Simultaneous determination of the major aniline metabolites in human urine by on-line turbulent flow chromatography HPLC-MS/MS

摘要

Aniline is used in many industrial processes (e.g. rubber, colorants, pesticides and Pharmaceuticals) and determining exposure to aniline is both of occupational and environmental relevance. In biological monitoring exposure to aniline has traditionally been assessed by measuring total aniline after hydrolysis in urine and/or covalent haemoglobin adducts in blood. However, we know from animal studies that the major urinary metabolite of aniline is N-acetyl-para-aminophenol (para-NAAP), representing 56%-76% of the dose. NAAP is also known as acetaminophen/paracetamol. Another urinary Aniline metabolite is N-phenylacetamide (NPA), representing approx. 3 % of the dose. Formation of other ring-hydroxylated and N-acetylated metabolites, e.g. N-acetyl-ortho-aminophenol (ortho-NAAP) has been reported. Human biomonitoring data on the body burden of the general population (and potentially exposed subpopulations) to para-NAAP, ortho-NAAP and NPA is scarce to nonexistent. Also, until now, no data on human metabolism of aniline is available. To be able to investigate human aniline metabolism and consecutively to determine the internal body burden of para-NAAP, ortho-NAAP and NPA in the general population, in workers exposed to aniline and users of Acetaminophen/Paracetamol, we developed a fast on-line turbulent flow HPLC-MS/MS-method with isotope dilution quantification in urine.For on-line extraction and enrichment of the analytes we used a Waters Oasis HLB precolumn. Chromatographic separation was performed on a Thermo Scientific Hypercarb column. Limits of quantification were 0.2-10 μg/L depending on the analyte. In a first application in urines of occupationally not aniline exposed volunteers we could quantify para-NAAP and ortho-NAAP in all samples analyzed, but NPA could not be quantified in any of the samples. However, in urine samples of persons exposed to aniline at levels typical for occupational settings we could quantify NPA, albeit at rather low levels.