A Reverse Phase LC Method Development and Validation for the Quantification of Acetazolamide and its Specified and Unspecified Degradation Products in Hard Gelatin Capsule Formulations

Acetazolamide, a carbonic anhydrase inhibitor, is used orally to reduce intraocular pressure in patients suffering from glaucoma. A simple, specific, accurate, precise and stability-indicating reverse-phase HPLC method has been developed and validated to identify and quantify acetazolamide and its specified and unspecified degradation products in hard shell capsules formulations. The chromatographic separation was achieved on Agilent Zorbax SB-CN, (4.6 mm × 150 mm, 3.5 μm) using mobile phase-A (methanol: water: phosphoric acid; 1:9:0.1 v/v/v) and mobile phase-B (methanol: water: phosphoric acid; 4:6:0.1 v/v/v). The flow rate was set at 1.0 mL min−1, and the column temperature was 40 °C. The wavelength 254 nm was used to detect and quantify acetazolamide and its related impurities with an injection volume of 30μL. The retention time for the acetazolamide and its impurities was found at 4.601 min (Acetazolamide), 4.221 min (Acetazolamide impurity A), 14.303 min (Acetazolamide impurity B), 8.342 min (Acetazolamide impurity C), 2.488 min (Acetazolamide impurity D) and 3.411 min (Acetazolamide impurity E), respectively. The linearity study was conducted in a range of 0.5 µg mL−1 to 82 µg mL−1 for acetazolamide and 0.1 µg mL−1 to 4 µg mL−1 for its related impurities. The proposed method was accurate, precise, stability indicating and convenient for the quantitative analysis of acetazolamide and related impurities in the drug product.