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.