Bioanalytical method development and validation of HPLC-VU method for quantification of Embelin from human plasma

Embelin is the main bioactive chemical in E. ribes berries and possesses various biological properties. Currently, no liquid chromatographic method is available for quantitative estimation of embelin from human plasma. The purpose of this study was to develop an accurate, precise, and simple reverse-phase high-performance liquid chromatographic method for measuring the amount of embelin in human plasma. The separation of embelin was achieved using a Waters C18 (150 x 4.6 mm i.d., 5µ particle size) column. A mixture of acetonitrile and phosphate buffer whose pH was adjusted to 3.6 in a 20:80 v/v ratio and at a flow rate of 1.4 ml/min was employed as a mobile phase. The detection was performed at 289 nm. The plasma extraction method was validated for various parameters, including precision, accuracy, and stability.The developed method using human plasma was linear over a range of 13.9–41.65 ng/µl concentrations with a regression coefficient of 0.984. The accuracy testing revealed the value of the mean percent recovery between 101.54 and 109.15. The mean intra- and inter-day precision of the assay ranged from 105.04 to 91.16% and 0.3628 to 1.4227% RSD, respectively. The extracted samples also showed bench-top and freeze-thaw stability over 72 hours. In human plasma, embelin was found to be stable. The method's validation parameters satisfied the required criteria for acceptance. From the results, we concluded that the developed method can be used for accurate and precise quantification of embelin from human plasma.