Separation of New Psychoactive Substances and Xylazine Metabolite Isomers using SLIM High-Resolution Ion Mobility-Mass Spectrometry (HRIM-MS)

Ralph Aderorho1, Shadrack Wilson Lucas2 and Christopher Donald Chouinard

Department of Chemistry

New psychoactive substances (NPS) such as synthetic cannabinoids (SC) are often laced with xylazine, a common and potent veterinary sedative. This adulteration increases potency thereby altering brain chemistry, causing hallucinations, euphoria, and potentially death. NPS and xylazine undergo metabolic transformations yielding complex and active metabolic isomeric mixtures. Traditional analytical methods face challenges in resolving these isomers, hindering accurate pharmacokinetic studies, and therefore it is critical to develop techniques for accurate identification. This study leverages Structures for Lossless Ion Manipulations (SLIM), a high-resolution ion mobility (HRIM) technique that enables improved separation of metabolite isomers. This innovative approach holds promise for advancing pharmacological studies, facilitating the identification and quantification of NPS and xylazine metabolites, and enhancing the understanding of their metabolic pathways.

Sample mixtures were prepared (1 µg/mL) and then derivatized using dansyl chloride to target hydroxyl and amine groups in different positions. The mixtures were analyzed by direct injection using an Agilent 1290 Infinity II UHPLC coupled to MOBILion Systems MOBIE HRIM SLIM and Agilent 6546 QTOF. The SLIM has a 13 m pathlength IM region containing nitrogen or helium buffer gas maintained at ~25 °C and 2.5 Torr. SLIM TW frequency/amplitude were optimized for isomer separations and sensitivity. Additionally, all samples were analyzed using an Agilent 6560 IM-QTOF, specifically for acquiring accurate reference CCS values. All data were subjected to PNNL Pre-Processor frame compression, and analysis was performed with Agilent MassHunter IM-MS Browser 10.0.

The collision cross sections (CCS) of all NPS compounds were measured in the range of 154.4 to 206.3 Ų for both protonated [M+H]⁺ and sodiated [M+Na]⁺ ions. Notably, a baseline separation was achieved for isomeric synthetic cannabinoid (SC) metabolites, including JWH-250 5-hydroxyindole (203.7 ± 0.1 Ų), JWH 250 N-(4-hydroxypentyl) (184.3 ± 0.1 Ų), and JWH 250 N-(5-hydroxypentyl) (183.8 ± 0.1 Ų), all observed as sodiated species at m/z 374.173. However, only partial separation was observed for JWH-018 4-hydroxyindole (206.4 ± 0.1 Ų), JWH-018 N-(5-hydroxypentyl) (193.9 ± 0.1 Ų), and JWH-018 6-hydroxyindole (206.3 ± 0.1 Ų) as sodiated species at m/z 380.163. In contrast, complete overlap was observed for 3-hydroxy xylazine (154.4 ± 0.1 Ų) and 4-hydroxy xylazine (154.4 ± 0.1 Ų), both as protonated species at m/z 237.105. These isomers differ only in the position of a hydroxyl group.

In an effort to improve IM separation, a derivatization reaction using dansyl chloride was employed to selectively target phenolic alcohols present in several of the metabolites. This led to a major product ion at m/z 591.233 for the JWH-018 metabolites and mono- and di-sulphonate esters with m/z 470.156 and 703.207, respectively, for the xylazine metabolites. Baseline separation of isomers with a peak-to-peak resolution of R_pp > 1.5 was achieved for both isomeric groups. However, the formation of a di-sulphonate ester with m/z 703.207, resulting from the reaction of xylazine metabolites with dansyl chloride, introduced plane chirality, leading to the formation of atropisomers exhibiting a unique structural characteristic.

The novel aspect of this research shows the first demonstration of SLIM-based separation of synthetic cannabinoids and xylazine metabolites using dansyl chloride derivatization.