Quantification of Hypocrellin B in Rabbit Ocular Tissues/Plasma by Spectrofluorimeter: Application to Biodistribution Study

A simple and sensitive red spectrofluorimetric method has been described for the estimation of hypocrellin B in rabbit plasma and ocular tissues such as retina, iris, aqueous humour and vitreous body. The fluorimetric estimation was achieved using black coloured 96- well plate at an excitation/emission wavelengths of 470/620 nm with a specific well volume of 100 µL/well. Hypocrellin B calibration curves were linear at a concentration ranging from 20 ng/mL to 1280 ng/mL with correlation coefficient between 0.86 and 0.99 in ocular tissues and plasma. Limit of detection and quantification was between 0.011 to 0.017 µg/mL and 0.034 to 0.054 µg/mL respectively for hypocrellin B in different ocular tissues and plasma. The percentage relative standard deviation observed for the validation parameters such as method precision, system precision, intra- inter day, analyst to analyst variations and robustness is found to be less than 12.5 % for the observed fluorescence intensity in different ocular tissues/plasma. The method has been applied to check the hypocrellin B concentration in plasma and ocular tissues after intravenous administration and found that hypocrellin B has some exposure to the posterior segment of eye without major intraocular presuure abnormalities. This selective and sensitive fluorimetric method will facilitate the future investigations of hypocrellin B on ocular delivery of hypocrellin B with less solvent consumption.