Controlled hydrolysis of ceftiofur sodium, a broad-spectrum cephalosporin; isolation and identification of hydrolysis products.

Ceftiofur sodium is the salt of (6R,7R)-7-{[(2-amino-4-thiazolyl)-Z-(methoxyimino)acetyl]amino}-3-{[(2-+ ++furanylcarbonyl)thio]methyl}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2- ene-2-carboxylic acid. This compound is very susceptible to acid, alkaline-, and enzyme-catalyzed hydrolysis, producing a number of unstable degradation products. In this report, we describe the preparation and identification of the hydrolysis products that are formed under controlled alkaline conditions. The primary hydrolysis product was desfuroyl ceftiofur, which is the most abundant metabolite in bovine blood. Desfuroyl ceftiofur was carefully oxidized with H2O2 to prepare the disulfide dimer, a urinary metabolite of ceftiofur sodium in the rat and cattle. Under acidic conditions, desfuroyl ceftiofur was converted to the corresponding thiolactone. The preparation of desacetyl cefotaxime, which is the oxygen analog of desfuroyl ceftiofur, is also described. Furoic acid was readily formed by hydrolytic cleavage of the thioester bond. Thiofuroic acid, formed by the less common cleavage on the alkyl side of the thioester bond, was also isolated.

Identification and determination of pirlimycin residue in bovine milk and liver by high-performance liquid chromatography-thermospray mass spectrometry.

Determinative and confirmatory methods of analysis for pirlimycin (I) residue in bovine milk and liver have been developed based on HPLC-thermospray (TSP) MS. Milk sample preparation consisted of precipitating the mill proteins with acidified acetonitrile followed by a solvent partitioning with a mixture of n-butyl chloride and hexane extraction of I from the aqueous phase into methylene chloride (MC), and solid-phase extraction clean-up. For liver, samples (2 g) were extracted with 0.25% trifluoroacetic acid in acetonitrile. The aqueous component was released from the organic solvent with n-butyl chloride. The aqueous solution was reduced in volume by evaporation, basified with ammonium hydroxide, then extracted with MC. The MC was evaporated to dryness and the dried residue reconstituted in 2.0 ml of 0.1 M ammonium acetate for analysis. A chromatographically resolved stereoisomer of I with TSP-MS response characteristics identical to I was used as an internal standard (I.S.) for quantitative analysis based on the ratio of peak areas of I to I.S. in the protonated molecular-ion chromatogram at m/z 411.2. The method for milk was validated by the analysis of control milk samples spiked with I at concentrations from 0.05 to 0.8 micrograms/ml. The overall recovery of pirlimycin across this concentration range was 95.4% +/- 8.7%. The limit of quantitation (LOQ) and limit of confirmation (LOC) of the method were validated to be 0.05 micrograms/ml and 0.10 micrograms/ml, respectively. The method for liver was validated by the analysis of control liver samples spiked with I at concentrations ranging from 0.025 to 1.0 micrograms/g. The overall recovery of pirlimycin was 97.6% +/- 5.1% in this concentration range. The validated limit of quantitation (LOQ) and limit of confirmation (LOC) of the method were 0.025 micrograms/g and 0.10 micrograms/g, respectively. Four diagnostic ions for I were monitored for confirmation: the pseudo-molecular ions (M+H)+ at m/z 411.2 (35Cl) and m/z 413.2 (37Cl), and fragment ions at m/z 375.2 and 158.1. Confirmatory criteria were defined for these assays.

HPLC-chemiluminescence and thermospray LC/MS study of hydroperoxides generated from phosphatidylcholine.

Lipid hydroperoxides generated from phosphatidylcholine (PC) by two commonly employed phosphatidylcholine hydroperoxide (PCOOH) generation methods were examined by HPLC-chemiluminescence (CL) and thermospray LC/MS assay. This HPLC-CL assay is specific for hydroperoxides. In the HPLC-CL chromatograms, a major peak eluted at 4.7 min for the samples generated by the photooxidation of PC in the presence of methylene blue. The direct LC/MS analysis of the hydroperoxides contained in this peak determined that the hydroperoxides are mono- and di-PCOOH. Quantitation showed that over 90% of the hydroperoxides generated by photooxidation are PCOOH. In contrast, a different major peak appeared at 3.7 min for the hydroperoxides generated by the incubation of PC with the azo compound AMVN. We determined by LC/MS analysis that the hydroperoxides contained in this peak were not equivalent to either mono- or di-PCOOH. Indeed, 70%-95% of the hydroperoxides generated by AMVN incubation were not PCOOH, but rather a large portion were AMVN-derived hydroperoxides. The hydroperoxides contained in the 4.7-min peak (i.e., PCOOH) were preferentially responsive to cytochrome c-luminol CL cocktail (about 100-fold more responsive than the hydroperoxides in the 3.7-min peak), whereas the hydroperoxides in the 3.7-min peak (including AMVN-derived hydroperoxide) were preferentially responsive to microperoxidase-isoluminol CL cocktail (about 20-fold more responsive than the PCOOH), suggesting a substrate specificity for the CL cocktail.

Kinetic evaluation of lipophilic inhibitors of lipid peroxidation in DLPC liposomes.

The authors have developed a kinetic method that allows one to obtain relative reactivity constants for lipophilic antioxidants in free radical systems. Two experimental model systems were developed: (a) a methanolic solution using AMVN as the free radical initiator and linoleic acid as the substrate, and (b) a multilamellar vesicle system composed of dilinoleoylphosphatidylcholine and AAPH as the substrate and the initiator, respectively. The use of these two systems allows researchers not only to determine the intrinsic reactivity of a potential antioxidant, but also to evaluate its potency in a membranous system where the contribution of the physical properties of the antioxidant to the inhibition of lipid peroxidation is important. These results show that all antioxidants tested acted in these systems as free radical scavengers, and they validate the synergism between intrinsic scavenging ability and membrane affinity and/or membrane-modifying physical properties in the inhibition of lipid peroxidation.

The use of salicylate hydroxylation to detect hydroxyl radical generation in ischemic and traumatic brain injury. Reversal by tirilazad mesylate (U-74006F).

Oxygen free radicals have been implicated as a causal factor in posttraumatic neuronal cell loss following cerebral ischemia and head injury. The conversion of salicylate to dihydroxybenzoic acid (DHBA) in vivo was employed to study the formation of hydroxyl radical (.OH) following central nervous system (CNS) injury. Bilateral carotid occlusion (BCO) in gerbils and concussive head trauma in mice were selected as models of brain injury. The lipid peroxidation inhibitor, tirilazad mesylate (U-74006F), was tested for its ability to attenuate hydroxyl radical formation in these models. In addition, U-74006F was studied as a scavenger of hydroxyl radical in an in vitro assay based on the Fenton reaction. For in vivo experimentation, hydroxyl radical formation was expressed as the ratio of DHBA to salicylate (DHBA/SAL) measured in brain. In the BCO model, hydroxyl radical formation increased in whole brain with 10 min of occlusion followed by 1 min of reperfusion. DHBA/SAL was also found to increase in the mouse head injury model at 1 h postinjury. In both models, U-74006F (1 or 10 mg/kg) blocked the increase in DHBA/SAL following injury. In vitro, reaction of U-74006F with hydroxyl radical gave a product with a mol wt that was 16 greater than U-74006F, indicative of hydroxyl radical scavenging. We speculate that U-74006F may function by blocking oxyradical-dependent cell damage, and thereby maintaining free iron (which catalyzes hydroxyl radical formation) concentrations at normal levels.

Two metabolites of anticonvulsant U-54494A: their anticonvulsant activity and interaction with sodium channel.

U-54494A, 3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzamide, has been shown to be a potent and long-acting anticonvulsant without analgesic or sedative effects on intact animals. The persistence of anticonvulsant activity after a decline in its concentration in the brain implies the conversion of the parent drug into active metabolites. In this study, two major metabolites of U-54494A, U-83892E [cis-N-(2-aminocyclohexyl)-3,4-dichlorobenzamide] and U-83894A [cis-N-(2-methylaminocyclohexyl)-3,4-dichlorobenzamide], were identified. The synthetic metabolites displayed anticonvulsant activity against electric shock in experimental animals and blocked voltage-gated sodium channel in N1E-115 neuroblastoma cells in voltage- and use-dependent manner by interacting with the inactivated channels as well as with the channels in the resting state (like the parent compound). These observations may provide one explanation for the long duration of the anticonvulsant activity of the parent compound U-54494A and further underscore the importance of voltage-dependent sodium channels in neuronal excitability, especially during seizures.