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. 2026 Jan;418(1):247-260.
doi: 10.1007/s00216-025-06217-z. Epub 2025 Nov 24.

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

Christoph Schmidtkunz  1 , Katja Küpper  1 , Frank Leßmann  2 , Volkmar Wendisch  2 , Oliver Seidelmann  2 , Edgar Leibold  3 , Melanie Flach  3 , Isabell Schönrath  4 , Gabriele Leng  1
Affiliations

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

Christoph Schmidtkunz et al. Anal Bioanal Chem. 2026 Jan.

Abstract

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.

Keywords: Antioxidants; Enzymatic hydrolysis; Online solid-phase extraction (SPE); Plastic additives; Sample preparation; Urinary metabolite.

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Conflict of interest statement

Declarations. Ethics approval and consent to participate: Ethics approval and source of biological material: We have used samples from an oral metabolism study to examine the deconjugation of the metabolites (3.5). This study conducted for the purpose of metabolism investigation and method development complied with the Code of Ethics of the World Medical Association (1964 Declaration of Helsinki, including its later amendments). It had been reviewed by the Ethics Commission of the Medical Faculty of the Ruhr-Universität Bochum under the reg. no. 19-6687 – BR. After receiving the study design in written form, each participant gave written informed consent. Apart from that, we sourced ten spot urine samples from persons without occupational exposure to 2,4-DTBP in the occupational biomonitoring programmes of Currenta to spike individual urine specimens for the robustness measurements (“Robustness and selectivity”). The participants in these programmes are comprehensively informed by occupational physicians of the Department of Health Protection about the biomonitoring examinations at the Institute of Biomonitoring. Participation is strictly voluntary for each worker and requires his or her informed consent. Competing interests: Edgar Leibold and Melanie Flach are employed by BASF SE. BASF SE is the registrant of the substance under EU REACH. All authors affiliated to Currenta or ChiroBlock declare no conflict of interest related to this work.

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