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

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. 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. 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. The procedure will be useful in future studies of the side-effects of these drugs, and their interactions with other compounds.