Development of a sensitive quantification method for STAT3 inhibitory molecule, Stattic, using high performance liquid chromatography

Abstract

Introduction: In the past decade, the general acceptance that signal transducer and activator of transcription 3 (STAT3) is a promising target for cancer therapy has led to the development of many agents that inhibit the activation and/or the gene transcription regulatory function of STAT3. One example for STAT3 inhibitory agents called Stattic, is a non-ionazable small molecule with the potency of blocking phosphorylation and dimerization of STAT3. The limitation for clinical use of this agent is due to its poor water solubility and low bioavailability. Therefore, developing of a valid analytical method is necessary for evaluation of new formulations in aqueous and plasma samples for in vitro and in vivo studies, respectively. Aim: In this study for the first time, a simple and accurate method has been developed for determination of Stattic in aqueous and plasma samples. Methods: Reverse phase high performance liquid chromatography (RP-HPLC) and C18 column as stationary phase and the mixture of acetonitrile (60%) and water (40%) as mobile phase were applied. The method was validated by Food and Drug administration (FDA) guideline. Results: The developed method provided a linear range between 1 to 40µg/ml for aqueous samples and 2.5 to 40µg/ml for plasma samples with a correlation coefficient of >0.999 for both analyses. The accuracy and precision (inter-day and intra-day) of the developed method was in the range of 95-105% and 85-115% for aqueous and plasma samples, respectively. Sensitivity based on FDA guideline was 1µg/ml and 2.5μg/ml and according to other guidelines, e.g. International Conference on Harmonization (ICH), LOQ=4.1µg/ml and LOD=1.3µg/ml for aqueous samples and LOQ=5μg/ml and LOD=1.5µg/ml for plasma samples were calculated. Conclusion: These results show that both established methods are separately a fast and accurate method for quantification of Stattic in aqueous and biologic samples.