Establishment of an LC-MS/MS method for quantification of lifitegrast in rabbit plasma and ocular tissues and its application to pharmacokinetic study

Lifitegrast, a lymphocyte function-associated antigen 1 antagonist, was approved by the FDA for the treatment of dry eye disease. Cornea and conjunctiva have been reported to be the sites of action of lifitegrast. To investigate the pharmacokinetics of lifitegrast, a sensitive analytical method for the determination of lifitegrast in various biological matrices such as plasma and ocular tissues is required. However, only limited information about the analytical method for lifitegrast in biological samples is available. In the present study, we aimed to develop a new liquid chromatography-tandem mass spectrometry method for the determination of lifitegrast in rabbit plasma, cornea, conjunctiva, and sclera. Lifitegrast-d₆ was used as an internal standard (IS). To prepare the biological samples, protein precipitation using acetonitrile was utilized. Analytes were separated from endogenous interferences on an Atlantis dC18 (5 µm, 2.1 × 150 mm), and a mixture of 0.1 % formic acid and acetonitrile was used as the mobile phase. The mass transition of precursor to product ion was monitored at 615.2 → 145.0 for lifitegrast and 621.2 → 145.1 for IS. The calibration curves were linear over the concentration range from 2 to 500 ng/mL for plasma and 5 to 500 ng/mL in ocular tissue homogenates. Intra- and inter-day accuracy ranged from 95.76 to 106.80 % in the plasma and 94.42 to 112.80 % in the ocular tissues. Precision was within 8.56 % in the plasma and 9.72 % in the ocular tissues. The short-term, long-term, auto-sampler, and freeze–thaw stabilities of lifitegrast were validated. The developed method was applied to a pharmacokinetic study of lifitegrast in rabbits. Following ophthalmic administration, only 3.26 % of administered lifitegrast was absorbed into the systemic circulation. Peak tissue concentrations were observed at 0.5 h after dosing, and topically administered lifitegrast was mainly distributed in the cornea and conjunctiva. The finding of this study is expected to be used in further pharmacokinetic studies and formulation development.