The most used positron emission tomography (PET) tracer, 2-[F-18]fluoro-2-deoxy-o-glucose ([F-18]FDG), is a glucose analogue that is used to measure tissue glucose consumption. Traditionally, the Sokoloff model is the basis for [F-18]FDG modeling. According to this model, [F-18]FDG is expected to be trapped in a cell in the form of [F-18]FDG-6-phosphate ([189FDG-6-P). However, several studies have shown that in tissues, [F-18]FDG metabolism goes beyond [F-18]FDG-6-P. Our aim was to develop radioHPLC and radioTLC methods for analysis of [F-18]FDG metabolites from tissue samples. The radioHPLC method uses a sensitive on-line scintillation detector to detect radioactivity, and the radioTLC method employs digital autoradiography to detect the radioactivity distribution on a TLC plate. The HPLC and TLC methods were developed using enzymatically in vitro-produced metabolites of ["HMG as reference standards. For this purpose, three [F-18]FDG metabolites were synthesized: [F-18]FDG-6-P, [F-18]FD-PGL, and [F-18]FDG-1,6-P2. The two methods were evaluated by analyzing the [F-18]FDG metabolic profile from rodent ex vivo tissue homogenates. The HPLC method with an on-line scintillation detector had a wide linearity in a range of 5 Bq-5 kBq (LOD 46 Bq, LOQ 139 Bq) and a good resolution (Rs > 1.9), and separated [F-18]FDG and its metabolites clearly. The TLC method combined with digital autoradiography had a high sensitivity in a wide range of radioactivity (0.1 Bq-2 kBq, LOD 0.24 Bq, LOQ0.31 Bq), and multiple samples could be analyzed simultaneously. As our test and the method validation with ex vivo samples showed, both methods are useful, and at best they complement each other in analysis of [F-18]FDG and its radioactive metabolites from biological samples. (C) 2017 Elsevier B.V. All rights reserved.