A validated high‐performance liquid chromatography method for the determination of brassinin, an indoleamine 2,3‐dioxygenase inhibitor in rat plasma

Brassinin, a phytochemical in cruciferous vegetables, is an indoleamine 2,3‐dioxygenase inhibitor and possesses a therapeutic opportunity in cancer immunotherapy. Here, we have developed and validated a novel, specific, and rapid bioanalytical method in rat plasma that is unavailable in the literature for preclinical studies. The method development involved single‐step protein precipitation as an extraction method using acetonitrile with an absolute and relative mean recovery of 79.32% and 93.59%, respectively. The method is accurate and precise over the linearity range of 1.0–50.0 μg/mL with a retention time of 5.3 and 3.5 min of brassinin and telmisartan, an internal standard. The intra‐day and inter‐day precision of the method expressed as a %coefficient of variation, ranging from 0.03% to 2.82% and 0.39% to 5.82%, whereas intra‐day and inter‐day accuracy ranged from 99.75% to 103.40% and 99.76% to 104.25%, respectively. The high‐performance liquid chromatography bioanalytical method was successfully validated as per Food and Drug Administration guidelines, and it can be applied in routine bioanalysis and pharmacokinetic studies.