Supercritical fluid chromatography for the separation of isoflavones.

The first protocol for the analysis of isoflavones by supercritical fluid chromatography is reported. Optimum results were obtained on an Acquity UPC(2) BEH 1.7 μm column, using a solvent gradient of supercritical carbon dioxide and methanol (with phosphoric acid as additive) for elution. The method enables the baseline separation of nine isoflavones (aglyca and glycosides) in 8 min, and is suitable for their quantitative determination in dietary supplements containing soy (Glycine max), red glover (Trifolium pratense) and kudzu (Pueraria lobata). Method validation confirmed that the assay is selective, linear (R(2)≥0.9994), accurate (recovery rates from 97.6 to 102.4%), as well as precise on the short- and long-term level (intra-day precision ≤2.1%), and shows an on-column detection limit of 0.2 ng and below. This, together with an excellent performance shown in the analysis of real samples, indicates that SFC is well suited for the fast and accurate determination of isoflavones in complex matrices. Disadvantages compared to the established approaches were not observed, so that SFC has to be considered in this case as an (at least) equivalent analytical alternative.