Decreasing the uncertainty of peak assignments for the analysis of synthetic cathinones using multi-dimensional ultra-high performance liquid chromatography

Abstract

Chromatographic techniques which are commonly employed in forensic analysis utilize retention time as an identification parameter. Conventional one-dimensional chromatographic techniques such as gas and liquid chromatography inherently lack the separation power required to resolve the multitude of combinations possible when analyzing emerging drugs. Multi-dimensional chromatography, which significantly increases resolving power, is a viable means to increase the utility of retention time measurements for compound identification. This study aims to demonstrate the utility of multi-dimensional ultra-high performance liquid chromatography (UHPLC) with at-column dilution for the screening and identification of synthetic cathinones with significantly decreased uncertainty. One-dimensional separations were conducted on mixtures of 16 controlled synthetic cathinones and 7 pentedrone positional isomers in order to determine the most orthogonal combination for multi-dimensional chromatography. The separations utilized several stationary phases for both reversed phase chromatography (RPC) and hydrophilic interaction chromatography (HILIC). Based on the separations performed, it was determined that a combination of a BEH C8 column (operated in RPC mode) and an HSS PFP column (operated in HILIC mode) as the first and second dimension columns, respectively, provided orthogonal separations with a decrease in peak assignment uncertainty of at least one order of magnitude. For at-column dilution, a HILIC trapping column was used with 0.025% formic acid in acetonitrile as the loading and diluting solvent. Multi-dimensional separations conducted on early, mid, and late eluting compounds showed excellent retention time repeatability, satisfactory recovery with excellent signal-to-noise, and good peak area repeatability for both the first and second dimensions. Ten simulated samples containing various adulterants and diluents were analyzed using multi-dimensional chromatography. All 10 samples showed excellent retention time matches between the sample and its corresponding standard. Thus, multi-dimensional UHPLC would significantly reduce peak assignment uncertainty leading to increased accuracy in the identification of seized drugs.