English 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.
