Description of analytical approaches to compare ketamine pharmacokinetics in feline dried blood spots and plasma

Introduction: Pharmacokinetic studies are usually based on measuring the target drug in plasma or serum; however, to obtain a sufficient amount of sample to perform analytical investigations, collecting at least 1 mL of blood at each time points is often required. For this reason, mini-invasive and ethically accepted alternatives to traditional sampling techniques are encouraged, especially in studies involving small size animals. Dried blood spot (DBS), first introduced to screen for diseases in new-borns, has recently gained relevance for pharmacokinetic and toxicokinetic studies in human medicine, but it has not been similarly considered in veterinary medicine so far. In this context, it would be interesting to explore the applicability of DBS for accurate quantification of selected drugs in companion animals. Objectives: The aim of the present work was to develop analytical methods for the correct quantification of ketamine in plasma and DBS samples collected from cats, allowing to compare its concentration/time profile in the two matrices. Materials and Methods: Ketamine was extracted from 100 μL of plasma through protein precipitation with acetonitrile and from 20 μL DBS using a 30:70 H2O:ACN solution. Chromatographic separation was obtained with a Waters Acquity UPLC pump equipped with a BEH C18 column, pumping a mixture of 0.1% formic acid in water and acetonitrile under programmed conditions. Quantification of the analyte was performed on a Waters XEVO TQ-S Micro, operating in positive electrospray ionization and monitoring two specific transitions (238.1>124.9 and 238.1>207.0). A preliminary application of the analytical approaches was performed on samples collected (T0, T10, T15, T20, T30, T45 and T60) from two male cats receiving 10 mg/kg of ketamine and 20 mg/kg of medetomidine intramuscularly for neutering. Results and Discussion: The proposed LC-MS/MS approach is under validation following the most recent European guidelines. In both matrices, linearity was assessed (R2>0.99) over the 250-5000 ng/mL range; accuracy and precision were calculated at four concentrations, providing values always within ±13% and below 11%, respectively. Samples analysed during the pilot study proved that the tested range was suitable for real plasma and DBS concentrations. Conclusions: We developed two quick and simple analytical approaches for the determination of the pharmacokinetic profile of ketamine in cat plasma and DBS. Their application to a larger number of patients will allow to calculate the main PK parameters in the two types of samples, and to confirm if DBS can represent a mini-invasive alternative to plasma during multiple time-point experiments.