Optimisation of collection, storage and preparation of rat plasma for 1H NMR spectroscopic analysis in toxicology studies to determine inherent variation in biochemical profiles.

Biofluid 1H NMR spectroscopy has been assessed as a tool for toxicological investigations for almost two decades, with most studies focussing on urinary changes. This study has examined variations in the 1H NMR spectroscopy spectra of plasma collected from control rats at different times of the day. The collection, preparation and storage of samples were optimised and potential sources of variation in samples taken for toxicology studies identified. Plasma samples were collected into heparinised containers and analysed following a standard dilution with D(2)O. The value of deproteinising plasma with acetonitrile to look at low molecular weight metabolites has also been assessed. Variations in lactate and citrate levels in whole blood plasma were found and are consistent with the observation that lactate is one of the most variable metabolites in human plasma. Lipids levels also varied, in particular higher levels of lipids were found in spectra from male rats compared to female rats, and in samples collected in the morning following the feeding period. No significant changes were identified in samples which were snap-frozen and stored for up to 9 months at -80 degrees C. More changes were observed after storage at 4 degrees C or room temperature, including an increase in glycerol and choline levels, which may have resulted from lipid hydrolysis.