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 o f 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 de tecting pyrene metabolites, this new method does not require an overni ght enzymatic hydrolosis step and is a much more rapid method of analy sis, The newly developed procedure involves solid phase extraction of pyrene metabolites followed by separation using HPLC with a phenyl mod ified reverse phase column and an acidic buffer and acetonitrile gradi ent elution system, Metabolites were detected using a fluorescence det ector with wavelength conditions optimized for each metabolite. This m ethod resulted in baseline separation of the glucuronic acid (1-OH P-G lcUA) 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 monit oring human exposure to mixtures of PAHs was evaluated by analyzing ur ine obtained from five individuals working in a coal gasification plan t, 1-OH P-GlcUA was detected as the major metabolite in the urine of a ll the five workers, This metabolite accounted for 80-100% of the tota l pyrene metabolites excreted in urine. 1-OH P-GlcUA levels ranged fro m 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 u sing 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 conju gate is 4-fold more fluorescent than l-hydroxypyrene, These results in dicate that conjugates of pyrene, specifically, 1-OH P-GlcUA can poten tially be used as a more sensitive biomarker of exposure to PAHs.