2.4. Urinary sampling and analysis

High performance liquid chromatography (HPLC)–grade acetonitrile was obtained from BDH chemical, UK. The metabolite, 1-hydroxypyrene, was obtained from Sigma–Aldrich, South Africa. Analysis was carried out using a Cecil 1100 series HPLC system at the Central Laboratory, University of Ibadan, Nigeria. Method of Choosong et al [18] was adopted with slight modification. Early morning urine samples (after charcoal pyrolysis and/or charcoal removal) were collected from all voluntary workers for the period of exposure. Early morning urine was used because the half-life of the parent congener benzo[a]pyrene is between 18 hours and 20 hours. An aliquot of 10 mL was separated into another tube to determine the urinary creatinine. The remaining spot urine sample was stored in a polypropylene tube and frozen at −20°C before sample preparation and analysis. A small portion of the urine sample (400 μL) was placed in an eppendorf tube. After adding acetate buffer (100 μL, 0.5M, pH 5) and β-glucuronidase (5 μL; 2000 unit), the sample was vortex mixed for about 30 seconds. The sample was then incubated at 37°C in a shaking bath for 16 hours (hydrolysis). Acetonitrile (700 μL) was added, and the sample was then vortexed for 10 seconds, centrifuged at 10,285 g, and incubated at 20°C for 10 minutes. Finally, a clear supernatant from the preparation was analyzed for 1-OHP using HPLC (Cecil 1100 Series). The prepared calibration concentration ranged from 5 ppb to 100 ppb, whereas the detection limit was calculated as three times the standard deviation of the lowest detectable concentration. It was calculated as 1.1 μgL⁻¹.