2.5. Enzymatic hydrolysis

To evaluate the effectiveness of enzyme hydrolysis (deconjugation) of the potential glucuronidated forms of isoprostanes in wastewater, 500 μL of β-glucuronidase type H-2 enzyme was added in each of three 100 mL raw wastewater samples and incubated at 37 °C for 1.5 h prior extraction. In addition, three raw wastewater samples not spiked with enzymes were also incubated at 37 °C for 1.5 h (to evaluate the effect of incubation) and three other raw wastewater samples not spiked with enzymes were placed at ambient lab conditions (to evaluate the effect of enzyme hydrolysis and incubation). After incubation, all samples were allowed to equilibrate to room temperature, spiked with the internal standard mixture, mixed thoroughly, extracted, and analyzed as described in section 2.2.