A novel reversed phase high performance liquid chromatography method to accurately determine low concentrations of curcumin in rat plasma

Abstract

Due to its lipophilic nature, curcumin levels in plasma are very low after oral administration, and therefore hard to detect. A number of chromatographic methods, including LC/MS have been developed. Although the LC/MS method is sensitive, the matrix effect can be difficult to handle. Furthermore, LC/MS equipment is relatively expensive compared to the conventional RP-HPLC. Therefore, the aim of this study was to develop a sensitive and reliable method for the determination of curcumin concentration in plasma using an RP-HPLC. Curcuma longa extract was used which contains three curcuminoids. The method started by selection of mobile phase to optimally separate the curcuminoids. The best mobile phase composition was used to analyze the plasma samples. Rat plasma was spiked with curcumin and processed for protein precipitation followed by liquid-liquid extraction of curcuminoids and an internal standard, emodin. Chromatogaphic separation of curcuminoids and emodin was achieved using a Knauer C-18 column (250 x 4.6 mm; particle size: 5µm) and a gradient program of mobile phase of three solvents, methanol -acetonitrile-1% acetic acid. A gradient elution was applied with increasing the ratio of the volume percentages of acetonitrile to acetic acid from 50/45 to 53/42 during 5 minute and thereafter elution was isocratic for 15 minute. The methanol concentration was kept constant at 5 vol-% during the whole run. The method was validated according to the FDA guidelines.The method was selective, with an excellent resolution (Rs value > 2.5). The peak shape of both curcumin and emodin were symmetric with a tailing factor of 0.9-1.1. The method linearity (correlation coefficient of 0.999) was demonstrated at 6 to 200 ng/mL. The intra and inter-day precision was 5.90-8.50% and 5.37-11.26%, respectively; the intra- and inter-day accuracy was 92.47-103.61% and 96.17-105.70%, respectively. In conclusion, the RP-HPLC method meets the validation requirements as described in the FDA guidelines and is applicable to accurately quantify curcumin concentrations as low as 6 ng/mL in rat plasma samples