Development and Validation of New LC-MS/MS Bioanalytical Method of Tremelimumab in Rat Plasma by Using Nivolumab as Internal Standard and Its Application with Pharmacokinetic Studies

Abstract

Nivolumab was decided to be used as the IS. To separate the drugs, an isocratic mobile phase of acetonitrile (ACN): Ammonium formate containing formic acid (70:30) was adopted and delivered at 1-mL/min, along with a 150 x 4.6 mm x 3.5 μm Alliance, e2695 Luna Phenyl Hexyl column. Drug and IS, both of which display proton adducts approximately m/z 146.3685 to 120.0638 and m/z 143.7695 to 76.7964, might be detected simultaneously using MRM-positive modalities. Over a linearity level of 2.00 to 40.00 ng/mL, the method had a correlation value (r2) of 0.99977. The accuracy and precision of this method during the day ranged from 89.85 to 102.89% and 0.19 to 2.81%, respectively. Tremelimumab was reported to remain stable during three freeze-thaw cycles, benchtop tests, and postoperative stability studies. Cmax and Tmax values were acquired directly from experimental data. Cmax and Tmax averaged 18.024 ng/mL and 6 hours, respectively. At t1/2 of 18 hours, plasma levels began to fall. The obtained AUC024 and AUC0 values were 257 and 257 ng h/mL, respectively.