Björn Norlander

Sensitive high-performance liquid chromatographic analysis of amlodipine in human plasma with amperometric detection and a single-step solid-phase sample preparation

A narrow-bore HPLC assay with electrochemical detection for the determination of the calcium antagonist amlodipine in human plasma samples is presented. By using a single-step solid-phase extraction procedure on Bond Elut C2 columns, the sample preparation step has been considerably simplified and less time-consuming compared to earlier presented works. With a linear and reproducible calibration curve over the range 0.5-20 ng ml-1 plasma, the assay has successfully been used in the analysis of more than 500 plasma samples from a multicenter trial.

Coupled-column chromatography on a Chiral-AGP phase for determination of amlodipine enantiomers in human plasma: An HPLC assay with electrochemical detection

A sensitive enantioselective high performance liquid chromatographic assay for determination of the dihydropyridine-type calcium antagonist amlodipine in human plasma samples is described. Chiral chromatography is performed on an alpha 1-acid glycoprotein column (i.e. Chiral-AGP) and the eluted enantiomers are trapped and compressed on two short columns before final achiral chromatography on a narrow bore column (i.e. Zorbax SB-Ph) using electrochemical detection. Both stereoselective quantitative analysis and enantiomeric ratio analysis, for samples with a known total concentration of amlodipine are described. The quantitative assay shows linearity over the range 0.5-10 ng ml-1 for the two enantiomers and the limit of detection is about 0.2 ng ml-1. The method has been applied to a pharmacokinetic study of the enantiomers of amlodipine in human subjects.

Solid-phase extraction with end-capped C2 columns for the routine measurement of racemic citalopram and metabolites in plasma by high-performance liquid chromatography

An assay based on solid-phase extraction followed by high-performance liquid chromatography (HPLC) was developed for the measurement of citalopram and its main metabolites desmethylcitalopram and didesmethylcitalopram. The best extraction procedure was performed with end-capped C2 column utilising secondary silanol interactions to obtain clean extract. The HPLC analysis was done on a phenyl column with a mobile phase without any amine additives. Fluorescence detection gave a limit of detection of 0.8 nmol/l plasma for the compounds analysed.

On-line extraction using an alkyl-diol silica precolumn for racemic citalopram and its metabolites in plasma. Results compared with solid-phase extraction methodology.

Sample preparation is usually the most critical and time consuming step when using HPLC for drug analysis in biological matrixes. Sample extracts have to be clean considering both chromatographic interferences and column maintenance. To meet some of these criteria a fully automated on-line extraction (OLE) analysis method was developed for the antidepressant drug citalopram and its two demethylated metabolites, using an RP-C4-ADS extraction column. A comparison between the new OLE method and an off-line solid-phase extraction method showed that the two methodologies were equal in analytical precision but that the OLE method was faster and therefore superior in sample capacity per day.

Optimization and Characterization of the Chiral Separation of Citalopram and its Demethylated Metabolites by Response Surface Methodology

SummaryResponse-surface modelling and sequential optimization have been used for optimization and characterization of the separation of the enantiomers of citalopram, desmethylcitalopram, and didesmethylcitalopram on an acetylated β-cyclodextrin column. In the model chosen the separation conditions mobile phase methanol content, buffer concentration, column temperature, and pH were varied to investigate their influence on the chromatography.It was found that what is good for selectivity within an enantiomer pair is bad for selectivity between enantiomer pairs. Because within-pair and between-pair selectivity does not reach its optimum at the same conditions, a middle course approach has to be followed.Use of an experimental design for this investigation enabled understanding of the mechanisms of within- and between-pair separation for citalopram, desmethylcitalopram, and didesmethylcitalopram. Sequential optimization can be a quicker means of optimizing a chromatographic separation; response-surface modelling, in addition to enabling optimization of the chromatographic process, also serves as a tool for leaming more about the separation mechanism.

Simultaneous determination of reboxetine and O-desethylreboxetine enantiomers using enantioselective reversed-phase high-performance liquid chromatography

Current knowledge of stereoselective pharmacokinetics and different potencies of drug enantiomers requires the performance of stereoselective analysis during therapeutic drug monitoring in clinical practice. However, in the case of the new antidepressant drug reboxetine, no effort has been made so far to find a such a suitable system. Therefore, as a step towards developing an enantioselective bioanalytical method for reboxetine and the O-desethylreboxetine metabolite, three stereoselective chromatographic approaches have been investigated. Several chiral columns were tested, among them Chiral-AGP, ChiraGrom 2 and Chiral-CBH, which were able to simultaneously separate the two compounds into enantiomers in total running times of 28, 18 and 12 min, respectively.

Determination of serum reboxetine enantiomers in patients on chronic medication with racemic reboxetine

The chiral compound reboxetine is used as a selective noradrenaline reuptake inhibitor (NARI) for the treatment of major depressive disorders. The pharmacokinetic variability of the enantiomers of the drug (S,S- and R,R-reboxetine) was studied using stereoselective high-performance liquid chromatography with mass spectrometric detection in a controlled clinical monotherapy situation (trial I) and a naturalistic clinical setting (trial II). Trial I included patients receiving racemic reboxetine as 6-month monotherapy for treatment of major depressive disorder. Trough level serum samples in steady state were analyzed for the concentration of the reboxetine enantiomers in study weeks 4, 12, and 24. In a therapeutic drug monitoring setting (trial II), 47 patients on doses ranging from 4 to 16 mg daily, including much polypharmacy, trough level steady-state serum samples were analyzed by the same bioanalytical method. Data from trials I and II were assessed to determine the inter- and intraindividual pharmacokinetic outcomes. The results showed that the median S,S/R,R ratio in steady state was 0.5 and ranged from 0.22 to 0.88. It was also shown that women have an approximately 30% higher S,S/R,R ratio than men. The S,S/R,R ratios of reboxetine were not found to correlate with reboxetine concentrations. To investigate the NARI activity of a circulating serum reboxetine concentration, a recalculation of the determined enantiomeric concentrations to previously demonstrated experimental NARI potencies of the drug enantiomers was performed. This partly novel concept of estimating pharmacodynamic activity showed that the serum NARI activity in women tended to be higher than in men at a given reboxetine concentration. In conclusion, the variability in the NARI activity per nmol/L reboxetine and the variability in the concentration outcome of the reboxetine enantiomers may justify the use of enantioselective drug monitoring in the clinic. The gender aspects of the drug have to be further assessed.

Exposure of rabbits to methylchloroform. Vestibular disturbances correlated to blood and cerebrospinal fluid levels.

SummaryA previously described experimental model for studying the effects on the central nervous system of rabbits, specifically the vestibular apparatus, has been applied to methylchloroform. To achieve a steady concentration the solvent was infused as a lipid emulsion. The blood and cerebrospinal fluid kinetics have been studied. The arterial blood level seems to be closely correlated to the concentration in the central nervous tissue. Vestibular function has been studied by recording the involuntary eye movements — nystagmus — which are elicited via central vestibulo-oculomotor connections. At blood levels of methylchloroform above 75 ppm a so called “positional nystagmus”, indicated vestibular disturbances, is demonstrated. The relationship between the present findings in rabbits and the reaction and blood concentrations in people exposed to industrial solvents, are discussed.

Chiral ion-pair chromatographic separation of two dihydropyridines with camphorsulfonic acids on porous graphitic carbon

Abstract The direct enantiomeric separation of the two racemic dihydropyridines amlodipine (AML) and UK52.829 (UK) with (1 S )-(+)-10-camphorsulfonic acid [(+)-CSA] as a chiral counter-ion, on porous graphitic carbon Hypercarb-S, is described. The enantiomers of AML and UK were separated in a mobile phase system consisting of 5 m M (+)-CSA in dichloromethane—methanol (25:75, v/v). When the enantiomeric separation of AML and UK was studied in a mobile phase system consisting of 5 m M (1 S )-(+)-3-bromo-10-camphorsulfonic acid [Br-(+)-CSA] in dichloromethane—methanol (25:75, v/v) the capacity factor, k ′, was markedly increased while the separation factor, α, was slightly decreased compared to the mobile phase with (+)-CSA as chiral counter-ion. No enantiomeric separation of AML or UK was seen in a chromatographic system with acetonitrile substituted for methanol as mobile phase solvent, neither with (+)-CSA nor Br-(+)-CSA as chiral counter-ion.

Fast chromatographic separation of (−)-menthyl chloroformate derivatives of some chiral drugs, with special reference to amlodipine, on porous graphitic carbon

SummaryEnantiomeric separation of (−)-menthyl chloroformate derivatives of some chiral cardioactive drugs, on porous graphitic carbon (PGC), Hypercarb-S, is described. Capacity and separation factors of derivatives of the calcium channel blockers; amlodipine and UK52.829, the β-blockers; atenolol, sotalol and propranolol, and mexiletine were studied in different chromatographic systems based on dichloromethane. A high content of a carboxylic acid in the mobile phase was found to decrease the retention and positively affect the stereoselectivity of the derivatives. A mobile phase with dichloromethane, acetonitrile and formic acid gave baseline separation of the enantiomers of amlodipine in less than 8 minutes. Results show that acetic acid and formic acid, may be regarded as strong organic solvents in PGC chromatography with nearly the same elution power as dichloromethane.