Quantification of fluorescent dyes in organ tissue samples via HPLC analysis

The determination of regional blood flow via the accumulation of fluorescent microspheres is a concept regularly used in medical research. Typically, the microbeads get extracted from the tissue of interest and are then quantified by measuring the absorption or fluorescence of the incorporated dyes without further separation from the medium. However, in that case the absorption spectra of different dyes can overlap when used simultaneously, leading to an overestimation of the concentration. Additionally, background absorption from the medium can be problematic. Therefore, a high performance liquid chromatography method for the simultaneous detection of four dyes (orange, crimson, yellow-green and red) incorporated in different microbeads in samples from biological media such as organ tissue (brain, heart and kidneys) was developed. Since for biological samples often a large sample size is required for sufficient statistics, the method was optimized to yield very short run times. With this method it was possible to detect very low concentrations of only one microsphere per gram of organ tissue. By applying this sensitive quantification technique, it was demonstrated that the application of microbeads for perfusion measurements might not be reliable due to different organ distributions in each animal.