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.

Reverse micellar aggregates: effect on ketone reduction. 2. Surfactant role.

Kinetics of the reduction of 3-chloroacetophenone (CAF) with sodium borohydride (NaBH(4)) were followed by UV-vis spectroscopy at 27.0 degrees C in different reverse micellar media, toluene/BHDC/water and toluene/AOT/water, and compared with results in an isooctane/AOT/water reverse micellar system. AOT is sodium 1,4-bis-2-ethylhexylsulfosuccinate, and BHDC is benzyl-n-hexadecyl dimethylammonium chloride. The kinetic profiles were investigated as a function of variables such as surfactant and NaBH(4) concentration and the amount of water dispersed in the reverse micelles, W(0) = [H(2)O]/[surfactant]. In all cases, the first-order rate constant, k(obs), increases with the concentration of surfactant as a consequence of incorporating the substrate into the interface of the reverse micelles where the reaction takes place. The reaction is faster at the cationic interface than at the anionic one probably because the negative ion BH(4)(-) is part of the cationic interface. The effect of the external solvent on the reaction shows that reduction is favored in the isooctane/AOT/water reverse micellar system than that with an aromatic solvent. This is probably due to BH(4)(-) being more in the water pool of the toluene/AOT/water reverse micellar system. The kinetic profile upon water addition depends largely on the type of interface. In the BHDC system, k(obs) increases with W(0) in the whole range studied while in AOT the kinetic profile has a maximum at W(0) approximately 5, probably reflecting the fact that BH(4)(-) is part of the cationic interface while, in the anionic one, there is a strong interaction between water and the polar headgroup of AOT below W(0) = 5 and, above that, BH(4)(-) is repelled from the interface once the water pool has formed. Application of a kinetic model based on the pseudophase formalism, which considers the distribution of the ketone between the continuous medium and the interface and assumes that reaction takes place only at the interface, has enabled us to estimate rate constants at the interface of the reverse micellar systems. At W(0) < 10, it was considered that NaBH(4) is wholly at the interface and, at W(0) >/= 10, where there are free water molecules, also the partitioning between the interface and the water pool was taken into account. The results were used to evaluate CAF and NaBH(4) distribution constants between the different pseudophases as well as the second-order reaction rate constant of the reduction reaction in the micellar interface.