Bioanalytical Method Development and Validation of Tofacitinib in Rat Plasma Using Green Solvent and Application to Pharmacokinetic Study by LC‐MS

ABSTRACT To reduce environmental harm, green chemistry principles were followed to develop a method for analyzing the drug tofacitinib in rat plasma. The commonly used but harmful solvent acetonitrile was replaced with an eco‐friendlier option, methanol, throughout the process. This method complies with the ICH M10 guidelines and uses mass spectrometry in positive‐ion mode. A quick, simple, and sensitive test was created and validated using liquid chromatography with tandem mass spectrometry (LC‐MS/MS) and electrospray ionization. The sample preparation involved a straightforward protein precipitation step using methanol. The separation was done using a mixture of 5 mM ammonium formate with 0.1% formic acid in water and methanol (15:85, v/v), running at 0.6 mL/min on an Ace Excel C18 column, with a total run time of 3.6 min. For detection, the MS/MS transitions were m/z 313→149 for tofacitinib and m/z 309→281 for the internal standard, ibrutinib used. The method showed good linearity across a concentration range of 1–1000 ng/mL. Precision was acceptable, with intra‐day variability between 2.17% and 12.34%, and inter‐day variability between 3.76% and 11.33%. This validated method was also used to carry out a pharmacokinetic study in rats.