Simultaneous determination of 1- and 2-naphthol in human urine using on-line clean-up column-switching liquid chromatography–fluorescence detection

We developed a new 3-D HPLC method for on-line clean-up and simultaneous quantification of two important naphthalene metabolites, 1-naphthol and 2-naphthol, in human urine. Except an enzymatic hydrolysis no further sample pre-treatment is necessary. The metabolites are stripped from urinary matrix by on-line extraction on a restricted access material pre-column (RAM RP-8), transferred in backflush mode onto a silica-based CN-(cyano)phase column for further purification from interfering substances. By another successive column switching step both analytes are transferred with a minimum of overlapping interferences onto a C12 bonded reversed phase column with trimethylsilyl endcapping where the final separation is carried out. The entire arrangement is software controlled. Eluting analytes are quantified by fluorescence detection (227/430 nm) after an external calibration. Within a total run time of 40 min we can selectively quantify both naphthols with detection limits in the lower ppb range (1.5 and 0.5 μg/l for 1- and 2-naphthol, respectively) with excellent reliability (ensured by precision, accuracy, matrix-independency and FIOH quality assurance program participation). First results on a collective of 53 occupationally non exposed subjects showed mean levels of 11.0 μg/l (1-naphthol) and 12.9 μg/l (2-naphthol). Among smokers (n=21) a significantly elevated mean level of urinary naphthols was determined (1-naphthol: 19.2 μg/l and 2-naphthol: 23.7 μg/l) in comparison to non smokers (n=32; 1-naphthol: 5.6 μg/l, 2-naphthol: 5.6 μg/l).