from the TEC in the back-flush mode by the LC mobile phase and transferred to the analytical column, packed with octyl silica. The chromatographic separation was performed at 35 degrees C and the analytes were monitored photometrically at 295 nm. Due to the differences in hydrophobic character between albendazole and its metabolites, a gradient elution was applied. The mobile phase consisted of a mixture of acetonitrile and pH 6.0 phosphate buffer. The proportion of organic modifier was increased from 10.0 to 50.1% in 12.30 min, then from 50.1 to 66.9% in 1.70 min. First, the gradient conditions and the temperature were optimised for the LC separation using the DryLab software. Then, the influence of some parameters of the dialysis process on analyte recovery was investigated. Finally, the method developed was validated. The mean recoveries for albendazole and its metabolites were about 70 and 65%, respectively. The limits of quantification for albendazole and its metabolites were 10 and 7.5 ng/ml, respectively.

Albendazole↗
PubMed1999

Elimination of adsorption effects in the analysis of water-soluble vitamins in pharmaceutical formulations by capillary electrophoresis.

A tendency to an increase in migration times was observed when different water-soluble vitamins were analysed repeatedly in pharmaceutical preparations by capillary electrophoresis. In order to better understand the origin of this effect, the influence of the vitamins and the excipients, such as cellulose derivatives, was investigated. These studies indicated that the increase in analyte migration times was most probably due to the adsorption of different kinds of constituents to the capillary wall. Different rinsing procedures were tested in order to eliminate these unfavourable effects. A rinse of the capillary with a 25 mM sodium dodecyl sulfate (SDS) solution in the running buffer between successive runs was found to be particularly effective when the analysis was performed by free solution capillary zone electrophoresis (CZE). When the vitamins were determined by micellar electrokinetic chromatography (MEKC) using SDS as surfactant, a short capillary rinse with the running buffer was sufficient to obtain reproducible migration times. The CZE and MEKC methods developed were validated and compared. Both methods could be applied to the determination of water-soluble vitamins in different multivitamin formulations.

Chemistry, Pharmaceutical↗
PubMed1999

Automated sample preparation techniques for the determination of drug enantiomers in biological fluids using liquid chromatography with chiral stationary phases.

The determination of drug enantiomers has become of prime importance in the field of pharmaceutical and biomedical analysis. Liquid chromatography (LC) is one of the most frequently used techniques for achieving the separation and quantitation of the enantiomers of drug compounds. In the bioanalytical field, the integrated systems present an interesting alternative to time-consuming sample preparation techniques such as liquid-liquid extraction. Solid phase extraction (SPE) on disposable cartridges, dialysis or column switching are sample preparation techniques that can be fully automated and applied to enantioselective analysis in biological fluids. The selection of the most appropriate LC mode and chiral stationary phase for enantioseparations in bioanalysis is discussed and some aspects of these automated sample preparation procedures are compared, such as selectivity, detectability, elution of the analytes from the extraction sorbent, sample volume and analyte stability.

Buffers↗
PubMed1998

Simultaneous determination of methylphenobarbital enantiomers and phenobarbital in human plasma by on-line coupling of an achiral precolumn to a chiral liquid chromatographic column.

A fully automated liquid chromatographic (LC) method for the simultaneous determination of methylphenobarbital enantiomers and phenobarbital in human plasma has been developed. The method is based on the use of a precolumn packed with an internal-surface reversed-phase packing material (LiChrospher ADS) for sample clean-up coupled to LC analysis on a cellulose tris(4-methylbenzoate) based chiral stationary phase (Chiralcel OJ-R). A 100-microliter plasma sample was injected directly on the precolumn packed with LiChrospher RP-18 ADS using a mixture of pH 5.0 phosphate buffer-methanol (97:3, v/v) as washing liquid. The analytes were then eluted in the back-flush mode with the LC mobile phase. The enantiomeric separation of methylphenobarbital was achieved on Chiralcel OJ-R). The retention times were modelled using a D-optimal design with ten experimental points in order to optimise the LC mobile phase for the separation of phenobarbital from the enantiomers of mephobarbital. The factors selected were the acetonitrile content, the pH and the sodium perchlorate concentration in the mobile phase. A Derringer's desirability function was used to find an optimal and robust solution within the experimental domain. The mobile phase selected consisted of a mixture of pH 7.0 phosphate buffer-acetonitrile (60:40, v/v). The elution profiles of phenobarbital, methylphenobarbital and blank plasma samples on the precolumn and the time needed for analyte transfer from the precolumn to the analytical column were then determined. Finally, the method developed was validated.

Chromatography, Liquid↗
PubMed1997

Automated determination of tramadol enantiomers in human plasma using solid-phase extraction in combination with chiral liquid chromatography.

A sensitive and automated method for the separation and individual determination of tramadol enantiomers in plasma has been developed using solid-phase extraction (SPE) on disposable extraction cartridges (DECs) in combination with chiral liquid chromatography (LC). The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with ethyl silica (50 mg) was first conditioned with methanol and phosphate buffer, pH 7.4. A 1.0-ml volume of plasma was then applied on the DEC. The washing step was performed with the same buffer. The analytes were eluted with 0.15 ml of methanol, and 0.35 ml of phosphate buffer, pH 6.0, containing sodium perchlorate (0.2 M) were added to the extract before injection into the LC system. The enantiomeric separation of tramadol was achieved using a Chiralcel OD-R column containing cellulose tris-(3,5-dimethylphenylcarbamate) as chiral stationary phase. The mobile phase was a mixture of phosphate buffer, pH 6.0, containing sodium perchlorate (0.2 M) and acetonitrile (75:25). The mobile-phase pH and the NaClO4 concentration were optimized with respect to enantiomeric resolution. The method developed was validated. Recoveries for both enantiomers of tramadol were about 100%. The method was found to be linear in the 2.5-150 ng/ml concentration range [r2=0.999 for (+)- and (-)-tramadol]. The repeatability and intermediate precision at a concentration of 50 ng/ml were 6.5 and 8.7% for (+)-tramadol and 6.1 and 7.6% for (-)-tramadol, respectively.

Analgesics, Opioid↗
PubMed1997

Enantioselective determination of oxprenolol in human plasma using dialysis coupled on-line to reversed-phase chiral liquid chromatography.

A fully automated method for the determination of the enantiomers of oxprenolol in human plasma was developed, involving dialysis through a cellulose acetate membrane, clean-up and enrichment of the dialysate on a short precolumn and subsequent chiral liquid chromatographic (LC) analysis. All sample handling operations were executed automatically by a sample processor equipped with a robotic arm (ASTED system). The trace enrichment column (TEC) was packed with octadecylsilica. After conditioning of the TEC with the LC mobile phase and pH 3.0 acetate buffer. After the enrichment step, the analyte was transferred by the LC mobile phase to the analytical column by means of a switching valve. The influence of different parameters of the dialysis process on the recovery of oxprenolol was first investigated using achiral LC conditions. The volume as well as the aspirating and dispensing flow rates of the acceptor solution were the main parameters studied. Oxprenolol was separated on a C18 stationary phase used for the enantioseparation of oxprenolol was a Chiralcel OD-R column which contained cellulose tris (3,5-dimethylphenylcarbamate) coated on silica as chiral selector. The corresponding mobile phase consisted of a mixture of pH 6.0 phosphate buffer containing NaClO4 at 0.45 M concentration and acetonitrile (70:30 v/v). UV detection was performed at 273 nm. The method developed was validated. Recoveries for each enantiomer of oxprenolol were about 80%. The method was found to be linear in the 50-2500 ng ml-1 concentration range (r2 = 0.999 for both enantiomers) and good results with respect to intra- and inter-day reproducibility as well as accuracy were obtained.

Adrenergic beta-Antagonists↗
PubMed1996

Automated determination of verapamil and norverapamil in human plasma with on-line coupling of dialysis to high-performance liquid chromatography and fluorometric detection.

A fully automated method for the simultaneous determination of verapamil and its main metabolite norverapamil in human plasma is described. This method is based on on-line sample preparation using dialysis followed by clean-up and enrichment of the dialysate on a precolumn and subsequent HPLC analysis with fluorometric detection. All sample handling operations were performed automatically by a sample processor equipped with a robotic arm (ASTED system). The plasma samples were dialysed on a cellulose acetate membrane (cut-off: 15 kD) and the dialysate was purified and enriched on a short pre-column filled with cyanopropyl silica. Before starting dialysis, this trace enrichment column (TEC) was first conditioned with the HPLC mobile phase and then with pH 3.0 acetate buffer. 370 microliters of plasma sample spiked with the internal standard (gallopamil) were dialysed in the static-pulsed mode. The solution at the donor side was pH 3.0 acetate buffer containing Triton X-100 while the acceptor solution was made of the same acetate buffer. When dialysis was discontinued, the analytes were desorbed from the TEC by the HPLC mobile phase and transferred to the C18 analytical column by means of a switching valve. This mobile phase consisted of a mixture of acetonitrile, pH 3.0 acetate buffer and 2-aminoheptane. The influence of different parameters of the dialysis process on the recovery of verapamil and norverapamil has been studied. The effect of the volume, the aspirating and dispensing flow-rates of the dialysis solution has been investigated. The recoveries of verapamil and norverapamil in plasma were close to 75% and the limits of quantification were 5 ng/ml for both analytes. The method was found to be linear in the concentration range from 5 to 500 ng/ml (r2: 0.9996 for both analytes). The intra-day and inter-day reproducibilities at a concentration of 100 ng/ml were 2.3% and 5.6% for verapamil and 1.7% and 5.1% for norverapamil, respectively.

Calcium Channel Blockers↗
PubMed1994

Knowledge-based system for the automated solid-phase extraction of basic drugs from plasma coupled with their liquid chromatographic determination. Application to the biodetermination of beta-receptor blocking agents.

Techniques for the preparation of biological samples are often based nowadays on solid-phase extraction (SPE). The different SPE steps can be performed automatically on disposable extraction cartridges (DECs) by means of a sample processor. A knowledge-based system was developed to facilitate the development of fully automated methods for the solid-phase extraction of relatively hydrophobic basic drugs from plasma, coupled with their determination by high-performance liquid chromatography (HPLC). The DEC filled with 50 mg of cyanopropyl-bonded silica phase is first conditioned with methanol and buffer solution (pH 7.4). After sample application, the DEC sorbent is washed with the same buffer. The analytes are then desorbed with an appropriate eluent and the eluate is finally diluted with the same buffer as used in the HPLC mobile phase before injection. Under these conditions, only three variables are still to be optimized: the composition and volume of the elution solvent and the volume of buffer to be added to the eluate. On the basis of this general strategy, a decision tree providing information about suggested starting conditions and guidelines for the optimization of the three variables was developed and implemented by use of a hypermedia software. This didactic expert system was evaluated using several beta-receptor blocking agents as model compounds and the operating conditions obtained for the automated SPE of these compounds are presented. A method for the determination of propranolol in plasma using the SPE conditions deduced from the knowledge-based system was validated. The absolute recovery of propranolol is ca. 93% and the limit of detection is 1.3 ng ml-1. The mean within-day and between-day reproducibilities are 2.3 and 3.6%, respectively.

Adrenergic beta-Antagonists↗
PubMed1993

Fully automated determination of sulfamethazine in ovine plasma using solid-phase extraction on disposable cartridges and liquid chromatography.

An automatic sample preparation procedure followed by on-line injection of the sample extract into a HPLC system has been developed for the quantitative analysis of sulfamethazine and its N4-acetyl metabolite in ovine plasma. The sample clean-up was performed by solid-phase extraction (SPE) on C18 disposable extraction cartridges (DECs). All the sample handling operations were effected by a robotic auto-sampler. The DEC was first conditioned with methanol and phosphate buffer pH 7.4. After loading 1.0 ml of plasma sample onto the DEC, the latter was washed with the same buffer. The elution step was performed with methanol (0.25 ml) and the eluate was then diluted by adding 0.75 ml volume of phosphate buffer pH 6.4. A 20-microliters volume of the resultant solution was injected onto an octadecyl silica column preceded by a short guard column. The HPLC mobile phase was methanol-phosphate buffer pH 6.4 (25:75, v/v). Sulfamethazine and N4-acetylsulfamethazine were determined photometrically at 262 nm. Under these conditions, linear calibration curves ranging from 2 to 250 micrograms ml-1 have been obtained for both compounds. Drug recoveries were higher than 90% and typical relative standard deviation values were 0.7% (within-day) and 2.0% (between-day) at a plasma concentration of 50 micrograms ml-1.

Animals↗
PubMed1991

Automatic determination of diltiazem and desacetyldiltiazem in human plasma using liquid-solid extraction on disposable cartridges coupled to HPLC--Part I: Optimization of the HPLC system and method validation.

A sensitive and automated method for the analysis of diltiazem and desacetyldiltiazem in plasma has been developed using liquid-solid extraction (LSE) on disposable extraction cartridges (DECs) in combination with HPLC. After isolation from plasma, the analytes are separated on a highly deactivated octyl silica column with a mobile phase of methanol-0.05 M phosphate buffer (pH 7.4) (62:38, v/v). The analytes are monitored photometrically at 238 nm. The complete preparation of the plasma sample as well as the injection of the final extract on to the analytical column are performed automatically by means of a sample processor equipped with a robotic arm to which is attached a needle dispensing the different liquids. The internal standard solution is first added to the plasma sample. The DEC is then conditioned successively with methanol and phosphate buffer (pH 7.4). A 1.0-ml volume of sample containing the internal standard solution is applied on an extraction cartridge filled with cyanopropyl silica (50 mg). After the DEC has been washed with the same buffer, the analytes are eluted with 0.16 ml of methanol. A 0.14-ml volume of buffer is then passed through the DEC and 0.25 ml of the final extract is injected onto the HPLC column. The absolute recoveries of the drugs are about 90% and the limit of detection for diltiazem is 0.8 ng ml-1. Relative standard deviations of 2.6% (within-day) and 3.7% (between-day) have been obtained for this compound at a plasma concentration of 50 ng ml-1.

Automation↗
PubMed1991

Automatic determination of diltiazem and desacetyldiltiazem in human plasma using liquid-solid extraction on disposable cartridges coupled to HPLC--Part II: Optimization of liquid-solid extraction.

An automatic liquid-solid extraction (LSE) procedure to be coupled to HPLC for the determination of diltiazem and desacetyldiltiazem in plasma has been developed. The LSE operations are performed on disposable extraction cartridges (DECs) by means of a sample processor equipped with a robotic arm holding a needle through which the different liquids are dispensed. The operating parameters of LSE have been optimized with respect to recovery, detectability and reproducibility by using, whenever possible, aqueous solutions of the analytes. Different kinds of DECs have been tested. For the compounds studied, DECs filled with 50 mg of cyanopropyl silica have been selected. The influence of the pH of the buffer used in the washing step has been studied, leading finally to the selection of the same phosphate buffer (pH 7.4) as in the HPLC mobile phase. The minimum volume of methanol which still gives a nearly complete elution of the analytes from the extraction cartridges has been determined. Under these conditions, a high sensitivity can be obtained without an evaporation step. Moreover, the volume of buffer to be added to the methanolic eluate before injection into the HPLC system has been optimized in such a way that a focusing effect is obtained at the top of the analytical column while the dilution of the extract is minimized.

Automation↗
PubMedDate not supplied

Determination of verapamil and norverapamil in human plasma by liquid chromatography: comparison between a liquid-liquid extraction procedure and an automated liquid-solid extraction method for sample preparation.

A conventional liquid-liquid extraction (LLE) procedure with high-performance liquid chromatography (HPLC) has been developed for the determination of verapamil and its main metabolite, norverapamil, in plasma. After addition of the internal standard, plasma samples were basified with phosphate buffer (pH 9.0) and extracted with a mixture of cyclohexane-dichloromethane. After centrifugation, the organic layer was separated and the analytes were extracted back into a 0.1 N sulphuric acid solution containing 2-aminoheptane. An aliquot of this aqueous phase was then injected directly onto the HPLC column. This LLE procedure has been compared with an automated liquid-solid extraction (LSE) method that has been developed in parallel. Good linearity was obtained using both extraction methods. The absolute recoveries for the two analytes were ca 95% with the automated LSE procedure and slightly lower (ca 84%) for the LLE method. The automated method gives better results with respect to detectability and precision, but the LLE procedure is simpler to develop, requires much less expensive equipment, and remains a useful alternative when the number of samples to be analysed is limited.

Buffers↗
PubMedDate not supplied

Evaluation of some parameters influencing the drug delivery from a dry powder inhalation device using an in vitro model of the horse airways.

The aim of this study was to determine the effect of breathing pattern, air humidity and position of the device on the delivery of an aerosol generated by a dry powder inhalation (DPI) device (Inhalator M). The in vitro inhalation study was performed using the cascade impaction method (Andersen Sampler) adapted to imitate nasal breathing. The amount of ipratropium found in the device, the artificial upper airways and the six stages of the Andersen Sampler was measured using high precision liquid chromatography. Stage 1 of the Andersen Sampler was considered to be the respirable fraction and stages 2 to 6 to be the non-respirable fraction. It was concluded that the theoretical respirable fraction of ipratropium obtained after DPI through Inhalator M was influenced by relative air humidity, air flow and the position of the device, whereas the number of successive inspirations and the duration of inspiration did not affect this fraction of the drug.

Aerosols↗