Profiling and quantitating the constituents of red clover extracts using UHPLC/UV/CAD/HRMS: A component of the safety assessment process

In order to support safety assessments without animal studies, we have developed a comprehensive approach to identify and quantitate the constituents of complex botanical mixtures. The methodology utilizes separation by UHPLC, followed by UV detection, charged aerosol detection (CAD) and high resolution mass spectrometry (with MS/MS and MS³). CAD provides quantitation of the analytes, facilitated by the addition of an inverse gradient stream which compensates for the known sensitivity of that general detector to mobile phase composition. The presentation will highlight the use of CAD for quantitation, as compared to quantitation using the UV or MS detectors with authentic standards, within the same analysis. The advantages and limitations of this multi-detector approach will be illustrated with results using extracts of red clover. Additionally, the use of MS³ for characterizing aglycones of isoflavone conjugates will be explained.