Directly coupled high-performance liquid chromatography and nuclear magnetic resonance spectroscopic with chemometric studies on metabolic variation in Sprague-Dawley rats

We report here the first combined use of NMR-PR (pattern recognition) analy sis and directly coupled HPLC-NMR analysis to identify metabolic subpopulat ions in normal laboratory animals and their discriminating endogenous urina ry biomarkers. Urine samples obtained from control Sprague-Dawley rats (n = 68) were analyzed using H-1 NMR spectroscopy and principal components (PC) analysis to investigate physiological variability. Two distinct subpopulat ions of animals were classified based on metabolite excretion profiles. Ana lysis of the PC loadings established the spectral regions that were respons ible for classification of the subpopulations and was used to direct the id entification of biomarkers using a directly coupled HPLC-NMR analysis. One population had low urinary hippurate levels together with an increased conc entration of 3-(3-hydroxyphenyl)propionic acid (3-HPPA) and 3-hydroxycinnam ic acid (3-HCA). The other subpopulation excreted high levels of hippurate. Thus, we report the bimodal occurrence of hippuric acid and chlorogenic ac id metabolites in a genetically homogeneous population of rats maintained u nder identical conditions, which may have significance in relation to the u nderstanding of the consequences of biochemical variation in animals used f or drug toxicity testing. (C) 2001 Academic Press.