Determination of trace amount of enantiomeric impurity in therapeutic nicotine derivative using capillary electrophoresis with new imaging technology detection

One of the many attempts to stop the danger of tobacco smoking is the development of an anti‐smoking vaccine using nicotine butyric acid (NBA) linked to a carrier protein to produce anti‐nicotine antibodies. NBA is a chiral molecule and there is a need to obtain a high degree of enantiomeric purity. The aim of this work is to develop a novel method for the enantioseparation of NBA and the determination of trace amounts of enantiomeric impurity required by regulatory authorities. This was achieved successfully using high‐performance capillary electrophoresis combined with label‐free intrinsic imaging as new imaging technology. A 50 μm id fused‐silica capillary was used with UV detection at λ₂₁₄ nm and label‐free intrinsic imaging. The background electrolyte consisted of highly sulphated β‐cyclodextrin 10% m/V as a chiral selector in 75 mM phosphoric acid–triethylamine at pH 7.0. Baseline separation and detection of 0.1% and possibly less of the unwanted impurity (R‐enantiomer) were achieved. Also, the detection limits were calculated for both enantiomers. The use of label‐free intrinsic imaging has improved the sensitivity, enabling us to detect trace amounts of enantiomeric impurities.