Efficient quantification of methazolamide in rat plasma using UHPLC-Q-Orbitrap MS for pharmacokinetic analysis

Methazolamide, a carbonic anhydrase inhibitor, is widely used in the treatment of acute angle-closure glaucoma, chronic open-angle glaucoma, secondary glaucoma, and for reducing intraocular pressure during ophthalmic surgeries. This study established and validated a highly selective and sensitive ultra-high-performance liquid chromatography coupled with quadrupole-orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS) analytical method for quantifying methazolamide in plasma. The separation of methazolamide and acetazolamide (internal standard) was conducted using a BEH Shield RP18 column. The mobile phase employed a gradient elution with acetonitrile (solvent A) and water containing 0.1% formic acid (solvent B). Data collection was executed in the positive ionization mode, employing comprehensive full MS/dd-MS2 on a Q-Orbitrap mass spectrometer. Methazolamide and acetazolamide (internal standard) were well resolved, with retention times of 2.63 min and 1.52 min, respectively. Methazolamide exhibited a robust linear correlation over a plasma concentration range of 10-5000 µg L^-1, with a lower limit of quantification established at 10 µg L^-1. The precision and accuracy were maintained below 8%, with methazolamide extraction recovery from plasma ranging between 86.16% and 96.06%. This research introduces an efficient method for the quantification of methazolamide. The validated method effectively assessed methazolamide pharmacokinetics in rat plasma following a 2 mg kg^-1 oral dose.