Development and validation of a UHPLC-MS/MS method for the quantitative analysis of trans-ISRIB in human plasma

The integrated stress response (ISR) is a crucial cellular defense mechanism activated under various stress conditions, leading to a reduction in global protein synthesis to maintain cellular homeostasis. Trans-integrated stress response inhibitor (trans-ISRIB) has emerged as a promising therapeutic candidate for neurodegenerative diseases by counteracting ISR-induced translational repression. However, its clinical and preclinical evaluation requires a sensitive and reliable analytical method for its quantification in biological matrices.

This study presents the development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the quantitative analysis of trans-ISRIB in human plasma, adhering to the U.S. FDA’s bioanalytical method validation guidelines. The analytical workflow incorporates a liquid-liquid extraction technique for sample preparation, where plasma samples are spiked with an internal standard (IS) to enhance analytical accuracy. Extracted samples are then dried under nitrogen, reconstituted in a mobile phase, and subjected to chromatographic separation on a Waters XSelect HSS T3 column under isocratic conditions. The mobile phase consists of 0.1% acetic acid in a 70% methanol aqueous solution, with a flow rate of 0.500 mL/min.

Detection and quantification are performed using a tandem mass spectrometer equipped with a positive electrospray ionization (ESI+) source, operating in multiple-reaction-monitoring (MRM) mode. The method demonstrates a linear calibration range spanning 0.500 to 1.00 × 10³ nM for trans-ISRIB, exhibiting high specificity, precision, accuracy, and recovery. This validated UHPLC-MS/MS method fills a critical analytical gap, providing a robust and reliable approach for trans-ISRIB quantification in human plasma, which is essential for its further pharmacokinetic and clinical studies.