Supercritical fluid chromatography for the separation of lipophilic tanshinones from Salvia miltiorrhiza Bunge.

This study systematically investigated the retention behavior of lipophilic tanshinones (Tans) on distinct stationary phases and developed a supercritical fluid chromatography (SFC) method under a binary mobile phase (CO2 and methanol). The non-polar VCC18 column showed extremely weak retention and virtually no separation selectivity for eleven standards. Despite some improvements observed with several typical polar columns (Viridis Silica, Diol, and NH2) and a SFC dedicated column (Torus 2-PIC), peak shapes and selectivity remained poor. However, a highly polar Click TE-Cys column exhibited good peak shapes and achieved complete separation of all standards, and was thus identified as the optimal stationary phase for Tans. These results indicate that despite the lipophilicity of Tans, their retention is primarily affected by internal polar oxygen atoms, requiring highly polar columns instead of C18 or conventional polar columns for adequate separation selectivity. In addition, the SFC separation method based on the Click TE-Cys column exhibited better separation selectivity and good orthogonality compared with RPLC. Its application potential was demonstrated by offline SFC × RPLC-MS/MS analysis, which annotated an additional 55 compounds in Tans, including 17 unknown compounds. Overall, this study provides mechanistic insights into the retention of oxygen-containing lipophilic compounds in SFC, as well as a methodological advance for Tans separation and a theoretical foundation for SFC method development of analogous compounds.