Analysis of ketamine and xylazine in complex matrices using two-dimensional liquid chromatography/tandem mass...

Analyzing tissue samples is routinely performed when liquid biological samples are not available for replicate analysis. Preparing complex matrices, such as tissue, for analysis can be time-consuming. Traditional sample preparation methods typically begin with homogenization followed by a sample clean-up step such as liquid-liquid or solid-phase extraction. Samples are typically eluted, evaporated and reconstituted prior to instrumental analysis. The aim of this project was to evaluate the utility of multi-dimensional chromatography in reducing the amount of time from sample acquisition to analysis.

Tissue specimens were homogenized using a ceramic beads shaker. Homogenates were then diluted and loaded onto a mixed mode solid-phase sorbent. The sorbent was washed, and the final eluate was transferred directly to vials without evaporation or reconstitution steps. Analysis was performed using a two-dimensional (2D) ultra-performance liquid chromatography (UPLC) configuration with an At-column dilution option coupled to a triple quadrupole mass spectrometer. The target analytes (xylazine and ketamine) were quantified under multiple reaction monitoring (MRM) using electrospray ionization (ESI) in positive mode.

The lowest limit of detection evaluated in this study was 0.01 ng/mL. The linear dynamic range utilized was 0.1 to 10 ng/mL. The concentrations for xylazine in their respective tissues ranged from 0 to 0.316 ng/mL. Ketamine concentrations ranged from 0 to 0.905 ng/mL. The overall time for sample preparation was reduced to 30 min. The total run time was 10 min.

The use of multidimensional chromatography with At-column-dilution allows for significant reduction in sample preparation time. The concentrations determined in these samples highlight the need for sensitive analytical techniques with the ability to detect analyte concentrations at sub ng/mL.