Human biomonitoring of 2,4-di-tert-butylphenol: determination of the parent substance and a novel, specific metabolite in urine by UHPLC-MS/MS.

2,4-Di-tert-butylphenol (DTBP) is a degradation product of the antioxidant plastic additive tris(2,4-di-tert-butylphenyl)phosphite and was shown to migrate from different plastics. Additionally, several natural sources for DTBP are known, together leading to exposure of the general population. Human biomonitoring (HBM) can be applied to determine the prevalence and also the magnitude of internal DTBP exposure. To this effect, we have developed a column-switching UHPLC-MS/MS method permitting the quantification of DTBP and a postulated metabolite, 2-tert-butyl-4-(2-hydroxy-1,1-dimethylethyl)phenol (DTBP-4-OH), in urine, after enzymatic cleavage of glucuronides. Since DTBP-4-OH was not available commercially, we synthesised it as a reference material, along with a deuterated analogue to be used as an internal standard for the analytical method. The method was validated, inter alia with regard to its precision, accuracy, robustness, and limit of quantification (LOQ). For both biomarkers, an LOQ of 1 µg l-1 was derived. Analysis of samples from a human metabolism study enabled the optimisation of the sample preparation and the confirmation of the metabolite DTBP-4-OH as a biomarker. As DTBP analysis is flawed by contamination due to the ubiquitous use of plastic materials, the analysis of a human-specific metabolite such as DTBP-4-OH can be particularly useful to increase the specificity of HBM.