A new, simple, and sensitive high-performance liquid chromatography method for the detection of ozanimod in mouse plasma

Abstract Ozanimod is a selective sphingosine-1-phosphate (S1P) receptor modulator used in the treatment of multiple sclerosis and ulcerative colitis; however, to the best of our knowledge, no published studies have yet described a validated method for the determination of ozanimod in plasma. Consequently, a sensitive, robust, and straightforward high-performance liquid chromatography method coupled with fluorescence detection was developed and validated for quantifying ozanimod in mouse plasma, employing nabumetone as the internal standard. Acetonitrile with 0.1% HCl was used to denature plasma proteins. After centrifugation, the supernatant was evaporated under nitrogen flow at 40 °C and finally dissolved in the mobile phase consisting of 16 mmol L −1 sodium acetate solution (pH 4.7) and acetonitrile (1.7/1; v/v). An isocratic mode with a 0.7 mL min −1 flow rate was used with a Chromolith HighResolution RP-18e monolithic column. Ozanimod and nabumetone were detected by fluorescent detection (with 278 nm excitation and 383 nm emission) at 8 and 18 min, respectively. An excellent linearity ( R 2 = 0.9996) was shown in the range of 5–500 ng mL −1 . The mean recoveries from spiked samples were higher than 75.4%. Relative standard deviation (RSD) in the interday assay was lower than 6.3%. The most important advantage of the presented method is its use of small amounts of mouse plasma, made possible by fluorescence detection.