Development of a novel method for polar metabolite profiling by supercritical fluid chromatography/tandem mass...

Supercritical carbon dioxide (scCO₂), the main fluid in the mobile phase for supercritical fluid chromatography (SFC), is non-polar. The majority of polar compounds are little soluble in scCO₂, thereby rendering them poor candidates for achieving separation by carbon dioxide-based SFC. There is no reported method for the comprehensive analysis of hydrophilic metabolites by SFC with mobile phases comprising a high CO₂ ratio. In this study, we investigated the effect of additives in the modifier for enabling the application of SFC to profile diverse polar compounds for metabolomics. Eleven types of columns were screened by using proteinogenic amino acids as the model compounds. The addition of water and acids (formic acid and trifluoroacetic acid (TFA)) to the modifier was also investigated to improve the solubility of the polar compounds and mitigate the unfavorable interaction between the stationary phase and the polar compounds. A significant improvement in the peak shapes of the amino acids was observed upon addition of TFA. The CO₂/modifier ratio and TFA concentration in the mobile phases were investigated using the CROWNPAK CR-I (+) column, which showed the best performance during the column-screening. The CO₂/methanol/water/TFA ratio of 70/27/3/0.15 (v/v/v/v) was determined as the optimized mobile phase composition. Furthermore, the applicability of the optimized analytical method to other polar compounds was examined; 100 cationic and amphoteric compounds with predicted logP_ow values that ranged from -5.9 to 1.7 could be simultaneously analyzed without derivatization. Anionic compounds such as organic acids, phosphates, and sugars were excluded from the target analytes. Most of the previously reported SFC methods for analyzing polar compounds employ a gradient elution and require the use of high modifier ratios at 40% or more. In the proposed method, the use of water and TFA enabled the rapid and simultaneous analysis under isocratic elution within 10 min, even with a high CO₂ ratio of 70%. Additionally, a rat serum extract was analyzed using the optimized conditions, and 43 polar metabolites were successfully detected. This result demonstrates the applicability of the SFC/tandem mass spectrometry method to real samples.