Enhancement of teprenone isomer separation by subcritical fluid chromatography using porous graphitic carbon column

Teprenone is a therapeutic anti-ulcer agent developed in Japan. As described in the Japanese Pharmacopoeia (JP) 17th Edition, gas chromatography/hydrogen flame ionization detection (GC/FID) and high-performance liquid chromatography/ultraviolet detection (HPLC/UV) have been used for the assay of active pharmaceutical ingredients (APIs) and teprenone capsules, respectively. The critical aspect of the assay is a separation of the structural isomers (mono-cis and all-trans) of teprenone. Herein, we propose an improved quantitative method for the quality control of teprenone in APIs and capsules via subcritical fluid chromatography/photo diode array detection (SubFC/PDA) using a porous graphitic carbon column. SubFC conditions, i.e., type and content of the organic modifier in the mobile phase, column temperature, injection volume, and flow rate, were optimized. The developed SubFC/PDA method was validated according to ICH guidelines Q2(R1) in terms of accuracy, precision (repeatability and intermediate precision), specificity, linearity, quantification range, robustness, and stability. Comparison of SubFC/PDA method with the GC/FID or HPLC/UV method (described in JP) revealed that the SubFC/PDA method gave better resolution and run time than the JP methods. The developed SubFC/PDA method is expected to be useful for pharmaceutical analysis or quality control of teprenone isomers.