A rapid and simple method for the analysis of 1-hydroxypyrene glucuronide: a potential biomarker for polycyclic...

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 1-hydroxypyrene in a single analysis. In comparison to other analytical methods for detecting pyrene metabolites, this new method does not require an overnight enzymatic hydrolysis step and is 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 1-hydroxypyrene and free 1-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 microgram/g creatinine. Low levels of the sulfate conjugate (0.002-0.06 microgram/g creatinine) were detected in four of the samples while free 1-hydroxypyrene (0.07-0.2 microgram/g creatinine) was detected in two of the five urine samples. Urine from occupationally exposed workers was also analyzed for 1-hydroxypyrene following enzymatic hydrolysis using the standard approach. Levels of 1-hydroxypyrene ranged from 0.51-1.17 micrograms/g creatinine. Comparison of the fluorescence intensities of 1-OH P-GlcUA and 1-OH P-Sul to 1-hydroxypyrene demonstrated that the glucuronide conjugate is 3-fold more fluorescent and the sulfate conjugate is 4-fold more fluorescent than 1-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.