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 0.5g of sample, and uses xylazine d6 as an internal standard. Results: The method was linear between 0.86 and 34.4μg/g of brain. Limits of quantification were 378 and 87ng (approximately 0.76 and 0.17μg/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 (