Quantification of isoflavones in nutritional supplements of red clover by HPLC and HPTLC analysis
Two methods for the quantification of the five main isoflavones in red clover containing nutritional supplements using high performance liquid chromatography and high performance thin layer chromatography were developed. Validation of both methods was performed according to the ICH guidelines. The HPLC method was found to be rapid (25min for a single run) and offered a good linearity, sensitivity and repeatability but caused high operating costs due to the sophisticated instrumentation and extensive amounts of solvent for the whole validation process. For the quantitative determination of the five isoflavones by HPTLC, a mobile phase of dichloromethane-acetic acid-ethyl acetate (12:2:1, v/v/v) was used which afforded sufficient separation. HPTLC allowed simultaneous fast quantification of the isoflavones comparable to the HPLC method and efficient distinction between different nutritional supplements. Therefore, this inexpensive method could be used in the quality control of nutritional supplements of red clover.