Analytical method development and validation of reverse-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous quantifications of quercetin and piperine in dual-drug loaded nanostructured lipid carriers.

Quercetin and piperine are often used as an add-on therapy for various diseases, however both drug exhibits poor aqueous solubility and photosensitivity issue. Therefore, the aim of the present study is to improve the pharmaceutical challenges by incorporating both the drugs in nanostructured lipid carriers (NLCs) and to develop a sensitive, selective, accurate and precise reverse-phase high performance liquid chromatography (RP-HPLC) method for the simultaneous analysis of both drugs in NLCs. Effective chromatographic separation of quercetin and piperine was achieved on Hypersil gold C-18 column and mobile phase consisting of a mixture of acetonitrile and HPLC grade water (pH 2.6, adjusted with 2%v/v glacial acetic acid) in an isocratic elution mode. The flow rate of the mobile phase was 1 mL/min, column temperature at 35 ± 0.2 °C and the injection volume was 20 μL. The retention time for quercetin and piperine were found to be at 2.80 min and 10.36 min, respectively and detected at an isobestic wavelength of 346 nm using a photodiode array (PDA) detector. The method was found to be specific for the simultaneous analysis of quercetin and piperine in presence of NLCs matrix, accurate (>90%) and precise (%RSD < 2%). The validated RP-HPLC method effectively utilised to determine the percentage drug entrapment efficiency cum percentage drug loading of quercetin and piperine in NLCs enriched formulations along with the secondary estimation of in vitro cumulative percentage drug release study. The results were found to be reliable, hence the validated RP-HPLC method could be further used for the simultaneous detection and quantification of both these drugs in other lipid-based nano-formulations such as solid-lipid nanoparticles, polymer-lipid hybrid nanoparticles, lipid drug conjugates, etc. in in vitro and in vivo.