Review of analytical methods for determining metabolites of polycyclic aromatic compounds (PACs) in fish bile.

Abstract

In fish, metabolites of polycyclic aromatic compounds (PACs) in gall bladder bile can be used as biomarkers for recent environmental exposure to PACs. These metabolites in the bile result from hepatic biotransformation processes whereby the lipophilic parent PACs are transformed in two steps (hydroxylation and subsequent conjugation) to more soluble forms and then passed to the gall bladder for elimination from the organism. As a biomarker of exposure, the determination of PAC metabolites in bile has several advantages over other assessment techniques. Several bile PACs are strong fluorophores and can thus be measured semi-quantitatively and very easily by means of straightforward fluorescence detection techniques. For example, fixed fluorescence detection and synchronous fluorescence scanning can be used for this purpose. These techniques are excellent for rapid screening of overall PAC exposure levels, but less suitable for the determination of individual compounds. The next level of resolution is reached with a high-performance liquid chromatographic (HPLC) separation of the conjugated bile metabolites prior to the fluorescence detection, so that individual metabolites and their patterns are discernable. Furthermore, PAC metabolites in bile can be enzymatically hydrolyzed to allow detection of free hydroxy PACs. After a centrifugation step the sample can be measured directly by HPLC/fluorescence (F). For analysis by gas chromatography/mass spectrometry (GC/MS) an extraction procedure is normally used to separate the hydroxy PACs from the bile matrix; derivatization can be used in order to increase separation and sensitivity. The latter set of methodological approaches can be used to determine individual metabolites, and a large number of hydroxy PACs are available as standards for accurate quantitation. In general, GC/MS methods are optimal for smaller compounds with 2–3 rings due to their better selectivity, while HPLC/F often provides better detection limits for larger metabolites with 4–5 rings. In the present review, the state-of-the-art for the various alternative methods is presented. Aspects of analytical quality control, interlaboratory comparability of data and the use of certified reference materials are also discussed. The advantages and limitations of each approach are discussed with respect to the use of PAC metabolites in bile as biomarkers of environmental PAC exposure in fish.