Hepatic disposition of ximelagatran and its metabolites in pig; prediction of the impact of membrane transporters through a simple disposition model.

PURPOSE: The double prodrug, ximelagatran, is bioconverted, via the intermediates ethylmelagatran and N-hydroxymelagatran, to the direct thrombin inhibitor, melagatran. The primary aim of this study was to investigate the hepatic metabolism and disposition of ximelagatran and the intermediates in pig. A secondary aim was to explore a simple in vitro methodology for quantitative investigations of the impact of membrane transporters on the disposition of metabolized drugs. METHODS: Porcine S1 (supernatant fraction obtained by centrifuging at 1,000 g for 10 min) liver fractions and hepatocytes were incubated in the absence and presence of known membrane transporter inhibitors. The in vitro kinetics and disposition were determined by simultaneously fitting the disappearance of ximelagatran and the appearance of the metabolites. RESULTS: In S1 liver fractions, the metabolism was significantly inhibited by co-incubation of verapamil or ketoconazole, but not by erythromycin, quinine or quinidine. The disposition of ximelagatran and the intermediate metabolites in hepatocytes were influenced, to various degrees, by carrier-mediated transport processes. CONCLUSION: This work demonstrates that it is possible to obtain profound information on the general mechanisms that are important in the drug liver disposition using the combination of common in vitro systems and the simple disposition model proposed in this study.

Quantitative aspects of analyzing small molecules - monitoring singly or doubly charged ions? A case study of ximelagatran.

Precision, reproducibility and lower limit of quantitation (LLOQ) are important characteristics of a quantitative method. We have investigated these properties for Ximelagatran (Xi), which has a high tendency to form doubly charged ions in electrospray ionization (ESI), by studying the percentage of doubly charged species formed when varying the formic acid (FA) concentration, analyte concentration, amount of organic modifier and flow rate. It was found that the percentage of [Xi + 2H](2+) can be controlled to be more than 90% or less than 10% by varying the amount of FA present, and that the change between these values is dramatic. Furthermore, the percentage of [Xi + 2H](2+) formed decreases with increased analyte concentration and increased flow rate. No apparent relationship with the amount of organic modifier was found. The results have the implication that, by carefully controlling the selected parameters, the LLOQ, precision and reproducibility can be improved. We have compared the fragmentation of the singly and doubly charged species and concluded that the [Xi + 2H](2+) ion is more inclined to undergo fragmentation than [Xi + H](+). As a consequence, unusual instrumental settings had to be used for the experiments. The fragmentation patterns are to a great extent similar, but the doubly charged species is more inclined to generate low-mass product ions.

Online capillary solid phase extraction and liquid chromatographic separation with quantitative tandem mass spectrometric detection (SPE-LC-MS/MS) of ximelagatran and its metabolites in a complex matrix.

This work presents the development and validation of a fully automated quantitative analysis method of melagatran, its prodrug ximelagatran, and its major metabolites for the study of drug behavior in biofluids. The method involves online sample clean-up and enrichment on a C(4) capillary column followed by separation on a capillary C(18) column. Electrospray ionization tandem mass spectrometric detection in positive ion mode was performed with multiple reactions monitoring of eight different transients, divided into two time segments with four transients each. The structural similarity, the complexity of the matrix (pig liver extract) and the formation of isobaric fragment ions, made efficient chromatographic separation necessary. The analysis method provides valid accuracy (<9%; RSD%), precision (<8%; RSD%), linearity (<1.2 nM-1 microM; R(2)>0.999), limit of quantitation (<3.6 nM), retention repeatability (<1.2%; RSD%), selectivity, as well as analyte and column stabilities over a wide concentration range.

Comparing capillary electrophoresis-mass spectrometry fingerprints of urine samples obtained after intake of coffee, tea, or water.

Metabolomic fingerprinting is a growing strategy for characterizing complex biological samples without detailed prior knowledge about the metabolic system. A two-way analysis system with liquid separation and mass spectrometric detection provides detail-rich data suitable for such fingerprints. As a model study, human urine samples, obtained after intake of coffee, tea, or water, were analyzed with capillary electrophoresis electrospray ionization time-of-flight mass spectrometry (CE-ESI-TOF-MS). In-house-developed software (in Matlab) was utilized to manage and explore the large amount of data acquired (230 CE-MS runs, each with 50-100 million nonzero data points). After baseline and noise reduction, followed by suitable binning in time and m/z, the data sets comprised 9 and 14 million data points in negative and positive ESI mode, respectively. Finally, a signal threshold was applied, further reducing the number to about 100 000 data points per data set. A set of interactive exploratory tools, utilizing principal component analysis (PCA) and analysis of variance (ANOVA) results based on a general linear model, facilitated visual interpretation with score plots (for group assessment) and differential fingerprints (for "hot spot" detection). In the model study highly significant differences due to beverage intake were obtained among the 10 first principal components (p < 10(-6) for two of the components in both ESI modes). Especially, the contrasts between "coffee" and "tea or water" indicated several "hot spots" with highly elevated intensities (e.g., for uncharged masses 93, 94, 109, 119, 123, 132, 148, 169, 178, 187, 190, and 193) suitable for further analysis, for example, with tandem MS.