Determination of Benzotriazole Metabolites in Urine

A list of chemical compounds and reagents used is available in SI (section Chemicals and reagents). Frequently used BTs and their potential metabolites were identified on the basis of a literature search and a total number of six BTRs (1-H-benzotriazole [1H-BTR], 4-OH-benzotriazole [4OH-BTR], 1-methyl-benzotriazole [1M-BTR], 4-methyl-benzotriazole [4M-BTR], 5-methyl-benzotriazole [5M-BTR] and xylyltriazole [XTR]) and two BTHs (2-hydoxy-benzothiazole [2OH-BTH] and 2-amino-benzothiazole [2NH2-BTH]) were analysed. The isomers 4M-BTR and 5M-BTR were expressed as their sum (4/5M-BTR). The sum of free and conjugated forms in urine was determined following a procedure reported in a previous study (Bláhová et al. 2023 (link)) with modifications. Urine samples were thawed and vortexed, and 500 µL of urine sample was introduced into a 2 mL plastic tube. 10 µL of a mixture of isotopically labelled internal standards (d4-1H-BTR and d5-atrazine) were added to achieve the concentration in samples 10 and 2 ng/mL, respectively. Next, the samples were spiked with β-glucuronidase (500 µL, 1000 U/mL in 1 M CH₃COONH₄, from Helix pomatia), vortexed, and incubated overnight (37 °C, 170 rpm) to release free forms via enzymatic de-conjugation. The enzymatic reaction was stopped by freezing at − 80 °C (6 h). The samples were then freeze-dried and extracted with 500 µL of isopropanol. Different types of β-glucuronidase (E.coli) and extraction solvents (based on literature search: acetonitrile (Bláhová et al. 2023 (link)), acetonitrile:dichloromethane (1:1) (Asimakopoulos et al. 2012 ), and methyl tert-butyl ether:ethyl acetate (5:1) (Li et al. 2017 (link))) were also tested. A better de-conjugation effect for BTs and lower concentrations of analytes in blanks (the contamination of blanks) were observed for β-glucuronidase from Helix pomatia when compared to E.coli (data not shown). All tested solvents resulted in similar recoveries. Insoluble particles were removed by centrifugation (12,000 rcf, 10 min, 10 °C); the clear supernatants were evaporated to dryness and then reconstituted in 10% methanol (v/v). Possible residual particles in final extracts were removed using microspin filters (0.2 µm, cellulose acetate, Fisher Scientific). Filtrates were stored in glass inserts at − 20 °C until instrumental analyses.