Polar Organic Phase Mode Research Articles

Enantiomer separation of a powerful chiral auxiliary, 2-methoxy-2-(1-naphthyl)propionic acid by liquid chromatography using chiral anion exchanger-type stationary phases in polar-organic mode; investigation of molecular recognition aspects

The enantiodiscriminating potential of the weak anion exchange-type quinine-based chiral stationary phases (CSPs) for direct enantiomer separation of racemic 2-methoxy-2-(1-naphthyl)propionic acid (selectand, SA) was studied. The influence of structure variations of the selector (SO) in the carbamate functional group and/or in the C6' position of quinoline moiety on retention and enantioselectivity was investigated. Systematic chromatographic studies were made to gain more insight into the overall chiral recognition mechanism for a given mobile phase. In this context, the tert-butylcarbamoyl quinine and the corresponding diisopropylphenyl-derived selector provided the highest resolution and enantioselectivity under polar-organic conditions with the elution order of (R) before the (S) enantiomer. When the bulkiness of the substituents in the C6' position of the SO was increased, the selectivity was decreased in all cases. Alkylation of the nitrogen atom in the carbamate functionality of the SO resulted in the complete loss of enantiomer separation, confirming the crucial importance of the hydrogen-bond formation involved in the stereodiscriminating events. In addition, ten different mono-, bi-, or trivalent acids, necessary as competitor molecules (counter-ions) of the mobile phase, were screened to judge their influence on retention and overall enantioselectivity. Among them, acetic acid, formic acid, N-acetylglycine, and glycolic acid proved to be the most promising counter-ions with R(S) values of 6.35, 6.81, 8.19, and 7.34, respectively. On the basis of chromatographic data, a tentative molecular recognition model was proposed. Simultaneous ion-pairing and hydrogen bonding, in concert with pi-pi stacking and steric interactions, were expected to be responsible for chiral recognition mechanism. This was partially corroborated by structural and/or conformational analysis of the tert-butylcarbamoyl quinine-2-methoxy-2-(1-naphthyl)propionic acid (SO-SA) complex.

Effect of the eluent on enantiomer separation of controlled drugs by liquid chromatography–ultraviolet absorbance detection–electrospray ionisation tandem mass spectrometry using vancomycin and native β-cyclodextrin chiral stationary phases

Enantioseparation of nine amphetamine derivatives, methorphan and propoxyphene was studied by comparing two different chiral stationary phases, macrocyclic antibiotic vancomycin and native β-cyclodextrin (β-CD). Effects of 46 eluent compositions on enantioseparation in reversed-phase (RP) and polar organic phase modes were investigated. β-CD was found to be more suitable to phenethylamines in general and vancomycin for methorphan and propoxyphene. An eluent system capable of separating the enantiomers of all phenethylamines in one run was developed. Also, systems providing competitive analysis times for enantioseparation of methorphan and propoxyphene were reported. The suitability of the eluent systems to electrospray ionisation (ESI) was discussed and methods using a tandem mass spectrometric (MS/MS) detection were developed. The suitability of chiral LC–ESI–MS/MS was tested with 14 seized drug samples. The results were in agreement with conventional non-chiral methods. Repeatability of the methods was good and limits of detection were 25–100 ng/ml for most compounds using mass spectrometric detection.

Synthesis and Electrophysiological Studies of a Novel Epibatidine Analogue

The new epibatidine analogue exo-2-(2-pyridyl)-7-azabicyclo[2.2.1]heptane (2PABH) was synthesised. Separation of enantiomers was performed on chiral HPLC chromatography in polar-organic phase mode at 0°C. Enantiomeric purity was greater than 99.8%ee for the (-)- and 90.5%ee for the (+)-enantiomer respectively. Optical rotation was determined to be [α]D23 = ±13°. Electrophysiological studies of 2PABH were carried out on α4β2. α3β4 and α7 nAChR subtypes cloned from rat and reconstituted in Xenopus oocytes. Both enantiomers could not significantly activate the heteromeric subtypes. The homomeric α7 nAChR displays a high sensitivity only towards (-)-2PABH. The EC50 for (-)-2PABH and ACh were determined (32.5 ± 9.5μM, 137.3 ± 16.5μM). (-)-2PABH was shown to be a partial agonist (80% of ACh). Thus the efficacy of 2PABH differs markedly from that of epibatidine. The intramolecular N-N-distance and the spatial pyridine nitrogen orientation play a central role in nAChR recognition.