Chiral separation and characterization of methoxphenidine – A popular street drug

Abstract

Methoxphenidine (MXP) belongs among dissociative anesthetics from the group of new psychoactive substances. According to the unofficial user experiences, MXP is less active than common dissociative anesthetics like ketamine, yet, several intoxications were associated with this substance up to this date. Within our previous work, we discovered that at least some MXP samples on the drug market may contain zincate complex which we believe may be responsible for the unusual properties of the purported HCl salts. MXP comprise a stereogenic center in its molecular structure, which is disregarded by the manufacturers and, therefore, MXP is available on the drug scene as a racemic mixture only. However, the differences between the effects and activity of the enantiomers remain to be discovered. Hereby we present a chiral resolution by crystallization, which we used to obtain both the enantiomers of MXP. To control their optical purity, we have developed a new supercritical fluid chromatography (SFC) method. Finally, a combination of electron circular dichroism (ECD) and vibrational circular dichroism (VCD) spectra supported by density functional theory (DFT) calculation has been used to assign the absolute configuration of both enantiomers. Racemic Methoxphenidine hydrochloride was prepared in-house. Then individual enantiomers were obtained by process of crystallization using chiral (−) or (+)-2,3-dibenzoyl-tartaric acid monohydrate. An Acquity UltraPerformance Convergence ChromatographyTM (UPC2) from Waters (Milford, MA, USA) was used for HPLC and SFC enantioseparation. For control of the purity of the obtained enantiomers after crystallization, a HPLC method has been used. To improve efficiency of the existing chiral HPLC method, an SFC enantioseparation method was develop. Four polysaccharide-based CSPs (Alcyon SFC CSP Amylose-SA, Cellulose-SB and Cellulose-SC and CHIRALPAK® IE-3) and CO2-containing mobile phases were employed. The ECD and UV absorption spectra were obtained on a Jasco J-815 spectrometer (Jasco, Japan) in a quartz cuvette (Hellma, Germany) with an optical pathlength of 1 mm. The VCD and IR absorption spectra were measured on Fourier transform infrared spectrometer IFS 66/S equipped with a PMA 37 VCD/IRRAS module (both Bruker, Germany), BaF2 polarizer, and MCT detector (InfraRed Associates, USA). Measurements were carried out in a BioCell cuvette with CaF2 windows (Biotools, Inc, USA) and an optical pathlength of 27.3 μm. DFT simulations were performed to find the stable structures of MXP in solution and to determine the absolute configuration of enantiomers. It was important to develop a fast and robust method, which would enable high-throughput screening of samples, e.g. in toxicology laboratories. Therefore, one of the goals of this study was to develop a novel SFC enantioseparation method for the chiral resolution of MXP enantiomers using a polysaccharide chiral stationary phase. In comparison, the SFC assay provides significant decreasing of retention time with satisfactory resolution. Repeatability of the method was proven by an intra- and an interday validation with respect to retention time and resolution factor. Using DFT calculation, we found two stable conformers in solution, calculated their relative abundances based on Boltzmann distribution and simulated their Boltzmann-weighted spectra. Their comparison with experimental spectra allowed us to determine the absolute configuration of enantiomers. We present an enantioseparation method for methoxphenidine using SFC and offer a comparison of four commercial chiral stationary phases. Using this method, we show that crystallization with a chiral acid provides pure enantiomers of MXP, which was confirmed by chiroptic methods supported with DFT calculations.