Development and Validation of a Liquid Chromatographic Method for Aroylhydrazones at Hydrolytic Conditions

Background: The indole-containing aroylhydrazone derivatives 3a-c with potent antimycobacterial activity against a referent strain M. tuberculosis H37Rv and low cytotoxicity were evaluated for their stability via the precise and accurate HPLC analytical method in aqueous media of different pH (2.0, 7.0, 9.0 and 12.0). Objective: The study describes the development and validation of a simple and reliable HPLC-UV procedure for the determination of aroylhydrazone derivatives and their hydrolytic stability. Additionally, to recognize if hydrolysis leads to generating undesired products, the degradation processes were identified. Method: The separation was achieved with a LiChrosorb®RP-18 (250 x 4.6 mm) column, at ambient temperature with isocratic mode with mobile phase containing mixture of component A (acetonitrile) and component B (0.001M NaH2PO4, with 5 mM 1-heptane sulfonic acid sodium salt, adjusted to pH 3.0) in a ratio 60:40 (v/v). The flow rate was 1.0 ml/min and the eluent was monitored at 297 nm. The proposed method was validated as per ICH guidelines. Result: The obtained results showed that the compounds were sensitive to hydrolytic decomposition in aqueous media, resulting in the splitting of the hydrazone bond. Rapid hydrolysis of substances was observed in the acid medium. The elevated temperature significantly accelerated the hydrolytic reaction. Relatively slow hydrolysis of 3a-c was observed in a neutral solution and aqueous solutions buffered to pH 9. The hydrolysis of 3a-c in neutral, alkaline and strong alkaline medium followed the pseudo- first-order reaction rate and showed a linear dependence of lnC versus time. Conclusion: A validated high-performance liquid chromatographic assay for the determination of the hydrolytic stability of a series of aroylhydrazones was developed and optimized for the first time. The methods devised are successfully applicable to the development of pharmaceutical formulations.