Analysis of ketamine and xylazine in fur and bones using multidimensional liquid chromatography tandem mass spectrometry

Abstract

While ketamine is traditionally administered for anesthesia or pain management, illicit usage is often seen in forensic cases either as a recreational drug or as a tool in drug-facilitated sexual assault. Xylazine is an anesthetic agent used in veterinary medicine and does not have FDA approval for use in humans. However, it has recently been observed as a cutting agent in heroin. Post-mortem specimens present many challenges when it comes to toxicological analysis. Due to compound degradation and decomposition factors, analytes present at trace levels may be missed in blood and urine. Hair, bone, and insects have recently been investigated as alternative matrices for postmortem analysis due to their increased durability compared to more traditional matrices. However, this durability increases the difficulties in extracting and isolating compounds of interest from these matrices via traditional extraction and chromatography methods. These methods require lengthy extraction times and extensive cleanup steps in order to obtain samples suitable for analysis. Utilizing multiple instrumentation combinations, analysts are able to detect compounds at trace levels. Through the use of multidimensional chromatography, several time-consuming extraction steps can be eliminated while still retaining the ability of trace level detection and quantitation. Using Waters Oasis® HLB PRiME solid phase extraction cartridges using a methanol pH10 loading and an acetonitrile pH3 elution, a solvent extraction yielded linear dynamic ranges of 2pg/mL-1ng/mL and 5pg/mL-1ng/mL for xylazine and ketamine respectively. Rat specimens utilized in this project were treated as per an Institutional Animal Care and Use Committee (IACUC) protocol. The test rodents received an acute dosage of 2mg/mL of xylazine and 24mg/mL of ketamine approximately half an hour prior to death. The 14 test samples were placed outside directly on the ground at the Boston University Forensic Anthropology Outdoor Research Facility (Holliston, MA, U.S.A.) for a period of 6 months. A 15th rat was kept in -20°C until analysis to serve as a Time=0 sample. The outdoor samples were recovered and de-fleshed along with the Time=0 sample manually. Drug-free hair samples were donated anonymously as per Internal Review Board (IRB) protocols.