Mass-balance data were acquired using fluorescence spectrometry for 2-naphthol and three polycyclic aromatic hydrocarbon (PAH) metabolites using liquid-liquid-liquid microextraction and liquid-liquid microextraction systems. The PAH metabolites are very important biomarkers, and there has been no previously reported mass-balance data on these compounds with microextraction systems. In addition, the effects of two solvent systems used in the preparation of donor and acceptor phases were investigated. The effects of the solvent systems on the partitioning process were compared. The mass balance results showed that significant amounts of the hydrophobic metabolites were held in the pores of the microfiber in both the three-phase and two-phase microextraction systems. Based on the mass-balance data, a new enrichment factor was defined as the concentration of the solute trapped in the pores of the fiber divided by the initial concentration of the solute in the donor phase. Because of the relatively large amount of the solute in the pores, it is envisioned that the solutes could be easily extracted from the pores, the extraction solvent concentrated, and further separated by capillary electrophoresis or characterized by solid-matrix phosphorescence, solution fluorescence, or mass spectrometry.