Abstract

The study aimed to investigate a novel approach by utilizing liquid chromatography (LC) and liquidchromatography-mass spectrometry (LC–MS) to separate, identify and characterize very nominal quantities ofdegradation products (DPs) of balsalazide along with its process related impurities without isolation from theirreaction mixtures. The impurities along with balsalazide were resolved on spherisorb ODS2 (250×4.6 mm, 5.0 μm)column at room temperature using 0.2 M sodium acetate solution at pH 4.5 and methanol in the ratio of 55:45(v/v) as mobile phase pumped isocratically at 1.0 mL/min as mobile phase and UV detection at 255 nm. Themethod shows sensitive detection limit of 0.003 μg/mL, 0.015 μg/mL and 0.009 μg/mL respectively for impurity1, 2 and 3 with calibration curve liner in the range of 50-300 μg/mL for balsalazide and 0.05-0.30 for itsimpurities. The balsalazide pure compound was subjected to stress studies and a total of four degradation products(DPs) were formed during the stress study and all the DPs were characterized with the help of their fragmentationpattern and the masses obtained upon LC–MS/MS. The DPs were identified as 3-({4-[(E)-(4-hydroxyphenyl)diazenyl]benzoyl}amino)propanoic acid (DP 1), 4-[(E)-(4-hydroxyphenyl)diazenyl] benzamide (DP 2), 5-[(E)-(4-carbamoylphenyl)diazenyl]-2-hydroxybenzoic acid (DP 3) and 3-({4-[(E)-phenyldiazenyl]benzoyl}amino)propanoicacid (DP 4). Based on findings, it was concluded that, the proposed method was successfully applicable forroutine analysis of balsalazide and its process related impurities in pure drug and formulations and also applicablefor identification of known and unknown impurities of balsalazide.