Binding of Clitoria ternatea L. flower extract with α-amylase simultaneously monitored at two wavelengths using a photon streaming time-resolved fluorescence approach.

The binding of an extract from the flowers of Clitoria ternatea L. to the digestive enzyme α-amylase was investigated. This extract is a mixture of flavonoids, including anthocyanins, and has been previously shown to inhibit the activity this enzyme. This has implications for modulating starch digestion. In order to investigate the kinetics, we made use of time-resolved fluorescence to simultaneously monitor two different emission bands emanating from the extract. This measurement was enabled by the use of a "photon streaming" approach and changes in fluorescence lifetime and intensity were used to follow the interaction. A longer wavelength band (655 nm) was ascribed to anthocyanins in the mixture and these were observed to bind at a rate an order of magnitude slower than other flavonoids present in the extract, monitored at a shorter wavelength (485 nm). Changes in the fluorescence emission of the extract upon binding were further assessed by the use of decay associated spectra.

Enrichment and quantification of monoacylglycerols and free fatty acids by solid phase extraction and liquid chromatography-mass spectrometry.

Quantification of monoacylglycerols (MAG) and free fatty acids (FA) is of interest in biological systems, in food, cosmetic and pharmaceutical products. This manuscript describes and validates a reversed phase liquid chromatography-tandem mass spectrometry based approach for simultaneous quantification of these analytes in fats and oils. Purification and concentration of MAG/FA were performed using cation exchange solid phase extraction, which allowed elimination of the abundant triacylglycerols. Following cleanup and concentration, the analytes were separated and detected with the aid of volatile ammonium-formate buffer. MAG were detected in positive ion mode, while FA were detected in negative ion mode. The method was validated by the method of standard additions and using stable isotope labeled internal standards. The results confirm the feasibility of quantifying these two classes of analytes simultaneously without any chemical derivatization. The obtained main quantitative features include: (1) lower limits of quantification 1-30ppm for MAG analytes, (2) lower limits of quantification 90-300ppm for FA analytes, (3) averaged inter-batch precision 6%, and (4) averaged bias -0.2% for MAG and 0.5% for FA. Various animal fat and vegetable oil samples were characterized for their MAG/FA profile indicating the usefulness of the method to address quality and authenticity of fats and oils.