LC–MS/MS assay for Acetazolamide, A Carbonic Anhydrase Inhibitor in Human Plasma and its Clinical Application

Background: Acetazolamide is a carbonic anhydrase inhibitor commonly used to treat glaucoma, epilepsy, mountain sickness, periodic paralysis, central sleep apnea and idiopathic intracranial hypertension. Objective: The objective of this research was to develop a novel liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the determination of acetazolamide in human plasma. Methods: An analytical method based on LC-MS/MS (API-4000) has been developed and validated for the quantitative determination of acetazolamide in human plasma using acetazolamide d3 as Internal Standard (IS). After Solid phase extraction (SPE), analyte and the IS were chromatographed on a C18 columns using a isocratic mobile phase composed of 0.1% formic acid buffer and acetonitrile (30:70, v/v) pumped at a flow rate of 0.80 mL/min. Results: Precision and accuracy of the method was determined using five analytical batches in the concentration range of 50.3–12046 ng/mL. All the validation experiments were carried out as per the US FDA guidelines and results met the acceptance criteria. Conclusion: The proposed LC–MS/MS assay method is simple, rapid and sensitive for the determination of acetazolamide in human plasma. A chromatographic run time of 2.0 min, allow us to analyze more than 300 samples in a day. Key words: Acetazolamide, Human plasma, LC–MS/MS, Method validation, Pharmacokinetics.