Levels of phenols (phenol and p-cresol) in urine were analyzed by high-performance lipid
chromatography (HPLC) according to a previously reported method [16 (link)].
In brief, a test sample, concentrated hydrochloride, and 4-chlorophenol (internal standard) were mixed and
heated at 100°C for 60 min to hydrolyze the phenol conjugates. After cooling in an ice bath, ether was added
to the mixture, and the mixture was then agitated on a vortex mixer. Then, the ether layer was taken,
neutralized, and dried with an N2 purge. The residue was dissolved in ethyl acetate and filtered
through a 0.45-μm Ultrafree-MC filter unit (Millipore, Billerica, MA, USA). Phenols were quantified by an HPLC
system (Alliance® 2695, Waters, Milford, MA, USA) equipped with an L-column ODS® (CERI,
Tokyo, Japan; 4.6 mm (i.d.) × 150 mm (length) and 5 μm particle size), eluted with 1% aqueous phosphoric acid
solution: acetonitrile (80:20 v/v) at a flow rate of 1 ml/min at 40°C, and monitored with a 474 fluorescence
detector (Waters, Milford, MA, USA; excitation at 260 nm and emission at 305 nm). Urine phenol levels were
normalized to urine creatinine levels, determined with a creatinine test kit (Wako Pure Chemical Industries),
and expressed as μM phenol per mM creatinine.
chromatography (HPLC) according to a previously reported method [16 (link)].
In brief, a test sample, concentrated hydrochloride, and 4-chlorophenol (internal standard) were mixed and
heated at 100°C for 60 min to hydrolyze the phenol conjugates. After cooling in an ice bath, ether was added
to the mixture, and the mixture was then agitated on a vortex mixer. Then, the ether layer was taken,
neutralized, and dried with an N2 purge. The residue was dissolved in ethyl acetate and filtered
through a 0.45-μm Ultrafree-MC filter unit (Millipore, Billerica, MA, USA). Phenols were quantified by an HPLC
system (Alliance® 2695, Waters, Milford, MA, USA) equipped with an L-column ODS® (CERI,
Tokyo, Japan; 4.6 mm (i.d.) × 150 mm (length) and 5 μm particle size), eluted with 1% aqueous phosphoric acid
solution: acetonitrile (80:20 v/v) at a flow rate of 1 ml/min at 40°C, and monitored with a 474 fluorescence
detector (Waters, Milford, MA, USA; excitation at 260 nm and emission at 305 nm). Urine phenol levels were
normalized to urine creatinine levels, determined with a creatinine test kit (Wako Pure Chemical Industries),
and expressed as μM phenol per mM creatinine.