A liquid chromatography with tandem mass spectrometry method for simultaneous determination of UTL-5g and its metabolites in human plasma

• UTL-5g is a novel small-molecule chemoprotector/radioprotector.
• The in vitro enzymatic products of UTL-5g under esterase were recently identified.
• The in vitro metabolites of UTL-5g in human plasma are identified for the 1st time.
• This is the 1st method validation utilizing LC-MS/MS for UTL-5g in human plasma.
• The method can be used to characterize PK profiles of UTL-5g and its metabolites.

UTL-5g is a novel small-molecule TNF-α inhibitor under investigation as both a chemoprotective and radioprotective agent. Animal studies showed that pretreatment of UTL-5g protected kidney, liver, and platelets from cisplatin-induced toxicity. In addition, UTL-5g reduced liver and lung injuries induced by radiation in vivo. Although a number of preclinical studies have been conducted, a validated bioanalytical method for UTL-5g in human plasma has not been published. In this work, a sensitive and reproducible reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) assay was developed and validated for the determination of UTL-5g and its metabolites, 5-methylisoxazole-3-carboxylic acid (ISOX) and 2,4-dichloroaniline (DCA), in human plasma. The method involves a simple methanol precipitation step followed by injection of the supernatant onto a Waters 2695 HPLC system coupled with a Waters Quattro Micro™ triple quadrupole mass spectrometer. Chromatographic separation was accomplished using a Waters Nova-Pak C18 column maintained at 30 °C, running at gradient mode with mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in methanol at a flow rate of 0.2 mL/min. The analytes were monitored under positive electrospray ionization (ESI). Quantitation of these compounds in plasma was linear from 0.05 to 10 μM. The lower limit of quantitation (LLOQ) was 0.05, 0.1, and 0.2 μM for UTL-5g, ISOX and DCA, respectively. The accuracy and intra-and inter-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method provides a practical tool to measure and characterize the plasma concentration-time profiles for UTL-5g and its metabolites, ISOX and DCA.