Development of a Novel Sequential Injection-Proton NMR Method for Determining Cathinones in Forensic Samples

Abstract

A novel method was developed for the determination of low levels of Cathinones in authentic samples by interfacing Sequential Injection Analysis (SIA) to Proton Nuclear Magnetic Resonance Spectroscopy (NMR). In the first part of this study, solid phase extraction (SPE) was studied by SIA to preconcentrate the sample and remove matrix interferences. Phenylpropanolamine (PPA), Phenylephrine (PEP), and Methylephedrine (MEP) were studied as simulants for the Cathinones, representing primary, secondary, and tertiary amines, respectively. A variety of stationary, mobile, and eluent phases for several column geometries were examined, and a wide cylindrical column (20 mm6.6 mm) with a bed volume of 2.7 mL with a strong cation exchange (SCX) resin was selected for further study. The method was applied to authentic samples at the Wisconsin State Crime Lab (WSCL) in Milwaukee. A range of samples were analyzed that differed in the Cathinones (Methcathinone, Methylone, and N-Ethylpentylone) and interferences (Mannitol and Caffeine) that were present. Measurements were done by SIA-NMR as well as directly by NMR. When SIA-NMR was performed, the interferences were efficiently removed. However, tertiary amines were found to be more difficult to quantitatively elute than primary and secondary amines. For the second part of the study, the SPE-SIA method was optimized by application of Plackett-Burman (P-B) screening (11 factor design) and Simplex optimization (7 factor design) methods. The optimized SPE-SIA method had the following conditions: 800 mL sample volume, 21 mL/s sample aspiration flowrate, 18 mL/s carrier load flowrate, 25 mL/s elution flowrate, 100 mM phosphate buffer (pH 6.00) mobile phase, 215 mM NaCl / 60 mM HCl eluent, and 1565 mL eluent volume. For the optimized method, the Limit of Detection was 0.023 mM (70% improvement) and the precision, measured for 0.25 mM PPA (n=3), was 0.10% (93% improvement). In the final part of the study, the optimized SIA-NMR method was applied to a new set of confiscated samples provided by the WSCL in which ( N-Ethylpentylone and N-Ethylhexedrone) were present. The optimized method was effective in the determination of low levels of Cathinones in the confiscated samples for all three types of amines. Predicted proton spectra of the Cathinones were then created using the MNova software program and compared to the observed spectra. The number of peaks in the observed and predicted spectra generally agreed, but differences in the chemical shifts and multiplicities of the protons were observed.