Archives of Toxicology

, Volume 77, Issue 4, pp 183–193 | Cite as

Simultaneous analysis of naphthols, phenanthrols, and 1-hydroxypyrene in urine as biomarkers of polycyclic aromatic hydrocarbon exposure: intraindividual variance in the urinary metabolite excretion profiles caused by intervention with β-naphthoflavone induction in the rat

  • Eivor ElovaaraEmail author
  • Virpi Väänänen
  • Jouni Mikkola
Toxicokinetics and Metabolism


Two fluorimetric HPLC methods are described for the quantification of naphthols, phenanthrols and 1-hydroxypyrene (1-OHP) in urine specimens obtained from male Wistar rats exposed to naphthalene, phenanthrene and pyrene. The polycyclic aromatic hydrocarbons (PAHs) were given intraperitoneally, either alone (1.0 mmol/kg body weight) or as an equimolar mixture (0.33 mmol/kg), using the same dosages for repeated treatments on week 1 and week 2. Between these treatments, PAH-metabolizing activities encoded by aryl hydrocarbon (Ah) receptor-controlled genes were induced in the rats with β-naphthoflavone (βNF). Chromatographic separation of five phenanthrols (1-, 2-, 3-, 4-, and 9-isomers) was accomplished using two different RP C-18 columns. Despite selective detection (programmable wavelengths), the quantification limits in the urine ranged widely: 1-OHP (0.18 µg/l) <phenanthrols (0.34–0.45 µg/l) <2-naphthol (1.5 µg/l) <1-naphthol (4 µg/l). The relative standard deviation of the methods was good, as also was the reproducibility. The molar fraction of the dose excreted in 24-h urine as naphthols (≤4.0%), phenanthrols (≤1.1%), and 1-OHP (≤2.4%) was low. Urinary disposition increased differentially in βNF-induced rats: naphthols, 9-phenanthrol (1- to-2-fold); 2-, 3-, and 4-phenanthrols (4- to 5-fold); 1-phenanthrol and 1-OHP (over 11-fold). The OH-metabolites were analyzed before and after enzymatic hydrolysis (β-glucuronidase/arylsulfatase). The percentage excreted as a free phenol in urine varied for 1-OHP (2–11%), 1-naphthol (36–51%), 2-naphthol (59–65%), and the phenanthrols (29–94%). 1-Naphthyl- and 1-pyrenyl β-d-glucuronide served as measures for the completeness of enzymatic hydrolysis. Characteristic differences observed in the urinary disposition of naphthalene, phenanthrene, and pyrene are described, as well as important factors (dose, metabolic capacity, relative urinary output) associated with biomarker validation. This intervention study clarifies intraindividual variation in PAH metabolism and provides useful information for the development of new methods applicable in the biomonitoring of PAH exposure in humans.


Monohydroxy polycyclic aromatic hydrocarbons Hydroxyphenanthrene isomers HPLC Fluorescence detection Urine Biomarkers 



We thank Ms Maria Pihlaja for valuable technical assistance, Dr Kaija Pekari for GC analysis of urinary naphthols, and Dr Pirjo Heikkilä for helpful co-operation. This work was conducted in association with the project BMH4-CT97-2621 supported by the Commission of the European Communities, and the Finnish Research Programme on Environmental Health (SYTTY) supported by the Academy of Finland and the Finnish Work Environment Fund.


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Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • Eivor Elovaara
    • 1
    Email author
  • Virpi Väänänen
    • 2
  • Jouni Mikkola
    • 1
  1. 1.Laboratory of Toxicokinetics and Metabolism, Department of Industrial Hygiene and ToxicologyFinnish Institute of Occupational HealthHelsinkiFinland
  2. 2.Chemistry Laboratory, Department of Industrial Hygiene and ToxicologyFinnish Institute of Occupational HealthHelsinkiFinland

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