P0105LC-MS/MS assay for etrasimod plasma concentration using phreephospholipid removal technology: Supporting pharmacokinetic variability and individualized dosing in hospital practice

Background Etrasimod, a oral, once-daily, selective sphingosine 1-phosphate receptor subtype 1, 4, and 5 (S1P1,4,5) modulator, represents a significant therapeutic advancement in the treatment of moderately to severely active ulcerative colitis [1,2]. The primary objective of this project was to develop, fully validate, and prepare for clinical implementation an ultra-sensitive LC-MS/MS method for the precise and selective determination of etrasimod concentrations in patient plasma, suitable for routine monitoring in a real-world hospital environment. Methods The bioanalytical method was developed and validated using human plasma. Quantification was achieved via the advanced LC-MS/MS technique utilizing an Agilent 1260 Infinity system coupled with the highly sensitive Ultivo Triple Quadrupol MS/MS detector using a Zorbax SB-C18 (3 × 100 mm, 2.7 μm) reversed phased column (Agilent Technologies). The column was maintained at 30 °C. Ultrapure-water and acetonitrile acidified with formic acid (0.1%) was used as mobile phase for gradient elution at a flow rate on 0.8 mL/min. The LC-MS/MS analysis, operated in positive electrospray ionization mode, was carried out in multiple-reaction monitoring mode, monitoring the characteristic mass transitions: m/z 485 → 159 for etrasimod and m/z 405 → 146 for the internal standard, ozanimod. Samples were efficiently processed using a streamlined PhreePhospholipid removal procedure prior to analysis (Phenomenex). The injection volume was 10 μL. The method validation was performed according to FDA guidelines for bioanalytical methods. Results Assay selectivity was assessed by employing six different human plasma and verifying for the presence of interference at the retention time and mass transitions of etrasimod and its internal standard. For linearity, a regression analysis was performed to determine the simplest regression algorithm that provided the best fit for calibrant response versus concentration data over ranges of 50 – 500 ng/mL using 100 µL of plasma. Accuracy across four quality control levels ranged from 90.1 to 99.5% while precision ranged from 1.9 to 11.7%. There was no cross-analyte interference, injection carryover, or matrix effect observed with this method. Conclusion The developed LC-MS/MS method and the PhreePhospholipid sample cleanup, demonstrated exceptional performance and robustness, fully meeting all regulatory requirements for bioanalytical validation. This method provides a reliable foundation for the pharmacokinetic variability and safety monitoring of etrasimod as well as patients adherence within our hospital. References: Lichtenstein GR, Allegretti JR, Loftus EV, et al. Assessment and Impact of Age on the Safety and Efficacy of Etrasimod in Patients With Ulcerative Colitis: A Post Hoc Analysis of Data From the ELEVATE UC Clinical Program. Inflamm Bowel Dis. 2025;31(9):2352-2362. doi:10.1093/ibd/izae308. Kovačić J, Amidžić Klarić D, Turk N, Krznarić Ž, Mornar A. Development and validation of stability-indicating method of etrasimod by HPLC/DAD/MS/MS technique with greenness profiling. Heliyon. 2024;10(13):e34066. doi:10.1016/j.heliyon.2024.e34066. Conflict of interest: Dr. Turk, Niksa: No conflict of interest Kovačić, Jelena: No conflict of interest Amidžić Klarić, Daniela: No conflict of interest Krznarić, Željko: Speakers honoraria (Abbott, Abbvie, Biocon, Celltrion/ Octal Pharma, Fresenius, Eli Lilly, Pharmas, Pfizer, Sobi, Takeda, Nestle, Nutricia, Baxter, Janssen, MSD). Advisory boards. Mornar, Ana: No conflict of interest