Laboratory analysts were blinded to

Laboratory analysts were blinded to the case-control status of
all samples. The applied methodology for the analysis of ZON and
its five metabolites is described in a previously published study
(Belhassen et al., 2014). In brief, urine samples were thawed completely
at room temperature, centrifuged at 4050g for 10 min to sediment particulate matter and 10 mL were taken to carry out the
analysis. Each sample was spiked with 100 lL of the surrogate
(400 ng mL1) and 100 lL of enzyme solution (b-glucuronidase/
sulfatase). The enzyme solution was prepared daily by dissolving
3.32 mg of b-glucuronidase/sulfatase (3  106 U g solid1) in 1mL
of 1 M ammonium acetate buffer (pH 5.0). 25 lL of 4-methylumbelliferyl
glucuronide/4-methylumbelliferyl sulfate/13C4-4-methylumbelliferone
standard mixture (4 lgmL1) was also added to
determine the extent of deconjugation. After mixing, the sample
was incubated at 37 C for 24 h. Next, analytes were extracted from
the samples by adding 15 mL of ethyl acetate/formic acid (99:1, v/
v) and shaking for 30 min. The mixture was centrifuged for 10 min
at 4050g. Subsequently, the extracts were evaporated to dryness at
room temperature under a nitrogen stream. The residue was dissolved
with 400 lL of a mixture of Milli-Q water/MeOH (50:50,
v/v) and placed in a 1.5 mL Eppendorf tube. 500 lL of hexane
was added and the mixture was centrifuged for 15 min at
24960g. Finally, the underlying aqueous phase was separated and
10 lL injected into the ultra high performance liquid chromatograph
(UHPLC) system.