Currently the use of a radiolabeled compound provides the most accurate assessment of the exposure of animals or humans to metabolites. In general, reverse phase HPLC coupled with radioactive detection is used to obtain metabolite profiles in different matrices in order to achieve the separation of all the major metabolites. However, one HPLC method may not be applicable for the separation of all the metabolites if the metabolite profile is complex. Sometimes, the major radioactive peak can contain multiple components, and the co-eluting metabolites can be isolated and subjected to secondary chromatography. The secondary chromatography may include the modification of HPLC conditions or the use of TLC instead of HPLC. However, TLC is not desirable to be used for quantitative analyses because many metabolites can undergo degradation on the TLC plate due to the direct exposure to air. Most co-eluting metabolites can be successfully separated by modification of HPLC conditions. However, when one or two major components in the region undergo conformational change, the change of HPLC condition may not be sufficient to separate the co-eluting compounds. Metabolites which contain the 1-hydroxy-tetrahydropyran moiety can exist in an open-chain (acyclic) form or a cyclic hemiacetal form in equilibrium, resulting in a broad peak (ca. 10 min) containing two or three different forms for each molecule. For the drug in this study, the broad peak region was isolated, and the isolate was subjected to derivatization using butyl PABA/sodium cyanoborohydride to prepare reduced Schiff base products of the two 1-hydroxy-tetrahydropyran-containing metabolites. Radioprofiles and LC/MS data before and after the derivatization were obtained. The derivatization was complete and RP-HPLC analysis of the derivatization mixture could successfully separate the five metabolites, which enabled the accurate assessment of their AUC values. The ID of each peak was then obtained using LC/MS. Since the reaction condition was mild, three other metabolites co-eluting in the same region remained intact. A TLC radiochromatogram of the isolate was also obtained, and as expected it showed degradation of some of the metabolites on the TLC plate.