Guilty by dissociation: Part C: Enantiomeric separation of diphenidine-derived new psychoactive substances (NPS) by polar organic chiral high performance liquid chromatography (HPLC) on polysaccharide-based stationary phases.

A practical HPLC method using six polysaccharide-based chiral stationary phases with a single polar organic solvent mobile phase separated the enantiomers and regioisomers of 32 novel diphenidine-derived psychoactive substances. The cellulose tris(3-chloro-4-methylphenylcarbamate) coated on silica gave baseline separation for 25 of 26 monosubstituted diphenidines with resolution values above 1.5, and optimum separation for 21 of them (resolution 2.9–22.4). The chiral selector type and the substituent position on the 1-phenyl ring strongly influenced chiral resolution, with 4-position substituents showing greater discrimination than 2-position ones. Enantiomer elution order reversed for 2-methoxphenidine depending on the stationary phase. Analysis of real samples confirmed 2-methoxphenidine and diphenidine were traded as racemates, and common adulterants did not interfere.

Study at a glance

Abstract

This study describes a simple and practical HPLC analysis for the direct enantiomeric separation of a range of 32 novel diphenidine derived psychoactive substances using a range of six polysaccharide-based chiral stationary phases employing a single generic polar organic solvent chromatographic mobile phase. Temperature was employed to optimize the chemo and enantiomeric selectivity. Baseline separation and differentiation of both the enantiomers and positional isomers (i.e., regioisomers) of the 2-, 3- and 4-methoxphenidines was achieved with the chiral selector cellulose tris(3-chloro-4-methylphenylcarbamate) coated onto silica. The latter proved to be the best of the six chiral stationary phases investigated in that it generated enantiomeric separation of 25 of the 26 monosubstituted diphenidines with resolution values > 1.5. It yielded the optimum separation for 21 of the 26 diphenidines (resolution values ranged from 2.9 - 22.4) including the 2-, 3- and 4-positional isomers of eight diphenidine derivatives. Excellent separation of all 26 monosubstituted diphenidines (i.e., resolution values > 1.5) and peak shape (i.e., typical tailing factors between 0.9 - 1.2) could be achieved by using Lux Cellulose-2 and Lux i-Amylose-3 columns. The nature of the polysaccharide-based chiral selector was demonstrated to be extremely important in determining the degree of chiral resolution. The location of the monosubstituent on the 1-phenyl ring of the diphenidine was shown to be important in promoting chiral resolution. Greater chiral discrimination was typically observed for substituents in the 4-position compared to those in the 2-position of the 1-phenyl ring. The chiral HPLC methodology displayed good chemo and enantiomeric selectivity of the mono-, di- and trisubstituted diphenidine regioisomers. Enantiomer elution order reversal was highlighted with 2-methoxphenidine enantiomers as a function of the chiral stationary phase. The (R)-enantiomer eluted before the (S)-enantiomer on cellulose-based chiral stationary phase whereas the reverse occurred with the amylose-based phases. Application of the methodology to the analysis of real-life samples of 2-methoxphenidine and diphenidine confirmed that these psychoactive substances were being traded as racemic products. Commonly used adulterants in powdered samples were shown not to interfere with the chiral analysis of 2-methoxphenidine and diphenidine.