Development and validation of a GC method for quantitative determination of enantiomeric purity of a proline derivative

Enantioselective HPLC, SFC and GC methods were evaluated for separation and quantitative determination of chiral purity of (2R,4R)-1-(1-tert-butoxyvinyl)-4-methoxypyrrolidine-2-carboxylic acid [(2R,4R)-TBMPCA], a common building block in organic synthesis. All three separation methods can provide baseline resolution of (2R,4R)-TBMPCA and its enantiomer (2S,4S)-TBMPCA; however, both enantioselective HPLC and SFC are unsuitable for quantitation of low levels of the undesired enantiomer in (2R,4R)-TBMPCA. Comparatively, the enantioselective GC method not only separates the derivatized enantioselective pair with resolution as high as 4, but also was shown to be sufficiently linear, precise, and accurate to enable quantitation of derivatized (2S,4S)-TBMPCA down to 2.4 μg/ml (0.04% of nominal concentration). The sample derivatization procedure is simple, and no sample clean-up is needed before injecting samples for enantiomeric GC analysis. Compared to the enantioselective HPLC and SFC methods, the enantioselective GC method is advantageous because of its high efficiency and high sensitivity.