Abstract

In the course of a bioavailability investigation of benzo(a)pyrene in the rat, an HPLC-method was developed to determine the concentration of this compound in plasma. In the clean-up procedure of plasma a small weighed amount of plasma is extracted with hexane. To compensate for losses during the clean-up procedure an internal standard is used. The internal standard is dissolved in the hexane used for extraction. Detection takes place with a fluorescence detector for benzo(a)pyrene as well as benzo(ghi)perylene (the internal standard) at an excitation wavelength of 296 nm and an emission wavelength of 415 nm. An eluens consisting of a mixture of 90% methanol and 10% water gives a retention time of about 9 minutes for benzo(a)pyrene on a reversed-phase C18/octodecyl column. The lowest measurable concentration of benzo(a)pyrene in plasma is about 0.2 ng per ml. The standard curve is linear for plasma concentrations up to 3 mug per ml. The within-day- and between-day reproducibility were studied at four concentration levels: 0.2, 20, 200 and 3000 ng per ml. The within-day coefficient of variation was between 21% and 33% for a concentration of 0.2 ng/ml, between 2% and 5% for 20 ng/ml, between 1% and 4% for 200 ng/ml, and between 1% and 3% for the concentration of 3000 ng/ml. Recovery experiments were performed at concentration levels of 3, 30, 300 and 3000 ng per ml; the recoveries were respectively 101%, 102% 95% and 103%. A stability experiment showed that samples are stable over a period of 6 weeks if stored at 4 degrees C or - 18 degrees C.