Matrix interferences in the analysis of benzene in urine

Abstract

The analysis of benzene in urine of the general population or of exposed workers can be performed with different methods using the ‘purge and trap’ or ‘solid-phase microextraction’ techniques in combination with gas chromatographic analysis and photoionisation or mass spectrometric detection. The published results, however, are deeply conflicting. Differences in sample preparation by different research groups and our own preliminary observations prompted us to investigate pre-analytical and analytical factors potentially capable of modifying the urinary benzene quantification results. Benzene concentrations were measured in 20 urine samples in relation to different conditioning conditions (at 24, 40 and 80°C) and at basic or acid pH. Urinary protein concentrations were measured in the same samples. Urine heating at 80°C yields benzene concentrations on average five times higher than at 24°C. On acidification of urine, the benzene released increases up to 28-fold in comparison to that obtained at uncorrected ‘physiological’ pH. Despite a widely scattered data distribution, a statistically significant linear correlation was found between ‘heat-released’ and ‘acid-labile’ benzene values. There was no correlation between total urinary proteins present in ‘physiological’ concentrations (between 12 and 110 mg/l) and the different kinds of benzene in urine. Our results could perhaps be explained if it is supposed that part of the benzene in urine is absorbed onto sediment, or bound to specific proteins, or derived from parent molecules and is released with pH modification or heat administration. Our observations may also help to explain why the urinary benzene concentrations reported by different investigators vary considerably even when environmental levels are comparable.

Introduction

Benzene is a carcinogenic aromatic hydrocarbon [1], [2] widely used as a basic component for chemical synthesis and as an organic solvent in many industrial applications; it is also added to gasoline as an antiknocking agent. Benzene has become a ubiquitous pollutant of urban air, mainly related to vehicle exhaust fumes [3]. Smoking is a further source of exposure to benzene, because the substance is released during the burning of tobacco [4]. Individual occupational or non-occupational exposure to benzene can be measured by individual samplers (though this is a very expensive method) or by biological monitoring. The concentration of benzene in blood can be used as a good parameter of the ‘body burden’ of the solvent, absorbed during work or from ubiquitous pollution [5]. The urinary concentration of trans,trans-muconic or phenylmercapturic acids, which are two specific metabolites of benzene, can also be used in the biological monitoring of very low-level exposure to benzene [6], [7]. Recent research suggests that urinary benzene is also a reliable biological index of exposure, but comparison of data reported in the literature shows wide differences between the urinary concentrations found in relation to similar exposure to benzene [8], [9], [10]. Urinanalysis of benzene has been performed using different versions of the ‘head-space’ technique: ‘dynamic head space’ [8], [11], [12] and solid-phase microextraction both combined with gas chromatography with mass spectrometry [13] or photoionisation [14]. During the pre-analytical phase, urine samples were added with different salts and conditioned at room temperature [8], [11], [13] or at 60°C [9], [14]. Bearing in mind these operating conditions and our own unpublished experience, in this paper we report the effects of a number of analytical or physiological factors such as acidic or alkaline pH, temperature of conditioning and sampling, and concentration of proteins, capable of substantially influencing the measurement of benzene in urine.