Development and validation of a RP-HPLC method for the estimation of netilmicin sulfate and its related substances using charged aerosol detection.

Netilmicin is a semi-synthetic aminoglycoside antibiotic used against a broad spectrum of Gram-negative bacteria. A reversed-phase high-performance liquid chromatographic method has been developed to determine the composition of netilmicin sulfate and to estimate its related substances without pre- or post-column derivatization. A UV detector cannot be used to detect low levels of known and unknown related substances of netilmicin, as it has only a weak UV chromophore. A charged aerosol detector was used instead to obtain the high sensitivity that was necessary for the intended purpose of the method. This method can separate all related substances of netilmicin. A (10 cm x 4.6 mm) pentafluorophenyl high-performance liquid chromatographic column from Restek was used with a mobile phase consisting of (A) pentafluoropropionic acid-water-acetonitrile (0.1:96:4, v/v/v) and (B) trifluoroacetic acid-water-acetonitrile (1:96:4, v/v/v).

Development and validation of a RP-HPLC method for the determination of gentamicin sulfate and its related substances in a pharmaceutical cream using a short pentafluorophenyl column and a charged aerosol detector.

Gentamicin sulfate is a potent broad spectrum aminoglycoside antibiotic which is used as an active pharmaceutical ingredient (API) against both Gram-positive and Gram-negative bacteria. A reversed-phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated to determine the composition of gentamicin sulfate and to estimate its related substances (without any pre- or post-column derivatization) in a pharmaceutical cream. As gentamicin has a weak UV chromophore, it is not possible to detect low levels of known and unknown related substances of gentamicin using a UV detector. In this method, a Charged Aerosol Detector (CAD) was used to obtain high sensitivity that was necessary for the intended purpose of the method. This method can separate all the analogues of gentamicin including all known and unknown related substances of the API. A short (5cmx4.6mm) pentafluorophenyl HPLC column from Restek (Allure PFP) was used with an ion-pair gradient mobile phase consisting of (A) heptafluorobutyric acid:water:acetonitrile (0.025:95:5, v/v/v) and (B) trifluoroacetic acid:water:acetonitrile (1:95:5, v/v/v).

Activity-based probes for the proteomic profiling of metalloproteases.

Metalloproteases (MPs) are a large and diverse class of enzymes implicated in numerous physiological and pathological processes, including tissue remodeling, peptide hormone processing, and cancer. MPs are tightly regulated by multiple posttranslational mechanisms in vivo, hindering their functional analysis by conventional genomic and proteomic methods. Here we describe a general strategy for creating activity-based proteomic probes for MPs by coupling a zinc-chelating hydroxamate to a benzophenone photocrosslinker, which promote selective binding and modification of MP active sites, respectively. These probes labeled active MPs but not their zymogen or inhibitor-bound counterparts and were used to identify members of this enzyme class up-regulated in invasive cancer cells and to evaluate the selectivity of MP inhibitors in whole proteomes. Interestingly, the matrix metalloproteinase inhibitor GM6001 (ilomastat), which is currently in clinical development, was found to also target the neprilysin, aminopeptidase, and dipeptidylpeptidase clans of MPs. These results demonstrate that MPs can display overlapping inhibitor sensitivities despite lacking sequence homology and stress the need to evaluate MP inhibitors broadly across this enzyme class to develop agents with suitable target selectivities in vivo. Activity-based profiling offers a powerful means for conducting such screens, as this approach can be carried out directly in whole proteomes, thereby facilitating the discovery of disease-associated MPs concurrently with inhibitors that selectively target these proteins.

Photoinduced electron transfer cleavage of oxetane adducts of uracil and cytosine.

Oxetane adducts of 1,3-dimethyluracil and 1,N4,N4-trimethylcytosine were prepared and their behavior under photoinduced electron transfer was examined by fluorescence quenching, laser flash photolysis and product analysis. The excited state electron donor, N,N,N',N'-tetramethylbenzidine, was shown to photosensitize a net cycloreversion of these oxetanes to give the pyrimidine derivative and benzophenone. It is demonstrated that this reaction occurs via the anion radical of the oxetane and that the latter cleaves very rapidly (>10(7) s(-1)).