Developing a quantitative method of mangiferin in rabbit lacrimal fluid as a tool for investigating pre-cornea drug residence

Abstract

Mangiferin (MGF) has been evidenced to effectively treat infectious diseases caused by Herpes simplex virus, especially on the acyclovir-resistant strains. For ophthalmic disease treatment, prolonging drug retention in the eyes plays an essential role in effectively delivering drugs, which are prone to elimination by eye blinking and tear fluid secretion. The objective of this study was to develop a method for analysis of MGF in tear fluid that can be further utilised to evaluate the kinetics of precornea drug concentration. For sample collection, lacrimal tear fluid was absorbed into pre-weighed absorbing paper strips, which were then immediately weighed again right after sampling to calculate the sample mass. Subsequently, MGF was extracted from the paper strips using a mixture of methanol : acetic acid 0.6% (35:65, v/v). The extracted solution was finally injected into an HPLC system for MGF analysis (RP-column C18 - 4.6 x 250 mm, 5 μm was stabilised at 40oC; moblie phase methanol : acetic acid 0.6%; 35 : 65 (v/v) at a flow rate of 1.0 mL/min; autosampler, injection volume 10 µL; detection wavelength: 258 nm). The developed methodology was validated following the U.S. FDA and ICH M10 guidances for bioanalytical method validation, with respect to system suitability, linearity, recovery, accuracy and precision, dilution integrity and stability. The method was also successfully employed to determine MGF concentration in the tear fluid collected from rabbit eyes at one hour after instilling with MGF suspension. The methodology herein provides a practical tool to investigate kinetics of MGF in tear fluid that can be applied in ophthalmic drug development as well as drug quality evaluation activities.