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Enantiomeric quality control of R ‐Tofisopam by HPLC using polysaccharide‐type chiral stationary phases in polar organic mode
Author(s) -
Foroughbakhshfasaei Mohammadhassan,
Szabó ZoltánIstván,
Mirzahosseini Arash,
Horváth Péter,
Tóth Gergő
Publication year - 2018
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201800220
Subject(s) - enantiomer , chemistry , chromatography , high performance liquid chromatography , amylose , methanol , cellulose , supercritical fluid chromatography , chiral resolution , analytical chemistry (journal) , organic chemistry , starch
Abstract A novel, fast and economic chiral HPLC method was developed and validated for the resolution of the four isomers of tofisopam. The separation capacity of eleven different chiral columns: six polysaccharide‐type including three amylose‐based (Chiralpak AD, Chiralpak AD‐RH and Chiralpak AS) and three cellulose‐based (Chiralcel OD, Chiralcel OJ and Lux Cellulose‐4); three cyclodextrin‐ (Quest‐BC, Quest‐C2 and Quest‐CM) and two macrocyclic glycopeptide antibiotic‐type (Chirobiotic T and Chirobiotic TAG) were screened using polar organic or reversed‐phase mode. Chiralpak AD, based on amylose tris(3,5‐dimethylphenylcarbamate) as chiral selector with neat methanol was identified as the most promising system. In order to improve resolution, an orthogonal experimental design was employed, altering the concentration of 2‐propanol, column temperature, and flow rate in a multivariate manner. Using the optimized method (85/15 v/v methanol/2‐propanol, 40°C, flow rate: 0.7 mL/min) we were not only able to separate the four isomers but also detect 0.1% S ‐enantiomer as chiral impurity in R ‐tofisopam. This is important since the latter is under development as a single enantiomeric agent. Thermodynamic investigation revealed an unusual entropy and enthalpy‐entropy co‐driven controlled enantioseparation on Chiralcel OJ and on Chiralpak AD column, respectively. Our newly developed HPLC method was validated according to the ICH guidelines and its application was tested on a pharmaceutical formulation containing the racemic mixture of the drug. As a further novelty, a separate circular dichroism method was applied for the investigation of the interconversion kinetics of tofisopam conformers, which proved to be crucial for sample preparation and method validation.