Glucuronidase from helix pomatia

Duplicate 2 mL aliquots of urine were mixed with 100 µL glucuronidase solution (250 µL glucuronidase from Helix pomatia (Sigma-Aldrich, Gillingham, UK) in 50 mL 0.1 M acetate buffer (pH 5) and 900 µL acetate buffer and incubated overnight at 37 °C to release free naphthols from conjugates. Calibration standards (0–200 nmol/L) were prepared in urine and analysed with each run.Quality control samples were prepared from a pool of urine obtained from naphthalene-exposed workers and characterised for acceptance criteria and analysed in duplicate after every five duplicate samples. Samples were extracted using solid phase extraction (C18, 1 mL, 100 mg) using the following conditions: condition with methanol (2 mL) and water (2 mL), load sample, wash with water (1 mL) and 30% acetonitrile (1 mL) then elute with 200 µL methanol. This extract was subsequently directly injected (100 µL) for analysis.

Plasma samples were thawed to 4 °C and transferred (150 µL) to an Eppendorf tube. Sulphatase from Helix Pomatia (S9626-5KU; 0.5 units in citrate phosphate buffer 0.3 mol L; pH 6; 120 µL), and glucuronidase from Helix Pomatia (G7017 136,000 units/mL; 30 µL) from Sigma Aldrich (Dorset, UK), were added. The samples were incubated for 18 h at 37 °C. Methanol (700 µL) containing hydrochloric acid (0.4 mol/L) was added and then the samples were mixed, centrifuged (12,500× g; 5 min; 4 °C) and analysed by LC-MS/MS.