Berthold Lausecker

Metabolic Profiles of Minimally Absorbed Orlistat in Obese/Overweight Volunteers

To determine the metabolic profile of minimally absorbed orlistat in obese/overweight patients, an open‐label, single‐dose study was performed in eight obese/overweight volunteers between 23 and 68 years of age. Each subject received a single oral dose of 360 mg orlistat containing approximately 400 μCi of 14C‐labeled orlistat. Serial blood samples were collected at specified times over 10 hours after administration of orlistat for determination of total radioactivity, unchanged orlistat, and major metabolites in the plasma. Urine samples were collected over 24 hours and analyzed to evaluate the urinary recovery of total radioactivity and the profile of orlistat metabolites in the urine. In addition, all fecal samples were collected and analyzed for total radioactivity. Urinary and fecal recovery of the administered dose of total radioactivity were 1.13 ± 0.50% (24‐hour data only) and 96.4 ± 18.1% (n = 7), respectively. Maximum observed concentration (Cmax) and time to Cmax (tmax) values of plasma total radioactivity were 150 ± 51 ng·eq/mL and 6.8 ± 1.5 hrs, respectively. All these parameters obtained in obese/overweight subjects were similar to those reported previously in healthy subjects. On the basis of the area under the concentration‐time curve from 0 to 10 hours (AUC0–10), two major metabolites comprise a total of ∼42% of the total radioactivity in plasma. The primary metabolite (M1) has a short half‐life (∼2 hours), whereas the secondary metabolite (M3) disappeared at a slower rate. No strikingly apparent difference in the urinary metabolic profile was observed between two gender groups. It is concluded that the disposition of orlistat appears to be similar between normal and obese/overweight subjects. Of the minimal fraction of the dose that was absorbed systemically, the presence of two major metabolites accounts for ∼42%.

Simultaneous determination of bosentan and its three major metabolites in various biological matrices and species using narrow bore liquid chromatography with ion spray tandem mass spectrometric detection.

An analytical method was developed for the determination of bosentan and its three main metabolites in various matrices and species with focus on robustness. The drug assay involved protein precipitation, followed by liquid-liquid extraction and column switching in combination with narrow bore HPLC-MS-MS. Deuterated analogues of the analytes were used as internal standards. The sample preparation procedure was optimised with respect to minimise the suppression effects from different matrices. The drug and its metabolites could be analysed in plasma, serum, bile, and liver samples from man, dog, and rat with a run cycle time of 10 min. The method used always calibration samples made up in human plasma, whereas quality control samples were prepared in human plasma as well as in the identical matrix as the unknown samples. Calibration graphs for the drug and for the metabolites were linear in the range from 1 or 2 to 2000 or 10,000 ng/ml using a sample volume of 0.25 ml. Mean inter-assay precision and accuracy were 3.0% and 98.4%, respectively. Two additional methods were derived from the main method for the analysis of plasma samples only with focus on reduced manual effort and instrumental run cycle time. The modified methods showed a mean inter-assay precision and accuracy of 5.0% and 99.9% for the method using column-switching, and 3.5% and 98.8% for the method using off-line SPE, respectively. All methods proved to be robust, sensitive, and selective during the analysis of several thousand samples.

Evolving bioanalytical methods for the cardiovascular drug bosentan

SummaryThe history of the development of analytical methods for the determination of the cardiovascular agent bosentan, and its three major metabolites in biological fluids is described. Before the advent of LC-MS-MS, conventional HPLC-UV methodology was adequate for the determination of the drug alone in samples generated from toxicology studies. The progressive changes in the methods described reflect improvements in MS instrumentation and software, the need for measuring metabolites, as well as the availability of deuterated internal standards. Finally, three LC-MS-MS methods were chosen as being suitable for routine use. The most sophisticated of these (E), involving protein precipitation, liquid-liquid extraction, and on-line solid phase extraction by automated column switching, allowed the simultaneous and sensitive determination of the drug and three metabolites in a variety of biological matrices from several species to support pharmacokinetic studies. Two further, simpler, MS methods were developed (F and G) which, compared with the sophisticated method, focussed on reduced manual effort and reduced instrumental run cycle times. The limits of quantitation were between 1 and 2 ng mL−1 for bosentan and the three metabolites.

Micro liquid chromatography—mass spectrometry with direct liquid introduction used for separation and quantitation of all-trans- and 13-cis-retinoic acids and their 4-oxo metabolites in human plasma

The separation and quantitation of the pentafluorobenzyl derivatives of all-trans- and 13-cis-retinoic acids and their 4-oxo metabolites in human plasma on micro high-performance liquid chromatographic columns (0.32 mm I.D.) is described. The column outlet was directly coupled to the source of a quadrupole mass spectrometer via a simple SFC-frit interface. Negative ion chemical ionization conditions were obtained by coaxial introduction of ammonia as a reagent gas. A signal-to-noise ratio well above 3 was obtained for 1 pg of each analyte injected. The limit of quantitation calculated from spiked biological plasma extracts was 0.3 ng/ml.

Determination of an endothelin receptor antagonist in human plasma by narrow-bore liquid chromatography and ionspray tandem mass spectrometry

A method is described for the determination of a new endothelin receptor antagonist, Bosentan Ro 47-0203, in human plasma using narrow-bore liquid chromatography and ionspray tandem mass spectrometry. After protein precipitation with acetonitrile, the compounds were extracted with dichloromethane at pH 11. The compounds were chromatographed on a 2 mm I.D. reversed-phase column and introduced into the mass spectrometer with an ionspray (pneumatically assisted electrospray) interface at a flow-rate of 170 microliters/min without postcolumn splitting. Two different internal standards were used for the assay: either a structural analogue or a deuterated analogue. The limit of quantification was 0.5 ng/ml using a 0.5-ml aliquot of plasma. Concentrations of the drug were determined in the range 0.5-200 ng/ml. The recovery from human plasma was 87%. The new API IIIplus collision cell was about five times more sensitive than the original API III cell. The assay was demonstrated to be sensitive, selective and robust for the analysis of over 1500 samples.

Workshop/Conference Report on EMA Draft Guideline on Validation of Bioanalytical Methods

This is a summary report of the workshop on the EMA Draft Guideline on Validation of Bioanalytical Methods held April 15-16th 2010 in Brussels (Belgium) and jointly organised by the European Bioanalysis Forum (EBF) and the European Federation for Pharmaceutical Sciences (EUFEPS). Aim of the workshop was to discuss the current scientific knowledge in the area of bioanalysis, the regulatory requirements with special focus on the new Draft Guideline and their subsequent implementation to the work in bioanalytical laboratories. Comments on the Draft Guideline were presented and discussed with representatives from regulatory authorities in Europe. The workshop started with discussions on the scope of the Guideline and the need for implementation of GLP. A special focus was set on method validation of chromatographic procedures and subsequent study sample analysis. In addition, requirements for ligand-binding assays were briefly addressed. Intention of this Conference Report is to summarise important aspects of the discussions in order to draw certain conclusions, and to identify points which remain open and may require clarification at a later stage.

Capillary electrophoresis-mass spectrometry coupling versus micro-high-performance liquid chromatography-mass spectrometry coupling: a case study.

Capillary zone electrophoresis (CZE) and micro-high-performance liquid chromatography (mu-HPLC) coupled to electrospray ionisation (ESI) mass spectrometry were compared with respect to their applicability to problems arising in pharmaceutical drug research and development. Both techniques, which are similar with regard to their operational parameters, were coupled to an API III plus triple quadrupole mass spectrometer using laboratory-built interfaces. The results achieved with the two combinations were compared for sensitivity and general applicability to the quantitative analysis of pharmaceuticals in biological fluids. Midazolam, the 8-chloro-6-(2-fluoro-phenyl)-1-methyl-4H-imidazo-[1,5-a][1,4]-benzodiaze pine, and three of its metabolites were used as test compounds, either as standard solution or after sample clean-up from human plasma. Following different sample preparation routes, liquid-liquid extraction or solid-phase extraction, differences in detection limits as well as robustness in CZE or mu-HPLC coupled with ion spray mass spectrometry (IS-MS) were investigated. Detection limits of about 500 pg/ml for the drug and 2 ng/ml for the metabolites were achieved, using 1 ml of human plasma, only when liquid-liquid extraction was used for sample preparation. Sample preparation using the simpler and faster solid-phase extraction route resulted in deterioration of the separation or clogging of the columns. In all cases, when standard solutions or sample extracts were used, CZE-ESI-MS provided both different selectivity and greater sensitivity.

Reduced exposure variability of the CYP3A substrate simvastatin by dose individualization to CYP3A activity

This study aimed to demonstrate that the dose of a CYP3A substrate (simvastatin) can be adapted individually on the basis of CYP3A activity as assessed by midazolam metabolic clearance. In 18 healthy participants individual CYP3A activity was quantified using midazolam metabolic clearance both alone and during CYP3A inhibition with 40 mg ritonavir. Thereafter, simvastatin acid exposure was determined after a simvastatin standard dose (40 mg) and doses adapted to individual CYP3A activity at baseline and during CYP3A inhibition. Interindividual variability of CYP3A activity and simvastatin acid AUC0–24 was large and both correlated (r2 = 0.745, P < .001). The adapted simvastatin doses ranged from 25 to 80 mg and their administration reduced simvastatin variability fivefold. Despite the low adapted simvastatin dose of 12 mg during CYP3A inhibition with ritonavir, exposure increased (point estimate of 4.2 [90% CI: 3.15–5.61]) probably caused by additional OATP1B1 inhibition. CYP3A activity‐based dose adaptation can be used to reduce interindividual variability in simvastatin exposure.