Virtual chromatographic resolution enhancement in cryoflow LC-NMR experiments via statistical total correlation spectroscopy.

A new approach to enhancing information recovery from cryogenic probe "on-flow" LC-NMR spectroscopic analyses of complex biological mixtures is demonstrated using a variation on the statistical total correlation spectroscopy (STOCSY) method. Cryoflow probe technology enables sensitive and efficient NMR detection of metabolites on-flow, and the rapid spectral scanning allows multiple spectra to be collected over chromatographic peaks containing several species with similar, but nonidentical, retention times. This enables 1H NMR signal connectivities between close-eluting metabolites to be identified resulting in a "virtual" chromatographic resolution enhancement visualized directly in the NMR spectral projection. We demonstrate the applicability of the approach for structure assignment of drug and endogenous metabolites in urine. This approach is of wide general applicability to any complex mixture analysis problem involving chromatographic peak overlap and with particular application in metabolomics and metabonomics.

Formation of toxic metabolites from thiabendazole and other thiazoles in mice. Identification of thioamides as ring cleavage products.

The metabolism of three nephro- or hepatotoxic thiazoles--2-(thiazol-4-yl)benzimidazole (thiabendazole) (1a), 4-tert-butyl-2-methyl-thiazole (1b), and 2-(p-methoxyphenyl)-4-methylthiazole (1c)--was examined in mice with special regard to the formation of ring cleavage products. By GC/MS analyses of derivatized metabolites and comparison with authentic samples, thioformamide and benzimidazol-2-ylglyoxal as the accompanying fragment were identified as urinary metabolites in mice dosed with 1a. Similarly, 1b produced thioacetamide and tert-butylglyoxal, and 1c produced p-methoxy-thiobenzamide (and its S-oxide) and methylglyoxal. These results could be explained by the postulated metabolic pathways where thiazoles would undergo microsomal epoxidation of the C = C double bond and, after being hydrolyzed, the resulting epoxide would then be decomposed to form the corresponding thioamides and alpha-dicarbonyl fragments.

Possible role of thioformamide as a proximate toxicant in the nephrotoxicity of thiabendazole and related thiazoles in glutathione-depleted mice: structure-toxicity and metabolic studies.

In mice depleted of GSH by treatment with buthionine sulfoximine (BSO), thiabendazole (TBZ) causes renal injury characterized by an increase in serum urea nitrogen (SUN) concentration and by tubular necrosis. Previous studies have shown that TBZ requires metabolic activation before it produces nephrotoxicity and that the structure contributing to the toxicity of TBZ is the thiazole moiety of the molecule. TBZ and its thiazole analogues were examined for the ability to increase SUN concentration and serum alanine aminotransferase activity in GSH-depleted mice. Unsubstituted thiazole and thiazoles with 4- and/or 5-, and no 2-, substituents caused marked increases in SUN concentration, suggesting nephrotoxicity. Furthermore, the nephrotoxic potency of these thiazoles decreased with the increasing number and bulk of the 4- and/or 5-substituents. On the other hand, the target organ (the kidney or liver) and the toxic potency of 4-methylthiazoles were markedly altered with the type of substituents at the 2-position. These observations and the known toxicity of thiono-sulfur compounds led us to the hypothesis that the nephrotoxic thiazoles, which lack 2-substituents, would undergo microsomal epoxidation of the C-4,5 double bond and, after being hydrolyzed, the resulting epoxide would then be decomposed to form thioformamide, a possibly toxic metabolite. Evidence for this hypothesis was provided by the results that thioformamide and tert-butylglyoxal as the accompanying fragment were identified as urinary metabolites in mice dosed with 4-tert-butylthiazole and that thioformamide caused a marked increase in SUN concentration when administered to mice in combination with BSO.