Dynamic high performance liquid chromatography on chiral stationary phases. Low temperature separation of the interconverting enantiomers of diazepam, flunitrazepam, prazepam and tetrazepam.

Diazepam and the structurally related 1,4-benzodiazepin-2-ones tetrazepam, prazepam and flunitrazepam are chiral molecules because they adopt a ground state conformation featuring a non-planar seven membered ring devoid of any reflection-symmetry element. The two conformational enantiomers of this class of benzodiazepines interconvert rapidly at room temperature by a simple ring flipping process. Low temperature HPLC on the Whelk-O1 chiral stationary phase allowed us to separate the conformational enantiomers of diazepam and of the related 1,4-benzodiazepin-2-ones, under conditions where the interconversion rate is sufficiently low, compared to the chromatographic separation rate. Diazepam, tetrazepam and prazepam showed temperature dependent dynamic HPLC profiles with interconversion plateaus indicative of on-column enantiomer interconversion (enantiomerization) in the temperature range between -10 °C and -35 °C, whereas for flunitrazepam on-column interconversion was observed at temperatures between -40 °C and -66 °C. Simulation of exchange-deformed HPLC profiles using a computer program based on the stochastic model yielded the apparent rate constants for the on-column enantiomerization and the corresponding free energy activation barriers. At -20 °C the enantiomerization barriers, ΔG(≠), for diazepam, prazepam and tetrazepam were determined to be in the range 17.6-18.7 kcal/mol. At -55 °C ΔG(≠) for flunitrazepam was determined to be in the 15.6-15.7 kcal/mol range. The experimental dynamic chromatograms and the corresponding interconversion barriers reported in this paper call for a reinterpretation of previously published results on the HPLC behavior of diazepam on chiral stationary phases.

Application of Empore C-8 extraction disks for screening urine in systematic toxicological analysis.

Solid-phase extraction (SPE) by means of disposable columns has become a widely accepted technique for sample pretreatment in toxicology, both for directed analyses and for screening analyses. However, the sample capacity in SPE is usually limited to a few millilitres. Therefore, we have investigated to what extent these problems can be overcome by using Empore extraction disks, consisting of chemically modified C-8 reversed-phase silica, embedded in an inert polytetrafluoroethylene (PTFE) matrix. Human urine was selected as the matrix and dexetimide and mepyramine were initially used as test drugs because these drugs were available in tritiated form. Additional drugs investigated included codeine, hexobarbital, imipramine, methamphetamine, and nitrazepam. In these investigations, the sample capacity for untreated urine was at least 25 mL, and analyte quantities up to 250 micrograms could be retained by these filters. Washing with water/methanol mixtures was successful in removing substantial amounts of endogenous interferences, and methanol proved to be an acceptable eluent. Thus, these disks seem to have interesting potential for toxicological analysis in that sample concentration and cleanup can be achieved at the same time.