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.