BIOCHEMICAL INVESTIGATION AND DEVELOPMENT OF HPLC METHOD FOR TACROLIMUS

The objective of this work was to develop a method for the quantification of tacrolimus in rabbit aqueous humor by UHPLC. UHPLC analysis was performed on a Waters Acquity UHPLC system (Milford, MA, USA). A 50 μL aliquot of rabbit aqueous humor was pipetted into a 2.0 ml Eppendorf tube and 100 μL of acetonitrile was added to precipitate the protein. The samples were vortex mixed for 2 min followed by filtration through 0.22 μm nylon filter. To this filtrate, 0.4 ml of 0.01M iodine was added and volume up to 1.0 ml was made with acetonitrile. Five microliter of this solution was injected into the UHPLC system. All the rabbit aqueous humor samples were stored at −20°C and were allowed to thaw at room temperature prior to sample preparation. Linearity was investigated by the assay in parallel of triplicate rabbit's aqueous humor samples spiked with TAC to concentrations of 10, 20, 50, 100, 200, 400, 600 and 800 ng/ml. Stability assessments under different conditions: bench-top, short-term, long-term storage stability and freeze–thaw were established. The results indicated that TAC had an acceptable stability under those conditions. A method was developed for quantification of tacrolimus in rabbit aqueous humor by UHPLC. This method with QL of 1.0 ng/ml was fast and just took 5 minutes. There were no interferences found from endogenous aqueous humor components or other sources. This assay has showed consistent precision and accuracy. The analytical method presented here will be useful for the determination of tacrolimus concentration in the ocular aqueous humor. Keywords: Tacrolimus, UHPLC, Validation, Stability Study, Ocular.