Measurement of ketamine and xylazine in rat brain by liquid-liquid extraction and gas chromatography-mass spectrometry.

INTRODUCTION: In human and veterinary medicine, the injectable drugs ketamine and xylazine are mainly used in combination to induce, and then maintain general anaesthesia; they also provide pain and stress relief. Some side-effects have been reported on the auditory brainstem response, a method is therefore required to determine their concentrations in the brain. METHODS: This paper presents a method to determine nanogramme quantities of ketamine and xylazine in rat brain using liquid-liquid extraction and gas chromatography-mass spectrometry in selective ion monitoring mode. The technique requires only 0.5 g of sample, and uses xylazine d6 as an internal standard. RESULTS: The method was linear between 0.86 and 34.4 µg/g of brain. Limits of quantification were 378 and 87 ng (approximately 0.76 and 0.17 µg/g of brain) for ketamine and xylazine, respectively. The reliability of the method in terms of accuracy, within-day and between-day precision was also demonstrated. For xylazine, bias and intra-day precision were good (<3.0%), as was between-day precision (<10.5%); the equivalent values for ketamine were 7%, 11.1% and 20.9%, respectively. Stability of the analytes in the matrix at -80 °C was assessed over five months; both compounds were found to be stable for at least 1 month, even at very low concentrations. The procedure was successfully applied to determine (for the first time) the in vivo brain levels of both drugs in animals following systemic administration. DISCUSSION: The procedure will be useful in future studies of the side-effects of these drugs, and their interactions with other compounds.

3-Hydroxybenzo(a)pyrene as a biomarker of dermal exposure to benzo(a)pyrene.

The aim of this study was to determine the percutaneous absorption flux of BaP (20 microg/cm(2) in ethanol) and the usefulness of urinary 3-OHBaP as a bio-indicator of dermal exposure to BaP. The percutaneous absorbed dose and absorption flux were estimated by comparison with intravenous administration of BaP (0.01 and 0.05 mg/kg in Cremophor) as reference way. A percutaneous absorption flux of 0.37 microg/cm(2)/h was determined by killing groups of rats, following exposure time of 4.5 and 24 h. [(14)C] skin content was 3.1 microg/cm(2), after 24 h exposure to BaP. Total urinary 3-OHBaP accounted for 0.4% of the real absorbed dose, which was fourfold higher than the percentage of an intravenous dose excreted as 3-OHBaP. This finding reveals that percutaneous absorption of BaP, based on the ratio of urinary excretion of 3-OHBaP following percutaneous exposure compared to percutaneous absorption following intravenous administration of BaP, is overestimated in the rat. In vitro, BaP was intensively metabolised by rat skin. Unchanged BaP and 3-OHBaP in receptor fluid accounted for 50 and 30% of the total radioactivity. This percutaneous first past effect of BaP in rats could, in part, explain the higher urinary excretion ratio of 3-OHBaP compared to the value based on intravenous administration of BaP. Conversely, BaP was largely lower metabolised as 3-OHBaP during percutaneous absorption by humans, so BaP absorption flux should be overestimated to a lesser extent in humans than in rats.