Nikalje & Choudhari: jourNal of aoaC iNterNatioNal Vol. 96, No. 3, 2013 593 Development and Validation of a Stability-Indicating HPLC-Photodiode Array Detector Method for Formulation Analysis and Degradation Kinetics and Dissolution Studies of Mycophenolate Sodium and HPLC/MS/MS Characterization of its Stress Degradation Products AnnA PrAtimA G. nikAlje Y.B. Chavan College of Pharmacy, Dr. Raiq Zakaria Campus, Postgraduate Department of Pharmaceutical Chemistry, Rauza Bagh, Aurangabad, 431001, Maharashtra, India Vishnu P. ChoudhAri Maharashtra Institute of Pharmacy, MIT Campus, Paud Rd, Kothrud, Pune-411038, Maharashtra, India DRUG FORMULATIONS AND CLINICAL METHODS A simple stability-indicating isocratic RP-HPLC method was developed and validated for the determination of mycophenolate sodium and its alkali degradation product. Forced degradation of the drug was carried out under thermolytic, photolytic, acid/base hydrolytic, and oxidative stress conditions. Alkali degradation product DP1 was isolated, and separation of stress degradation products was achieved on a Symmetry C18 (250 × 4.6 mm × 5.0 µm) column using the mobile phase methanol–acetate buffer adjusted with acetic acid to pH 6.0 (76 + 24, v/v) at a 0.55 mL/min low rate and 50°C. Data were integrated at the detection wavelength of 251 nm. The method validation characteristics included accuracy, precision, linearity, range, speciicity, and sensitivity per International Conference on Harmonization guidelines. Robustness testing was conducted to evaluate the effect of minor changes in the chromatographic conditions and to establish appropriate system suitability parameters. Structural elucidation of degraded products was performed by HPLC/MS/MS. The method was used successfully for drug product analysis, dissolution study, and determination of the drug’s acid, alkali, and oxidative degradation kinetics. M ycophenolate sodium (MPS), chemically sodium 4(E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1, 3 dihydroisobenzofuran-5-yl) -4-methylhex-4- enoate, is an inhibitor of nucleic acid synthesis and immunomodulator (1). It is not included in any pharmacopoeia. There are no methods reported for its determinations, although there are a few HPLC bioanalytical methods (2–11) available for determination of mycophenolic acid (MPA) in plasma and other biological luids by using luorometric (12, 13) and MS (14–16) detectors alone or with mycophenolate mofetil (MMF) and its gluconoride derivative. To our knowledge, there is no stability-indicating HPLC method reported for either MPS or MPA, and no oficial method for MPS. Accordingly, the aim of the present study was to establish inherent stability of MPS through stress studies under a variety of the International Conference on Harmonization (ICH)- recommended test conditions (17, 18), and to develop a stability- indicating assay method and application of the proposed method to study the degradation kinetics and dissolution. Experimental Reagent and Chemicals HPLC grade methanol was purchased from Merck (Mumbai, India). Acetate buffer (25 mM), pH 6.0, was prepared by dissolving and diluting 1.93 g ammonium acetate to 1000 mL with distilled water; pH was adjusted with 5 M acetic acid. Analytical grade acetic acid and ammonium acetate were purchased from LOBA Chemie Pvt. Ltd, Mumbai, India. Tablets analyzed were Renodapt S-180 (enteric coated, manufactured by Biocon Ltd, Bangalore, India, Batch No. BLMP09019) containing MPS equivalent to 180 mg MPA. Pure drug sample of MPS (purity = 99.8%) was kindly supplied as a gift by Panacea Biotech Ltd, Solan, India, and standard hydrochlorothiazide (HTZ; purity = 99.78%) was a gift from Cipla Ltd, Mumbai, India; these were used without further puriication. Chemicals were of HPLC grade and were obtained from commercial sources in the local market. Instrumentation The Waters Inc. (Milford, MA) HPLC system consisted of a binary pump (Model 515), autosampler (Model 717 plus), column heater (Model CHM, Sr. No. A08CHM 289M), and photodiode array (PDA) detector (Model 2998). Data collection and analysis were performed using Empower Version 2 software (Waters Inc.). Separation was achieved on Symmetry C18 (250 × 4.6 mm id, 5.0 µm particle size; Waters Inc.) and Kromasil C18 (250 × 4.6 mm id, 5.0 µm particle size; Sigma-Aldrich, St. Louis, MO) columns maintained at 50°C using the column oven. The column was supported with a Waters Inc. Symmetry C18 (3.9 × 20 mm id, 5.0 µm particle size) guard column. A calibrated dissolution apparatus (U.S. Pharmacopeia) was used for the dissolution study. Received February 12, 2011. Accepted by SW April 17, 2011. Corresponding author’s e-mail: ana@k.st DOI: 10.5740/jaoacint.11-063