The major challenges encountered for the successful radioprofiling and identification are (1) chromatography for separation of major/minor metabolites (2) sufficient radioactivity in the separated peak (3) reliable mass spectral data of metabolites for structural elucidation. At times, the conventional methods (i.e., solvent extraction or SPE clean-up) wont be able to provide an appropriate concentrate to provide a reliable data. This presentation shares the results from case studies providing the desirable application of a large volume technique for radioprofiling and ID. Two injection valves (6-port and 10-port valves) were utilized to assist in the step-wise procedure: sample clean-up/enrichment on a trap column (TC; C18 as a stationary phase), back loading of sample onto an analytical column (AC; Same C18 as a stationary phase when used for radioprofiling or phenyl hexyl as a stationary phase when used for the isolation purpose), and sample analysis using the same gradient program as used for the initial radioprofiling. Flow was split with 80% of the effluent collected in the SSC well plates, and the plates were dried and counted to obtain reconstructed radiochromatograms. The results provided qualitatively and quantitatively comparable profiles for three test matrices (i.e., plasma, urine, and hepatocyte incubation). The peak integrity was not compromised up to 9 mL of plasma extract (3-mL plasma), which is equivalent to 60-fold concentration of plasma or 12-fold greater than that of on-column injection when prepared using a conventional method. Comparable profiles were obtained up to 20-fold for hepatocyte incubation sample and 400-fold for rat urine. However, the method didnt show much advantage for the fecal sample analysis due to its major biological matrices being non-polar. In addition, it showed that polar metabolites (e.g., un-retained compounds on the RP-LC column) may be lost during the loading step. It also showed that the recovery and peak shape of those metabolites with ionic properties (i.e., carboxylic acid) were shown to be more sensitive to the total amount of injected sample matrix. Thus, the LC column recovery as well as the peak shape comparison need to be determined with a representative sample prior to the use of the methodology for radioprofiling of all the samples within a study. Even with the large-volume injection, some compound still didnt yield a corresponding molecular ion peak, likely attributable to suppression of ionization by co-eluting matrices. Thus, the wells containing the radiopeak of interest collected from the SSC well plates, after being dried and counted, was reconstituted and utilized for the secondary LC (using a different stationary phase AC, pH and organic mobile phase). Secondary LC of the isolate successfully provided a distinctive molecular ion, demonstrating a significant reduction/removal of the co-eluting matrices, and enabled ID of the metabolite. The result demonstrated that the large-volume method, utilizing different types of TC and AC in conjunction with off-line radioactive detection, can be utilized for isolation/purification of metabolite(s) of interest for secondary analysis (i.e., derivatization, secondary LC, or NMR), providing additional clean-up without introducing unknown impurities as often experienced using a prep-HPLC approach.