The application of microbore UPLC/oa-TOF-MS and 1H NMR spectroscopy to the metabonomic analysis of rat urine following the intravenous administration of pravastatin.

The metabonomic effects of hepatotoxic doses of pravastatin on the urinary metabolic profiles of female rats have been investigated using ultra performance liquid chromatography (UPLC)-oa-TOF-MS and, independently, by (1)H NMR spectroscopy. UPLC was performed using a 1 mm microbore column packed with 1.7 microm particles. Examination of the data obtained from the individual animals, aided by statistical interpretation of the data, made it possible to identify potential markers for toxicological effects, with both NMR and UPLC-MS analysis highlighting distinct changes in the urinary metabolite profiles. These markers, which included elevated taurine and creatine, as well as bile acids, were consistent with hepatotoxicity in some animals, and this hypothesis was supported by histopathological and clinical chemistry findings. The analytical data from both techniques could be used to define a metabolic "trajectory" as toxicity developed and to provide an explanation for the lack of hepatotoxicity for one of the animals. The two analytical approaches (UPLC-MS and NMR) were found to be complementary whilst the use of a 1mm i.d. x 100 mm column reduced the amount of sample required for analysis to 2 microL, compared with 10 microL for a 2.1mm i.d. x 100 mm column. The 1mm i.d. column also provided increased signal-to-noise without loss of chromatographic efficiency.

Methyl propionate radical cation.

Examination of the reactions of the long-lived (>0.5-s) radical cations of CD3CH2COOCH3 and CH3CH2COOCD3 indicates that the long-lived, nondecomposing methyl propionate radical cation CH3CH2C(O)OCH 3 (+·) isomerizes to its enol form CH3CH=C(OH)OCH 3 (+·) (ΔH isomerization ≃ -32 kcal/mol) via two different pathways in the gas phase in a Fourier-transform ion cyclotron resonance mass spectrometer. A 1,4-shift of a ß-hydrogen of the acid moiety to the carbonyl oxygen yields the distonic ion (·)CH2CH2C(+) (OH)OCH3 that then rearranges to CH3CH=C(OH)OCH 3 (+·) probably by consecutive 1,5- and 1,4-hydrogen shifts. This process is in competition with a 1,4-hydrogen transfer from the alcohol moiety to form another distonic ion, CH3CH2C(+)(OH)OCH 2 (·) , that can undergo a 1,4-hydrogen shift to form CH3CH=C(OH)OCH 3 (+·) . Ab initio molecular orbital calculations carried out at the UMP2/6-31G** + ZPVE level of theory show that the two distonic ions lie more than 16 kcal/mol lower in energy than CH3CH2C(O)OCH 3 (+·) . Hence, the first step of both rearrangement processes has a great driving force. The 1,4-hydrogen shift that involves the acid moiety is 3 kcal/mol more exothermic (ΔH isomerization=-16 kcal/mol) and is associated with a 4-kcal/mol lower barrier (10 kcal/mol) than the shift that involves the alcohol moiety. Indeed, experimental findings suggest that the hydrogen shift from the acid moiety is likely to be the favored channel.