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