A sensitive UPLC-APCI-MS/MS method for the determination of dexamethasone and its application in an ocular tissue distribution study in rabbits following topical administration

Abstract

A sensitive, specific and rapid ultra-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (UPLC-APCI-MS/MS) method has been developed and validated for the determination of dexamethasone (DEX) in ocular tissues and biofluids of rabbits. Dexamethasone-d₅ (DEX-d₅) was used as an internal standard. Protein precipitation was used to extract the drug from ocular tissue homogenates, biofluids and plasma. Chromatographic separation was achieved on an Acquity C₁₈ column (100 × 2.1 mm, 1.7 μm) using a gradient run with a mobile phase composed of methanol and 5 mM ammonium formate in 0.1% formic acid in water buffer at a flow rate of 0.5 mL min⁻¹. A detailed method validation in two matrices (bovine serum albumin and phosphate buffer) was performed as per the FDA guidelines and the calibration curve obtained was linear (r² = 999) over the concentration range 200–100 000 pg mL⁻¹. A Sciex 6500+ coupled with a Waters Acquity UPLC (Ultra Performance Liquid Chromatography) system was operated in the multiple monitoring mode (MRM) during the analysis. The proposed method was applied to a rabbit ocular tissue distribution study after topical ophthalmic administration of dexamethasone and tobramycin suspension.