Development and Validation of RP-HPLC Method for Simultaneous Quantification of Tacrolimus (TAC) and Thymoquinone (THQ)

Abstract

The development and validation of analytical methods are essential for ensuring the quality of pharmaceutical formulations. This study focuses on the development of a reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous quantification of tacrolimus (TAC) and thymoquinone (THQ) in combined formulations. A robust method was optimized using a C18 column with a mobile phase enabling efficient separation and quantification. The method was validated following ICH guidelines, assessing parameters such as linearity, accuracy, precision, robustness, and specificity. The results demonstrated excellent linearity for both TAC and THQ over a wide concentration range, with correlation coefficients exceeding 0.999. Recovery studies confirmed the method’s accuracy, showing a recovery rate within 98–102%. Precision studies indicated low intra- and inter-day variability, while robustness tests highlighted the method’s reliability under minor experimental variations. The validated RP-HPLC method was successfully applied to quantify TAC and THQ in pharmaceutical formulations, offering a simple, rapid, and reliable tool for quality control and routine analysis. This work provides a significant contribution to pharmaceutical research, facilitating the development of combination therapies involving TAC and THQ for inflammatory and autoimmune disorders.