Development and Validation of Reversed Phase HPLC Method for the Simultaneous Detection of Lactone and Carboxylate Forms of Topotecan Along with Thymoquinone: Application to Nanoparticulate Anticancer Formulation System

Nanomedicine combination therapy, a strategy to delivery multiple drugs via a single nanosystem has opened new avenues to treat cancer and prevent side effects and overcome chemoresistance. The present work utilizes a chemotherapeutic agent topotecan hydrochlorideand a natural chemosensitizer (thymoquinone, TQ) incorporated into poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) to target tumor. The objective of the present work is to develop a reversed phase HPLC method for simultaneous estimation of TQ along with the pH dependent lactone and carboxylate forms of topotecan and validate its applicability. Chromatographic separation was obtained on Agilent 1200 Infinity series C18 analytical column using gradient elution with acetonitrile and 1% triethylamine acetate (TEAA) buffer (pH 5.5) as mobile phase and a flow rate of 0.8 mL/min. Analytical detection was done at 253 nm. Validation was performed as per ICH guidelines and characterization and stability of the optimized nanoparticles were evaluated. The retention time of 5.93, 8.73, and 19.76 min was obtained for lactone, carboxylate and TQ, respectively. Linearity response was established in the range of 5‒45 µg/mL. The limits of detection and quantification were found to be 15 and 50 ng/mL for lactone, 18 and 59 ng/mL for carboxylate, and 5 and 18 ng/mL for TQ, respectively. The method was validated in terms of robustness, precision, accuracy, specificity and percent loading, entrapment efficiency, and in vitro drug release. The stability of the nanoparticles was determined using the above validated method. The method was successfully validated and applied for routine analyses indicating its use as a suitable analytical tool to determine efficiency of the proposed therapy.