Cleys_2025_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_1256_124569

Tri-(2-ethylhexyl) trimellitate (TEHTM or TOTM) is an alternative plasticizer used as replacement for conventional phthalates, such as bis-(2-ethyl-hexyl) phthalate (DEHP) in plastic materials. Migration studies show a lower leaching potential from the polymer matrix, compared to DEHP. Additionally, toxicological studies showed lower negative health effects of TOTM. A sensitive bioanalytical method is necessary to further enable the exposure assessment of TOTM. In this study, we developed a quantitative method based on SPE and LC-MS/MS for analysis of ten urinary metabolites of TOTM, such as 1,4-di-(2-ethylhexyl) trimellitate (1,4-DEHTM), 2,4-di-(2-ethylhexyl) trimellitate (2,4-DEHTM), 1-mono-(2-ethylhexyl) trimellitate (1-MEHTM), 2-mono-(2-ethylhexyl) trimellitate (2-MEHTM), 1-mono-(2-ethyl-5-carboxypentyl) trimellitate (5Cx-1-MEPTM), 2-mono-(2-ethyl-5-carboxypentyl) trimellitate (5Cx-2-MEPTM), 2-mono-(2-ethyl-5-hydroxyhexyl) trimellitate (5OH-2-MEHTM), and 1-mono-(2-ethyl-5-hydroxyhexyl) trimellitate (5OH-1-MEHTM), and 1-mono-(2-carboxymethylhexyl) trimellitate (2Cx-MMHTM). The method was validated according to the current 'Bioanalytical Method Validation guidelines' from European Medicines Agency (EMA). All validation criteria were successfully passed, except for 2Cx-MMHTM and 5Cx-2-MEPTM, which were discarded due to unsatisfactory validation results and low clinical relevance. Additionally, 1,4- and 2,4-DEHTM were successfully validated as a sum of two isomers. The validated method was applied to a pilot set of 30 urine samples from newborn patients. Significant higher urinary concentrations were found for 5Cx-1-MEPTM, 5OH-1-MEHTM, 5OH-2-MEHTM, 5oxo-1-MEHTM and 2-MEHTM in prematurely born babies from the neonatal intensive care unit (NICU) compared to term-born newborns. The current study expanded the range of analytes to eight TOTM metabolites which can be simultaneously analysed in urine samples. It also showed its clinical importance by analysing urine samples from newborns admitted to the NICU.