Development and Validation of a Novel Gradient LC Method for Simultaneous Determination of Isoniazid and Acetylisoniazid in Human Plasma

The goal of this study was to develop and validate a new gradient high-performance liquid chromatography method for the simultaneous determination of isoniazid (INH) and acetylisoniazid (Ac-INH) in human plasma samples. A C18 reversed-phase column was employed for separation followed by UV detection at 266 nm. The calibration involved the use of five concentration levels ranging from 1 to 20 μg mL^?1 for both analytes. The developed method was validated using ICH guidelines. The calibration curve was found to be linear with correlation coefficient values (r ²) above 0.9991 and the highest RSD% values for intra-day assays were found to be 6.34 and 2.57% for INH and Ac-INH, respectively. The highest RSD% values for inter-day assays were 9.31 and 10.17% for INH and Ac-INH, respectively. LOD was calculated to be 0.1 and 0.15 μg mL^?1 for INH and Ac-INH, respectively. LOQ was calculated to be 0.33 and 0.5 μg mL^?1 for INH and Ac-INH, respectively.