Liquid chromatography-positive ion electrospray mass spectrometry method for the quantification of citalopram in human plasma

A rapid, sensitive and novel narrow-bore liquid chromatography-mass spectrometric method was developed and fully validated for the quantification of citalopram in human plasma. The analyte and internal standard (imipramine) were extracted by liquid-liquid extraction with a mixture of hexane-heptane- isopropanol (88:10:2, v/v/v). The use of a Hypersil BDS C8 micro-bore column (250 mm × 2.1 mm i.d.; 3.5 μm particle size), results in substantial reduction in solvent consumption. The mobile phase consisted of 10 mM ammonium formate-formic acid (pH 4.5) and acetonitrile (30:70, v/v), pumped at a flow rate of 0.15 ml min-1. The analytes were detected after positive electrospray ionization using the selected ion-monitoring mode of the species at m/z 325 for citalopram and m/z 281 for imipramine. The method had a chromatographic run time of 10.0 min and a linear calibration curve over the range 0.50-250 ng ml-1 (r2 > 0.996). The limit of quantitation was 0.50 ng ml-1. Accuracy and precision were below the acceptance limits of 15%. © 2004 Elsevier B.V. All rights reserved

Development and validation of a liquid chromatographic/electrospray ionization mass spectrometric method for the quantitation of prazepam and its main metabolites in human plasma

A method was developed and fully validated for the quantitation of prazepam and its major metabolites, oxazepam and nordiazepam, in human plasma. Sample pretreatment was achieved by solid-phase extraction using Oasis HLB cartridges. The extracts were analysed by high-performance liquid chromatography (HPLC) coupled with single-quadrupole mass spectrometry (MS) with an electrospray ionization interface. The MS system was operated in the selected ion monitoring mode. HPLC was performed isocratically on a reversed-phase XTerra MS C 18 analytical column (150 × 3.0 mm i.d., particle size 5 μm). Diazepam was used as the internal standard for quantitation. The assay was linear over a concentration range of 5.0-1000 ng ml-1 for all compounds analyzed. The limit of quantitation was 5 ng ml-1 for all compounds. Quality control samples (5, 10, 300 and 1000 ng ml-1) in five replicates from three different runs of analysis demonstrated an intra-assay precision (CV) of ≤9.1%, an inter-assay precision of ≤6.0% and an overall accuracy (relative error) of <4.6%. The method can be used to quantify prazepam and its metabolites in human plasma covering a variety of pharmacokinetic or bioequivalence studies. Copyright © 2005 John Wiley & Sons, Ltd

HPLC method with UV detection for the determination of trans-resveratrol in plasma

The main representative of phytoalexins (biologically active substances produced by the plant as an immediate immunological response to infections or abiotic stresses) in wine grapes (Vitis vinifera) is the stilbene trans-resveratrol. The proposed method for the determination of trans-resveratrol in blood plasma is based on reversed phase HPLC utilizing UV detection (310 nm), under isocratic conditions (1.0 mL/min), with mobile phase consisting of acetonitrile-phosphate buffer pH 4.8 (30 mM) 25:75 v/v, a C 18 Novapack 150 x 4.0 mm column (4 μm particle size), and carbamazepine as internal standard (5 μg/mL). Plasma samples (1 mL) are buffered with phosphate buffer (0.4 mL) pH 6.0 and extracted with 3 x 3 mL of ethyl acetate, trans-resveratrol is eluted at around 4.6 min, whereas carbamazepine at approximately 9.6 min, yielding a resolution of 4.4. The method appears to be linear within a range of 0.15-4.0 μg/mL (r = 0.9998), with good repeatability (%RSD = 0.86) and reproducibility (%RSD = 2.2). Detection and quantification limits were found equal to 0.10 and 0.33 μg/mL, respectively. Accuracy, expressed as recovery determined at two concentrations of 0.3 and 3.0 μg/mL (6-replicates) were found to be 88.3 ± 7.5% and 100.7 ± 0.7%, respectively. The reported method is simple, rapid, accurate, and intended for use to further bioavailability studies, aiming to the development of pharmaceutical dosage formulations of resveratrol for oral administration. Copyright © Taylor & Francis, Inc

Development of a potentiometric kinetic method for drug adsorption studies: The chlorpromazine-charcoal model case

A potentiometric method for the in vitro adsorption kinetic study of an ionic micromolecule to charcoal, based on the continuous direct monitoring of the micromolecule free concentration by means of an ion-selective electrode (ISE), has been developed. A chlorpromazine ISE was constructed and used to study the adsorption kinetics of the drug on pure activated charcoal and two commercial formulations (Ultracarbon tablets and Carbomix powder). The method consists of the rapid addition of a slurry containing the charcoal into the drug solution under stirring at pH 1.2 (to simulate a gastric fluid environment) and continuous recording of the electrode potential until the establishment of equilibrium. The drug free concentration at appropriate time intervals was calculated from the recorded adsorption curve and the apparent adsorption rate constant was estimated assuming first order kinetics. Within run RSD of the estimates ranged from 0.3 to 12% (mainly less than 5%), while between run RSD (n = 3) ranged from 1 to 19% (mainly less than 10%). A linear relationship was found between the apparent adsorption rate constants and the amount of charcoal used with slopes following the rank order activated charcoal > Ultracarbon tablets > Carbomix powder. These results were explained on the basis of different surface areas of the adsorbents. The work proved the usefulness of ion-selective potentiometry in adsorption studies and can be extended to other ionic drugs for which selective electrodes can be constructed

Simultaneous determination of oleuropein and tyrosol in plasma using high performance liquid chromatography with UV detection

Oleuropein (O) and Tyrosol (T) are polyphenolic compounds with potent biological activities including, but not limited to, an antioxidant activity. An increased interest by many pharmaceutical companies has been shown to develop a formulation of O and/or T. In this research effort, a fast, simple, and reliable analytical method for the determination of O and T in plasma is required in order to carry out bioavailability studies. In this work, a reversed-phase isocratic high-performance liquid chromatographic (HPLC) method has been developed and validated for the simultaneous determination of O and T in plasma. The isolation of the polyphenolic analytes from plasma was carried out by liquid extraction with ethyl acetate after the addition of Na2SO4 to the sample (salting out effect), followed by evaporation to dryness at 50°C, and reconstitution with mobile phase (4 x preconcentration). Chromatographic analysis was performed using a C8 column with MeOH/CH3COOH 2% in water, 45: 55 v/v as mobile phase, and UV detection at 280 nm. Vanillin (V) was used as internal standard. The recovery of the isolation procedure was 80% for O (CV = 6-18%) and 98% for T (CV = 3-8%). Retention times (min) were 4.6 for T, 6.2 for internal standard, and 8.9 for O, while endogenous plasma components were eluted before 4.3 min. Calibration curves of O and T in plasma were linear from 1-20 and 0.1-2.0 μg/mL at least, respectively (r > 0.9996 and 0.999, correspondingly). The detection limits of the method were 0.36 μg/mL for O and 0.09 μg/mL for T (4 x preconcentration). Precision and accuracy were determined from spiked plasma samples and were, for O CV= 5.7-8.3% and Er=1.3-6.1% and for T CV=6.1-0% and Er=1.5-5%. The analytical methodology developed in this report is simple, rapid, accurate, and sensitive enough to be used in bioavailability studies

Development and validation of liquid chromatographic method for the determination of lycopene in plasma

An isocratic reversed-phase liquid chromatographic method with spectrophotometric detection (472nm) was developed and validated for the determination of lycopene in isolated lycopene crude material and in plasma. The method utilizes a Nova Pak C-18 column (3.9mm×150mm), 5μm particle size, with a pre-column of the same type and a mobile phase consisting of methanol:acetonitrile:methylene chloride (55:30:15 (%, v/v)) at a flow rate of 1.0mlmin-1. The rentention time of lycopene was 4.8min and the total run time was 8min. Experiments were carried out at room temperature in the absence of direct sunlight. The isolation of lycopene from plasma was achieved with both liquid-liquid extraction (LLE) and solid-phase extraction (SPE) with recoveries of 94.5±5.1% and 62.3±2.6%, respectively. Calibration graphs from plasma standards were linear in the range 0.2-2.5μM with a detection limit of 0.13μM for the LLE procedure and 0.067-0.83μM with a detection limit of 0.043μM for the SPE procedure. Relative standard deviations were <5% for both procedures. © 2002 Elsevier Science B.V. All rights reserved

In vitro adsorption study of fluoxetine onto activated charcoal at gastric and intestinal pH using high performance liquid chromatography with fluorescence detector

Background: This in vitro investigation was performed to study the adsorption characteristics of fluoxetine to activated charcoal and its commercial formulation Carbomix® powder in simulated gastric (pH = 1.2) and intestinal (pH = 7.2) fluid environments. Methods: Solutions containing fluoxetine and charcoal were incubated at 37°C for one hour. Reversed phase high performance liquid chromatography was used for the determination of free fluoxetine concentrations (range 0.2-8 μg/mL) in the diluted filtrate. Results: The maximum adsorption capacities at pH 1.2 for activated charcoal and Carbomix were 223 and 333 mg/g, respectively; at pH 7.2 they were 301 and 453 mg/g, respectively. The affinity constant values at pH 1.2 of activated charcoal and Carbomix were 441 and 122 L/g, respectively, while at pH 7.2 they were 482 and 589 L/g, respectively, indicating a strong binding of fluoxetine onto charcoals. Conclusions: Relative to the toxic and, lethal doses in cases of fluoxetine intoxications, both types of charcoals tested were found effective for adsorption at gastric and intestinal pH. Adsorbed fluoxetine was significantly increased at intestinal pH, consistent with predominant adsorption of the undissociated form of the drug. We conclude that activated charcoal and Carbomix have adsorptive properties appropriate to medical treatment in cases of fluoxetine overdose

The effect of polyethylene glycol on the charcoal adsorption of chlorpromazine studied by ion selective electrode potentiometry

Background: This investigation was undertaken to study: a) the adsorption characteristics of chlorpromazine to activated charcoal and its formulations Carbomix® powder and Ultracarbon® tablets at gastric pH; b) the effect on chlorpromazine adsorption of polyethylene glycol and its combination with electrolyte lavage solution; c) the effect of the order of addition of polyethylene glycol-electrolyte lavage solution. Method: ion selective electrode potentiometry, based on the selective, direct and continuous response of a chlorpromazine-ion selective electrode to the concentration of the free drug, was used. Successive additions of microvolumes of a chlorpromazine solution were made into a charcoal slurry in acidic medium of pH 1.2 with measurement of the chlorpromazine-ion selective electrode potential at equilibrium. Results: The maximum adsorption capacity values of activated charcoal, Carbomix and Ultracarbon, were 297, 563, and 382 mg/g respectively, while the affinity constant values were 40.2, 70.4, and 40.5 L/g, respectively. The adsorption of chlorpromazine to each of the Ultracarbon and Carbomix components was compared to the total adsorption of the formulations. The addition of polyethylene glycol-electrolyte lavage solution causes a slight desorption of chlorpromazine from activated charcoal at gastric pH, more pronounced when polyethylene glycol-electrolyte lavage solution follows the addition of activated charcoal, suggesting the possibility of a nonspecific binding of chlorpromazine to polyethylene glycol. The amount of chlorpromazine adsorbed to Carbomix and Ultracarbon was not significantly affected at gastric pH by the presence of polyethylene glycol or polyethylene glycol-electrolyte lavage solution added either concurrently sequentially to these formulations

Liquid chromatography-positive ion electrospray mass spectrometry method for the quantification of citalopram in human plasma

A rapid, sensitive and novel narrow-bore liquid chromatography-mass spectrometric method was developed and fully validated for the quantification of citalopram in human plasma. The analyte and internal standard (imipramine) were extracted by liquid-liquid extraction with a mixture of hexane-heptane-isopropanol (88:10:2, v/v/v). The use of a Hypersil BDS C-8 micro-bore column (250 mm x 2.1 mm i.d.; 3.5 mum particle size), results in substantial reduction in solvent consumption. The mobile phase consisted of 10 mM ammonium formate-formic acid (pH 4.5) and acetonitrile (30:70, v/v), pumped at a flow rate of 0.15 ml min(-1). The analytes were detected after positive electrospray ionization using the selected ion-monitoring mode of the species at m/z 325 for citalopram and m/z 281 for imipramine. The method had a chromatographic run time of 10.0 min and a linear calibration curve over the range 0.50-250 ng ml(-1) (r(2) > 0.996). The limit of quantitation was 0.50 ng ml(-1). Accuracy and precision were below the acceptance limits of 15%. (C) 2004 Elsevier B.V. All rights reserved

In vitro adsorption study of fluoxetine onto activated charcoal at gastric and intestinal pH using high performance liquid chromatography with fluorescence detector

Background: This in vitro investigation was performed to study the adsorption characteristics of fluoxetine to activated charcoal and its commercial formulation Carbomix® powder in simulated gastric (pH = 1.2) and intestinal (pH = 7.2) fluid environments. Methods: Solutions containing fluoxetine and charcoal were incubated at 37°C for one hour. Reversed phase high performance liquid chromatography was used for the determination of free fluoxetine concentrations (range 0.2-8 μg/mL) in the diluted filtrate. Results: The maximum adsorption capacities at pH 1.2 for activated charcoal and Carbomix were 223 and 333 mg/g, respectively; at pH 7.2 they were 301 and 453 mg/g, respectively. The affinity constant values at pH 1.2 of activated charcoal and Carbomix were 441 and 122 L/g, respectively, while at pH 7.2 they were 482 and 589 L/g, respectively, indicating a strong binding of fluoxetine onto charcoals. Conclusions: Relative to the toxic and, lethal doses in cases of fluoxetine intoxications, both types of charcoals tested were found effective for adsorption at gastric and intestinal pH. Adsorbed fluoxetine was significantly increased at intestinal pH, consistent with predominant adsorption of the undissociated form of the drug. We conclude that activated charcoal and Carbomix have adsorptive properties appropriate to medical treatment in cases of fluoxetine overdose