Investigators who study functional foods, nutraceuticals, and other natural products are faced with a formidable challenge. The bioactive compounds are often present at low abundance in extremely complex mixtures. Advances in liquid chromatography and mass spectrometry techniques have greatly enhanced our ability to characterize such mixtures. However, the ideal analytical technique would go beyond detection and enable the active components to be distinguished from those that are inactive. Our laboratory is engaged in the development of mass spectrometry strategies to achieve this goal. In particular, we have focused on identifying natural product compounds or mixtures useful for the treatment of infection.As a first step towards identifying bioactive compounds, active crude extracts are fractionated into 96 well plates, creating simplified pools of compounds compatible with biological assays. The biological assays are then conducted in these very same well plates. Importantly, the bioassays have been designed so that mass spectrometry can be used to detect the desired biological endpoints. Thus, using ultrahigh performance liquid chromatography coupled to an LTQ-Orbitrap mass spectrometer, it is possible to both tentatively identify the constituents present in the wells (based on molecular formula) and evaluate their activity. Using this approach, we have identified a series of compounds from the botanical medicine goldenseal (Hydrastis canadensis) with diverse chemical structures and several different biological activities. These compounds appear to contribute via several different mechanisms to the overall effectiveness of crude H. canadensis extracts against bacterial infections such as methicillin-resistant Staphylococcus aureus (MRSA). We have identified compounds that act as quorum quenching agents, efflux pump inhibitors, and antimicrobials.  Overall, our investigations demonstrate the complexity of the interactions that occur between complex botanical extracts and biological systems. High resolving power mass spectrometry is a powerful tool for unraveling this complexity.

Mass spectrometry based strategies to distinguish bioactive and inactive components of complex mixtures

246th National Meeting (2013)

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