A New UHPLC-UV-MS Method for Detection and Identification of Bioactive Compounds

Broad nutrient and bioactive compound screening can be challenging due to the vast differences in molecular properties of the compounds (e.g. capturing data on hydrophilic organic acids as well as hydrophobic sterols and vitamins). Therefore, the present work aimed to develop an liquid chromatography-mass spectrometry (LC-MS) analytical method to simultaneously identify and quantify more than 91 compounds in a single method. This chromatographic separation was tailored for detection of hydrophilic as well as hydrophobic compounds, with special attention towards the separation of phenolic compounds, of which there are many dozen mass isomers. The chromatographic separation was performed in 45 min on a C18-PFP phase, enhancing the resolution of isomers. In addition, the presence of an ultraviolet (UV) detector before the MS detector allowed the confirmation of the identities of many species by their UV absorbance profile. The developed method was applied in this work to measure different types of compounds recognized to have potential health benefits: 29 flavonoids, 16 phenolic acids, 15 vitamins, eight tocochromanols, eight steryl ferulates, six phytosterols, and many other small molecules. Extracts from wheat bran and lemon peel were used to illustrate the advantages of this methodology. Raw wheat bran contains many steryl ferulate compounds, which release ferulic acid upon hydrolysis. This method successfully quantified the precursors and the liberated ferulic acid, revealing its potential to assess and determine the underlying chemistries at play during food and feed processing and digestion. Additionally, the lemon peel extract demonstrated the efficiencies of separating the multitude of phenolic species. This methodology will allow for a more accurate and comprehensive analysis of the main bioactive constituents in raw materials and by-products of economically relevant crops. This data enables discoveries of new commercial value in the food processing industry and can be leveraged to promote health and well-being of consumers.