Development and Validation of Stability Indicating LC-PDA Method for Mycophenolate Mofetil in Presence of Mycophenolic Acid and Its Application for Degradation Kinetics and pH Profile Study

Factorial design tool applied for development of isocratic reversed-phase stability-indicating HPLC method for the analysis of mycophenolate mofetil (MMF) and its degradation products. MMF stress degradation products mycophenolate acid (MPA) and DP3 (USP impurity H) were isolated and used for quantitation. Separation achieved on a Symmetry C18 (250？mm × 4.6？mm, 5.0？μ) column using a methanol: acetate buffer (75？:？25？v/v), pH 6.0 (adjusted with acetic acid), at 0.5？mL flow rate, column maintained at 55°C, and data integrated at 251？nm. MMF is subjected to hydrolysis, oxidation, heat degradation, and so forth; under all these conditions degraded products are well separated. The method validation characteristics included accuracy, precision, linearity, range, specificity, and sensitivity. Robustness testing is conducted to evaluate the effect of minor changes to the chromatographic conditions and to establish appropriate system suitability parameters. The proposed method is used to investigate kinetics of acid, alkali hydrolysis and oxidation process. Major degradation products MPA and DP3 were isolated and quantitated. Characterization of MPA by NMR and LC-MS/MS and other degraded products by LC-MS/MS is attempted successfully. The method is used successfully for the quality assessment of three MMF drug commercial formations and its acid, alkali, and oxidative degradation kinetics study. 1. Introduction Mycophenolate mofetil (MMF) is chemically 2-(morpholin-4-yl) ethyl (4E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoate. It is inhibitor of nucleic acid synthesis. It is the ester moiety of mycophenolic acid (MPA). It is a white or almost white crystalline powder and is used as immunosuppressive [1]. HPLC and HPTLC analytical methods for dissolution study of MMF in capsules, for its assay in bulk and dosage forms, [2–6] are reported. Bioanalytical and immunoassay methods are available for its determination individually or in presence of MPA [7–9] and one UV dissolution method is reported [10]. Its degradation products in aqueous solution are studied [11]. Stability-indicating HPLC and LC-MS/MS methods [12, 13] are reported where structural elucidation of degraded product and separation of the stress degraded product is lacking. MMF is included in BP, EP, and USP and assayed by potentiometry [14, 15]. Isocratic LC method was described in proposed USP monograph [16, 17] for MMF tablet and capsule assay using mobile phase acetonitrile？:？water containing 0.3% triethylamine (30？:？20？v/v), pH 5.3 (adjusted with