A comparison of HPLC with fluorescence detection and fixed wsavelength fluorescence methods for the determination of polycyclic aromatic hydrocarbon metabolites in fish bile

Fish exposed to polycyclic aromatic hydrocarbons (PAHs) accumulate only a small amount of PAH in their tissues. Hydrophobic PAHs are readily transformed into hydrophilic metabolites and excreted in bile. Bile samples of five fish species were collected from the Baltic Sea and analyzed using two methods: high-performance liquid chromatography (HPLC) with fluorescence detection and a fixed wavelength fluorescence method. With the HPLC method PAH compounds were quantitated after hydrolysis, and with the fluorescence method without enzyme-assisted hydrolysis. In HPLC analysis the major metabolite in all fish species, 1-hydroxypyrene, was the only metabolite detected in perch and salmon samples. 1-Hydroxyphenanthrene was detected in a few flounder and in most eelpouts. The correlation between the two methods for pyrene-type metabolites was good (r2 = 0.773). Polycyclic aromatic hydrocarbons (PAHs) are a group of organic compounds that are present in air, soil, and water as pollutants. PAH contamination is a matter of concern for marine ecosystems, especially near urban and industrialized areas. Fish absorb these hydrophobic compounds through the gills and body surface but also may be exposed to PAHs by ingestion of food or through contaminated sediment. Fish exposed to PAHs show only trace amounts of these compounds in their tissues (1), because PAHs are quite rapidly transformed into more hydrophilic metabolites that are excreted. Therefore, PAH metabolites are usually determined in fish bile, where they are concentrated and stored prior to subsequent excretion. Bile PAH metabolite analysis provides information about the actual exposure of fish to PAH compounds, and reveals the state and suitability of the marine environment for fish. Several analytical techniques are available for estimating fluorescent aromatic compounds (FAC) in fish bile. For biomonitoring purposes a fast and cost-effective method is required, since monitoring often involves analysis of large numbers of samples. Krahn et al. (2) introduced a high-performance liquid chromatography (HPLC) method for quantifying PAH metabolites in fish bile. Later, a method based on synchronous fluorescence spectrometry (SFS) was described (3). Ariese et al. (4) compared SFS and HPLC as methods for analyzing PAH metabolites in bile samples from flounder exposed to contaminated sediments in a mesocosm study. Lin et al. (5) made a comparison between HPLC and a fixed wavelength fluorescence (FF) method for benzo[a]pyrene- and naphthalene-type metabolites. Aas et al. (6) have used the latter for detecting metabolites of polyaromatic hydrocarbons in fish bile. The FF method involves diluting bile with ethanol or water followed by fixed wavelength fluorescence detection andwas presented as a promising tool for PAH analysis because of its simplicity and low cost. The method is considered semiquantitative because, at a certain wavelength pair, there may be several interfering compounds that contribute to the fluorescence instead of one particular PAH metabolite. In this article,wecompare a modifiedHPLCmethod with fluorescence detection (HPLC-F) against the FF method for detecting pyrene-type metabolites in fish bile. Over 250 bile samples from five fish species caught in different areas of the Baltic Sea were analyzed using the two techniques. In the HPLC-F method, PAH compounds were quantitated as hydroxy-PAH after the sample had been enzymatically hydrolyzed. With the FF method, PAH compounds were detected without hydrolysis.