The lack of authentic reference standards has hindered the definitive identification and quantitative determination of tiletamine metabolites in forensic specimens. To address this limitation, synthesized reference standards of nortiletamine and reduced nortiletamine were prepared and used to develop and validate a ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for their simultaneous determination in human hair.

The collected hair samples were washed with a detergent solution, followed by with deionized water and acetone. After air-drying, the samples were cut into 2 mm segments and then ground to powder by a low-temperature grinder. For extraction, precisely 20 mg of homogenized hair sample was spiked with deuterated internal standard and subjected to methanol extraction with vortex mixing followed by centrifugation. The resulting supernatant was filtered through a 0.22 μm membrane filter prior to UPLC-MS/MS analysis.

All validation parameters met the pre-defined acceptance criteria. The applicability of the method was confirmed through the analysis of authentic hair specimens obtained from 114 abusers. Tiletamine was detected in all samples, whereas reduced nortiletamine and nortiletamine were detected in 64.9% and 3.5% of the samples, respectively. The concentration ranges detected in the hair of drug abusers were 33.3–242,375 pg/mg for tiletamine, 162–986 pg/mg for nortiletamine, and 33.4–26,489 pg/mg for reduced tiletamine.

This study presents a validated analytical method for the simultaneous determination of tiletamine and its metabolites in authentic hair samples. To the best of our knowledge, this is the first reported methodology that enables both qualitative identification and quantitative analysis of tiletamine metabolites in biological specimens based on reference standard solutions, which has been successfully applied to forensic case samples.