Abstract
Thymoquinone (THY) is a bioactive phytochemical constituent found in Nigella saliva (NS) seed. The purpose of this research is to determine THY with high precision and accuracy using HPLC by Quality by Design (QbD) approach. To improve chromatographic conditions, response surface approach with central composite design (CCD) was employed, with flow rate and lambda(mas) as independent factors, and retention time and tailing factor as dependent variables. The mobile phase composition was 2-propanol: acetonitrile: buffer (2 mM ammonium formate) in the ratio of 45:40:15 v/v/v. The separation was achieved on Symmetry (R) C18 (5 mu m, 3.9x150 mm) column, as stationary phase. The flow rate of mobile phase was 1 mL/min in isocratic mode, and the eluted analyte was detected at 254 nm by UV-Visible detector. The developed HPLC method was validated according to ICH guidelines. A calibration curve of eight dilutions over the range 6.25-100 mu g/mL with r(2) value of 0.9957 was obtained. The limit of detection (LOD) and limit of quantification (1.0Q) obtained were 2.08 and 6.25 pg/mL, respectively. The percentage relative standard deviation (%RSD) of system suitability (retention time 0.883, and tailing factor 0.512), and other validation parameters like precision and accuracy, solution stability, and robustness were determined. The developed HPLC method is simple, accurate, quick, and robust, and it can be used for the routine analysis of THY in different kinds of formulations.