Determination of Evans blue as a blood–brain barrier integrity tracer in plasma and brain tissue by UHPLC/UV method

Blood–brain barrier changes are an integral part of many neurodegenerative diseases. Evans blue is an intravital dye that binds to albumin and can therefore be used to monitor extravasation of this plasma protein across blood–brain barrier in animal models of neurodegeneration. To monitor extravasation of albumin across blood–brain barrier, we developed and validated an ultrahigh-performance liquid chromatography method for the analysis of Evans blue in rat plasma and brain samples. Analyte was separated on ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm) using a 5-min gradient run and detected by a UV detector. The limits of quantification (LOQ) were 10 µg/mL in plasma and 0.5 µg/g in brain samples. The limits of detection (LOD) were 1 µg/mL in plasma and 0.015 µg/g in brain samples. The method showed excellent linearity with regression coefficients higher than 0.99. The accuracy was within the range of 91–105%. The inter-day precision was in the range of 1.3–8%. The benefits of using UPLC are selectivity, short analysis period, and thus, a very good sample throughput. Using this method, we analyzed albumin extravasation across blood–brain barrier in transgenic rat model for tauopathy SHR-72 and age-matched control animals.