METHOD DEVELOPMENT AND VALIDATION OF NORETHINDRONE ACE-TATE ASSAY AND ITS RELATED IMPURITIES IN API AND PHARMACEUTICAL FORMULATION WITH ORTOGONAL DETECTOR TECHNQUES

Abstract

Objective: To develop and validate a sensitive and stability indicating gradient reverse phase ultra-high performance(UHPLC-PDA) liquid chromatography with photodiode array(PDA) and mass spectroscopy (MS) of Norethindrone Acetate (NA) assay and organic impurities (OI) in active pharmaceutical Ingredient(API) and Pharmaceutical Formulation (PF).

Methods: The chromatographic conditions were optimized using Zorbax SB-C18 analytical UHPLC column with the dimensions (100 x 2.1) mm and 1.8 μm particle sizes. The mobile phase consisted of water (solution A) and acetonitrile (solution B) with gradient elution as mentioned time (min)/% Solution B: Initial/40,0.2/40, 9.2/55,12.0/55,12.2/90,15.5/90, 15.8/40 and 18.0/40. The flow rate was at the rate of 0.4 ml/min and the detection wavelengths were 254 nm and 210 nm. The column was kept at 40 °C and the injection volume was 5 μL. Stability of NA sample in different conditions was investigated by exposing the drug to stress study utilizing acid, base, oxidation, thermal, Humidity and photolytic.

Results: There was no interference from excipients, impurities or degradation products at the retention time of NA about 9.1 min indicating the specificity of the method.

The drug showed good stability under oxidation, thermal, humidity and photolytic conditions, but significant degradation was observed under acid and base conditions. The procedure was validated for specificity, linearity, accuracy, precision and robustness. The degradation products were well resolved from NA and its impurities. The obtained LOD (Limit of detection) values are 0.001% to 0.015% and LOQ (Limit of quantification) values are 0.003% to 0.05% of impurities.

Conclusion: A sensitive, rapid, specific and stability indicating gradient Reverse Phase UHPLC-PDA-PDA with MS (Orthogonal detectors) method for the determination of NA for the assay and organic impurities was successfully developed. The developed method was validated to be specific, linear, accurate, precise and robust. The peak purity and LC-MS test results confirmed that the NA peak was homogenous in all stress samples and the mass balance was found to be more than 99%, thus proving the stability indicating power of the method