J.B. Lecaillon

Determination of cefsulodin, cefotiam, cephalexin, cefotaxime, desacetyl-cefotaxime, cefuroxime and cefroxadin in plasma and urine by high-performance liquid chromatography

Closely related methods for the determination of several cephalosporins in plasma and urine are described. Deproteinized plasma or diluted urine is directly injected on a RP-8 or RP-18 bonded-material column. Chromatography is performed either in the reversed-phase or the ion-pair mode. The limits of sensitivity range from 0.4 to 2 mumol of cephalosporins per liter of plasma, and from 20 to 100 mumol per liter of urine. The sensitivity may be improved two to five times by using precolumn loading, direct sample clean-up and automatic injection. The stability of the cephalosporins in plasma, urine and water and the reproducibility and accuracy of the methods are reported.

Automated microanalysis of oxcarbazepine and its monohydroxy and transdiol metabolites in plasma by liquid chromatography

An automated high-performance liquid chromatographic method for the simultaneous determination of oxcarbazepine and its monohydroxy and transdiol metabolites in plasma is described. 5,6-Dihydro-11-oxo-11H-dibenz[b,e]azepine-5-carboxamide was used as internal standard. Liquid-solid extraction from plasma (100 microliters) on 50 mg Bond-Elut C18 cartridges was automatically performed by the Automatic Sample Preparation with Extraction Columns (ASPEC) system. A reversed-phase column (ODS Hypersil, 3 microns particle size, 4 cm x 4.6 mm I.D.) was used with acetonitrile-methanol-0.01 M potassium dihydrogenphosphate as mobile phase. The eluted compounds were detected at 210 nm. The limit of quantitation was 0.2 mumol/l for oxcarbazepine and 0.1 mumol/l for its metabolites. No interference with concomitantly administered anti-epileptic drugs such as phenobarbital, phenytoin, valproic acid or carbamazepine, was found.

The influence of food on the disposition of the antiepileptic rufinamide in healthy volunteers

The effect of food on the pharmacokinetics of the antiepileptic rufinamide was investigated in healthy volunteers. Twelve subjects were treated with single per‐oral doses of 600 mg of rufinamide after overnight fasting or a fat and protein rich breakfast. Mean (±S.D.) areas under the plasma concentration–time curves (AUCs) of the unchanged compound were 57.2 (16) μg mL−1 h when given to the fasted volunteers and 81.7 (22.2) μg mL−1 h (p = 0.0001) when given after the breakfast. The average AUC was increased by 44% when rufinamide was given with food and the maximum concentration (Cmax) by about 100%. The time at which Cmax was reached (tmax) was shorter (8 h in fasted conditions and 6 h in fed after breakfast); the terminal half‐life was not influenced by concomitant intake of food.

Automated and sensitive method for the determination of formoterol in human plasma by high-performance liquid chromatography and electrochemical detection

An automated high-performance liquid chromatography (HPLC) method for the determination of formoterol in human plasma with improved sensitivity has been developed and validated. Formoterol and CGP 47086, the internal standard, were extracted from plasma (1 ml) using a cation-exchange solid-phase extraction (SPE) cartridge. The compounds were eluted with pH 6 buffer solution-methanol (70:30, v/v) and the eluate was further diluted with water. An aliquot of the extract solution was injected and analyzed by HPLC. The extraction, dilution, injection and chromatographic analysis were combined and automated using the automate (ASPEC) system. The chromatographic separations were achieved on a 5 microm, Hypersil ODS analytical column (200 mm x 3 mm I.D.), using (pH 6 phosphate buffer, 0.035 M + 20 mg/l EDTA)-MeOH-CH3CN (70:25:5, v/v/v) as the mobile phase at a flow-rate of 0.4 ml/min. The analytes were detected with electrochemical detection at an operating potential of +0.63 V. Intra-day accuracy and precision were assessed from the relative recoveries of calibration/quality control plasma samples in the concentration range of 7.14 to 238 pmol/l of formoterol base. The accuracy over the entire concentration range varied from 81 to 105%, and the precision (C.V.) ranged from 3 to 14%. Inter-day accuracy and precision were assessed in the concentration range of 11.9 to 238 pmol/l of formoterol base in plasma. The accuracy over the entire concentration range varied from 98 to 109%, and precision ranged from 8 to 19%. At the limit of quantitation (LOQ) of 11.9 pmol/l for inter-day measurements, the recovery value was 109% and C.V. was 19%. As shown from intra-day accuracy and precision results, favorable conditions (a newly used column, a newly washed detector cell and moderate residual cell current level) allowed us to reach a LOQ of 7.14 pmol/l of formoterol base (3 pg/ml of formoterol fumarate dihydrate). Improvement of the limit of detection by a factor of about 10 was reached as compared to the previously described methods. The method has been applied for quantifying formoterol in plasma after 120 microg drug inhalation to volunteers. Formoterol was still measurable at 24 h post-dosing in most subjects and a slow elimination of formoterol from plasma beyond 6-8 h after inhalation was demonstrated for the first time thanks to the sensitivity of the method.

Quantitative assay of sulphinpyrazone in plasma and urine by high-performance liquid chromatography

A method for the quantitative determination of sulphinpyrazone in plasma and urine is described. The drug is extracted from the acidified aqueous phase with 1-chlorobutane-ethylene dichloride (4:1) and separated from its metabolites by high-performance liquid chromatography on 5-mum LiChrosorb using dichloromethane-ethanol-water-acetic acid (79.1:19:1.9:0.002) as the mobile phase. The sensitivity limit is 0,2mug/ml using a 1-ml sample. Examples of applications are given.

Determination of metoprolol and its α-hydroxylated metabolite in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed for the simultaneous determination of metoprolol and its alpha-hydroxylated metabolite in plasma, Metoprolol, alpha-hydroxymetoprolol and alprenolol (internal standard) are extracted from plasma at alkaline pH with diethyl ether-dichloromethane (4:1, v/v) and back-extracted with 0.01 N sulfuric acid. A 100-microliter volume of the acidic extract is injected into the chromatographic system. The compounds are eluted in about 12 min with acetonitrile-acetate buffer (75:25, v/v) on a LiChrosorb RP-8 (5 micron) column. The quantitative determinations are made fluorometrically. Concentrations down to 35 nmol/1 (10 ng/ml) of metoprolol base and 30 nmol/1 (8 ng/ml) of alpha-hydroxymetoprolol base in plasma can be determined with good precision and accuracy.

Automated analysis of a novel anti-epileptic compound, CGP 33 101, and its metabolite, CGP 47 292, in body fluids by high-performance liquid chromatography and liquid-solid extraction

Automated procedures for the determination of CGP 33,101 in plasma and the simultaneous determination of CGP 33,101 and its carboxylic acid metabolite, CGP 47,292, in urine are described. Plasma was diluted with water and urine with a pH 2 buffer prior to extraction. The compounds were automatically extracted on reversed-phase extraction columns and injected onto an HPLC system by the automatic sample preparation with extraction columns (ASPEC) automate. A Superlosil LC-18 (5 microns) column was used for chromatography. The mobile phase was a mixture of an aqueous solution of potassium dihydrogen phosphate, acetonitrile and methanol for the assay in plasma, and of an aqueous solution of tetrabutylammonium hydrogen sulfate, tripotassium phosphate and phosphoric acid and of acetonitrile for the assay in urine. The compounds were detected at 230 nm. The limit of quantitation was 0.11 mumol/l (25 ng/ml) for the assay of CGP 33,101 in plasma, 11 mumol/l (2.5 micrograms/ml) for its assay in urine and 21 mumol/l (5 micrograms/ml) for the assay of CGP 47,292 in urine.

Fully automated analytical system using liquid—solid extraction and liquid chromatography for the determination of CGP 6140 in plasma

Liquid-solid extraction on disposable extraction columns (DECs) and liquid chromatography can be combined in a completely automated analyser. The Gilson ASPEC system was used to develop a procedure for the determination of CGP 6140 in plasma. Both sample preparation via C8 Bond-Elut DECs and injection were fully automatic. The fully automated system prepared the samples by performing the same operations as for a manual procedure. The DEC was first wetted with methanol, then with water. A 400-microliters volume of plasma and 40 microliters of the internal standard solution, diluted with 1 ml of water, were applied to the DEC, rinsed with 10(-2) mol/l dipotassium hydrogenphosphate and eluted from the DEC with 300 microliters of acetonitrile-methanol (50:50, v/v). The eluting strength of the eluate was reduced by dispensing 1 ml of water into each vial prior to direct injection into a Spherisorb ODS column via a 1-ml loop. This allowed the reconcentration of the extracted compounds on the top of the column, as they were injected in a large volume of solvent of lower eluting strength than the mobile phase [acetonitrile-methanol-4 x 10(-3) mol/l ammonia solution (54.5:5:40.5, v/v/v)]. Reproducibility results are presented.

Determination of metoprolol in human plasma by column switching high-performance liquid chromatography

SummaryRetention characteristics of metoprolol have been studied in reversed phase mode on RP2, RP8 and CN columns. The plots of retention time as a function of the acetonitrile content and of the ionic strength of the mobile phase permitted the choice of the best conditions to separate metoprolol from plasma components by switching of these three types of columns.Human plasma (0.5–1 ml) diluted with water is first injected on a RP2 column (25–40 μm particle diameter, prepared by dry packing) and rinsed with water. The sample is then back eluted with acetonitrile-0.022 M acetate buffer (75∶25, v:v) and switched to a CN column (10 cm long, 5 μm particle diameter). The heart cut of the eluate is selected and loaded on a RP8 analytical column (25 cm long, 5 μm particle diameter) with acetonitrile-0.088 M acetate buffer (75∶25, v:v) as mobile phase.Auto-sampler and switching valves are actuated automatically by a computing integrator based on a fixed time schedule. The duration of one cycle is about 30 min, but the last analytical step is about 15 min and represents the time interval between two injections. Metoprolol, its alpha-hydroxy metabolite and the internal standard are detected by fluorescence (λex= 225 nm; λem > 320 nm).

Automated microanalysis of carbamazepine and its epoxide and trans-diol metabolites in plasma by column liquid chromatography

A fully automated high-performance liquid chromatographic procedure for the simultaneous determination of carbamazepine and its main metabolites, epoxycarbamazepine and dihydroxycarbamazepine, in plasma is described. Liquid-solid extraction on disposable C18 columns and reversed-phase chromatography on a 3 microns particle size C18 column were combined and automated by using the Automatic Sample Preparation with Extraction Columns system. Ultraviolet detection was performed at 210 nm. 5,6-Dihydro-11-oxo-11H-dibenz[b,e]azepine-5-carboxamide was used as internal standard. A small plasma volume (100 microliters) was required. The total run time for the assay of one sample was about 10 min. The assay demonstrated good reproducibility. The limit of quantitation was 0.1 mumol/l (about 25 ng/ml).