A rapid and simple method for the analysis of 1-hydroxypyrene glucuronide: A potential biomarker for polycyclic aromatic hydrocarbon exposure

The present study describes a simple method of analyzing metabolites of pyrene in urine, This method is capable of detecting the glucuronic acid and sulfate conjugates of pyrene as well as free l-hydroxypyrene in a single analysis, In comparison to other analytical methods for detecting pyrene metabolites, this new method does not require an overnight enzymatic hydrolosis step and is a much more rapid method of analysis, The newly developed procedure involves solid phase extraction of pyrene metabolites followed by separation using HPLC with a phenyl modified reverse phase column and an acidic buffer and acetonitrile gradient elution system, Metabolites were detected using a fluorescence detector with wavelength conditions optimized for each metabolite. This method resulted in baseline separation of the glucuronic acid (1-OH P-GlcUA) and sulfate conjugate (1-OH P-Sul) of l-hydroxypyrene and free l-hydroxypyrene (1-OH P), The potential of this method for use in monitoring human exposure to mixtures of PAHs was evaluated by analyzing urine obtained from five individuals working in a coal gasification plant, 1-OH P-GlcUA was detected as the major metabolite in the urine of all the five workers, This metabolite accounted for 80-100% of the total pyrene metabolites excreted in urine. 1-OH P-GlcUA levels ranged from 0.31-0.94 mu g/g creatinine. Low levels of the sulfate conjugate (0,002-0,06 mu g/g creatinine) were detected in four of the samples while free l-hydroxypyrene (0.07-0.2 mu g/g creatinine) was detected in two of the five urine samples, Urine from occupationally exposed workers was also analyzed for l-hydroxypyrene following enzymatic hydrolysis using the standard approach, Levels of l-hydroxypyrene ranged from 0.51-1.17 mu g/g creatinine, Comparison of the fluorescence intensities of 1-OH P-GlcUA and 1-OH P-Sul to l-hydroxypyrene demonstrated that the glucuronide conjugate is 3-fold more fluorescent and the sulfate conjugate is 4-fold more fluorescent than l-hydroxypyrene, These results indicate that conjugates of pyrene, specifically, 1-OH P-GlcUA can potentially be used as a more sensitive biomarker of exposure to PAHs.