Background: A simple, precise, selective and Reproducible Reversed Phase Ultra-High Performance Liquid Chromatography (RP-UHPLC) method has been developed and validated for the simultaneous determination of two plant constituents, Curcumin (Cur) and Thymoquinone (THQ) in lipid based self-nanoemulsifying formulation and commercial products (Turmeric 500mg capsules and black seed oil 500ml).
Method: The chromatographic separation was carried out on a reversed-phase Acquity® BEH C18 column using methanol: 0.25% formic acid solution in water (60:40, v/v) as the mobile phase. The isocratic flow rate was 0.3 ml/min with a rapid run time of 4 min. The UV detections were at 254 nm for THQ and 428 nm for Cur. The method was validated over a concentration range of 100-50,000 ng mL-1 (r2= >0.9999 for both Cur and THQ).
Result: The selectivity, specificity, recovery, accuracy and precision were validated for determination of Cur and THQ in lipid based formulation. The lower limits of detection and quantitation of the method were 25 and 75 ng mL-1 for Cur and 35 and 100 ng mL-1 for THQ, respectively. The within and between-day coefficients of variation were less than 5%. The validated method has been successfully applied to quantify both Cur and THQ in self-nanoemulsifying formulation and commercial products. In addition, the proposed method was also applied in ex-vivo (permeability) study using an animal model (rat) to illustrate further the scope and application of the method.
Keywords: Curcumin, thymoquinone, UHPLC-UV-Vis, method validation, self-nanoemulsifying lipid based formulation, simultaneous determination.