Development and Validation of UPLC–MS/MS Method for Quantitative Analysis of 5-Fluorouracil in Aqueous Humor of Rabbits

5-Fluorouracil (5-FU) is now used in eye drops for the management of conjunctival malignant melanoma, intraepithelial neoplasia, and corneal squamous cell carcinoma. The previously methods 5-FU quantification AqH were time-consuming less sensitive. Herein, a highly perceptive bioanalytical UPLC–MS/MS method was developed quantitative determination aqueous humor (AqH) rabbits using allopurinol as internal standard (IS). IS well separated an Acquity™ HILIC column. Acetonitrile 10 mM ammonium acetate at 95:5 (v/v) isocratically pumped 0.3 mL/min flow rate with total runtime 2.5 min. samples processed liquid–liquid extraction ethyl acetate. identified negative mode electrospray ionization. parent to daughter ion transitions occurred m/z 128.92→41.68 134.80→64.10, respectively, quantified multiple reaction monitoring mode. validated ICH-Harmonized Guideline Bioanalytical Method Validation, parameters within acceptable limits. calibration curve linear 10.5–2000 ng/mL concentration range, correlation coefficient (R2) 0.9946, lower limit detection 3.55 ng/mL. rapid, sensitive, accurate robustly able quantify rabbit AqH. effectively applied establish ocular pharmacokinetics following topical instillation 5-FU-containing preparations rabbits.