LC-Q-TOF-MS/MS determination of darunavir and its metabolites in rat serum and urine: application to pharmacokinetics.

A simple, rapid and reliable liquid chromatography coupled with quadrupole time of flight mass spectrometry (LC-Q-TOF-MS/MS) method was developed and validated for simultaneous determination of darunavir and its metabolites in rat serum and urine. The separation was accomplished on an Agilent RP-18 (250×4.6mm, 5µm) column using 20mM ammonium acetate and methanol (40:60, v/v) as a mobile phase at a flow rate of 1.0mL/min in an isocratic mode. The [M+H](+) ions of darunavir (m/z 548) and metabolites-I (m/z 392) were monitored in positive mode of ionization, while [M-H](-) ion of metabolite-II (m/z 172) in negative mode selectively. The matrix effects of rat serum and urine were found to be negligible and the recoveries were 87-93% for all the analytes. The short and long term stability of darunavir and its metabolites was within acceptable limits and the lower limits of quantification were in the range of 3.63-5.24ng/mL with a linear range of 5-5000ng/mL in rat serum as well as urine. The method exhibited good intra- and inter-day performance in terms of 2.54-8.92% precision and 0-5% accuracy. The method was successfully applied to a single-dose pharmacokinetic study of darunavir boosted with ritonavir in Wistar rats.

Development and validation of an LC-MS/MS method for determination of methanesulfonamide in human urine.

A sensitive and selective liquid chromatographic method coupled with tandem mass spectrometry (LC-MS/MS) was developed and validated for the quantification of methanesulfonamide (MSA) in human urine. MSA is a potential in vivo metabolite of reparixin, a specific inhibitor of the CXCL8 biological activity. In this study, a simple derivatization procedure with a new reagent, N-(4-methanesulfonyl-benzoyl)-imidazole, was set up to enable MSA and the internal standard (I.S.), ethanesulfonamide (ESA), to be analysed by LC-MS/MS. After derivatization, samples were evaporated and reconstituted in 30% acetonitrile, aq. MSA and I.S. derivatives were separated by reversed phased HPLC (high performance liquid chromatography) on a Luna 5micro C18 column and quantitated by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MR M) in the negative ion mode. The most intense [M-H](-) MRM transition of derivatized MSA at m/z 276.2-->197.2 was used for quantitation and the transition at m/z 290.2-->211.2 was used to monitor derivatized ESA. The method was linear over the concentration range from 1 to 100 microg/ml, with a lower limit of quantitation of 1 microg/ml. The intra- and inter-day precisions were less than 5.5% and 10.1%, respectively, and the accuracies were between -4.0% and +11.3%. The method was successfully applied to quantify levels of MSA in human urine after intravenous administration of reparixin to healthy volunteers.

Metabolism distribution and excretion of a matrix metalloproteinase-13 inhibitor, 4-[4-(4-fluorophenoxy)-benzenesulfonylamino]tetrahydropyran-4-carboxylic acid hydroxyamide (CP-544439), in rats and dogs: assessment of the metabolic profile of CP-544439 in plasma and urine of humans.

The metabolism and disposition of 4-[4-(4-fluorophenoxy)-benzenesulfonylamino]tetrahydropyran-4-carboxylic acid hydroxyamide (CP-544439), a selective inhibitor of matrix metalloproteinase-13, was investigated in rats and dogs following oral administration of [(14)C]CP-544439. Both species showed quantitative recovery of the radiolabel, and feces was the major route of excretion. Whole-body autoradioluminography study in rats suggested distribution of CP-544439 in all tissues except central nervous system. The radiolabel was rapidly eliminated from most tissues except the periodontal ligament. Metabolism of CP-544439 was extensive in both species. Only 8.4 and 1.5% of the total dose constituted unchanged CP-544439 in the rat and dog, respectively. Similarly, pharmacokinetic analysis of [(14)C]CP-544439 and unchanged CP-544439 indicated that the exposure of the parent drug was 16 and 6.5% of the total radioequivalents in rat and dog, respectively. Metabolic profiling revealed that CP-544439 was primarily metabolized via glucuronidation, reduction, and hydrolysis. Glucuronidation was the primary route of metabolism in dogs, whereas reduction of the hydroxamate moiety was the major pathway in rats. Human plasma and urine obtained from a dose escalation study in healthy human volunteers were also analyzed in this study to assess the metabolism of CP-544439 in humans and ensure that selected animal species were exposed to all major metabolites formed in humans. Analysis suggested that CP-544439 was metabolized via all three pathways in humans consistent with rat and dog; however, the glucuronide conjugate M1 was the major circulating and excretory metabolite in humans. Preliminary in vitro phenotyping studies indicated that glucuronide formation is primarily catalyzed by UGT1A1, 1A3, and 1A9.