The detection of aminorex in biological fluids from horses, humans, and other species is a complex issue due to various sources: the administration of the anthelmintic drug levamisole, designer drug aminorex and its analogs, and newly identified plant products containing barbarin precursors. Given the complexity of these sources, distinguishing between them is crucial. Historically, analyses for marker compounds, along with chiral analysis, have been employed to differentiate the administration of a single enantiomer of levamisole from racemic aminorex. However, with the identification of the third, plant-derived source, there is an evident need for a more sensitive and specific method.
We are excited to introduce a novel and highly sensitive method that not only resolves aminorex and rexamino enantiomers simultaneously but also significantly enhances the accuracy of differentiating these sources. Our innovative approach involves the liquid-liquid extraction of analytes from biological matrices after adjusting the pH to 12. The recovered analytes are then derivatized using a chiral derivatizing agent, Nα-(2,4-Dinitro-5-fluorophenyl)-L-valinamide. This derivatization process produces diastereoisomers, which are subsequently resolved using a stable-bond phenyl liquid chromatographic column.
The robustness of our method is demonstrated through full validation in horse blood plasma and urine matrices, showcasing high sensitivity with a limit of quantitation at 50 pg/mL in horse plasma and 200 pg/mL in horse urine. Furthermore, this method holds potential for adaptation across various matrices and species, broadening its applicability and utility in diverse toxicological contexts. Our goal is to provide a reliable and precise solution to the challenges posed by the detection of aminorex, thereby advancing the field of toxicology and enhancing the accuracy of our analyses.