Simultaneous determination of hydrazinocurcumin and phenol red in samples from rat intestinal permeability studies: HPLC method development and validation

Hydrazinocurcumin (HZC) is a patented multiactivity compound and is a potent derivative of curcumin which is not yet explored for further development as formulation and requires the determination of biopharmaceutical suitability of the molecule. Intestinal permeability and log P of a compound are two vital biopharmaceutical properties by which, any “hit” molecule proceeds towards NCE (new chemical entity) and govern formulation design of bioactive molecules. In this report, a simple, precise and accurate isocratic reverse phase (RP) liquid chromatography method for simultaneous analysis of HZC and phenol red, for the application in rat in situ single-pass intestinal perfusion (SPIP) was developed and validated using FDA bioanalytical guidelines. RP-HPLC method was developed on C-18 column with UV detection at 332 nm for both the compounds. Isocratic run with the mobile phase consisting of organic phase (methanol:acetonitrile:: 50:20 v/v) and water in the ratio of 80:20 v/v provided a short run time of 7 min with resolution of more then two without interference of blank matrix. The working/expected concentration range for HZC and phenol red were 0.5–50 and 2–200 μg/ml. LOQs for HZC and phenol red of the method was found to be 0.167 and 0.271 μg/ml respectively. The validation parameters indicate that method was suitable for the intended purpose. Permeability, considering water flux with the help of non-permeable marker phenol red was calculated to be 0.34 × 10−4 cm/s. Along with other descriptors, log P (1.78) and MW (<500) of HZC makes it a potential candidate for oral formulation.