2 naphthol 2 nap

OH-PAH standards including 1-hydroxypyrene [1PYR (MW: 218.26 g/mol, purity 98%)] and 2-naphthol [2NAP, (MW: 144.17 g/mol, purity 99%)] were purchased from Sigma-Aldrich Inc., (St Louis, MO). HPLC-grade reagents including acetonitrile, methanol, pentane, isooctane and ethyl acetate were obtained from Fisher Scientific (Pittsburgh, PA). Sodium acetate (reagent grade, >99%), potassium dihydrogen phosphate (ACS reagent, ≥99%) and the enzyme β-glucuronidase (from Helix pomatia, type HP-2) was purchased from Sigma-Aldrich. Type I ultrapure water was obtained from Milli-Q water purification system (Millipore, Bedford, MA). All stock solutions were prepared by dissolving 1 mg of the native metabolites in 100 mL of methanol to obtain a 10 µg/mL concentration. Two working solutions of 2NAP and 1PYR were prepared by diluting the stock solution in methanol to yield a final concentration of either 0.5 µg/mL or 0.05 µg/mL. Calibration solutions (6 levels) were prepared in blank pooled urine matrix by spiking appropriate volumes of the working solutions to yield the following concentration ranges: 2NAP, 1–25 ng/mL and 1PYR, 0.1–6 ng/mL. All stock and working standards were prepared in amber vials and stored at −20 °C until further use.