A new sensitive derivatization assay of PrC-210 in plasma and tissues using liquid chromatography coupled with tandem mass spectrometry

BACKGROUND: PrC-210 is a direct-acting reactive oxygen species (ROS) scavenger. It is active when administered orally, IV, or subcutaneously. Its profound efficacy in preclinical models makes it a candidate to prevent human ROS-induced i) ionizing radiation toxicities, ii) neurodegenerative disease, and iii) organ ischemia-reperfusion injuries. RESEARCH DESIGN AND METHODS: A simple, highly sensitive, specific LC-MS/MS method was developed to measure the PrC-210 molecule in mouse plasma and tissue homogenates. To prevent oxidative degradation or disulfide formation of the PrC-210 thiol form, its free sulfhydryl group was protected by derivatization with N-ethylmaleimide (NEM), which produced the stable, quantifiable, PrC-210-NEM thioether conjugate. The PrC-210-NEM conjugate, and acyclovir used as an internal standard, were extracted from plasma or tissue homogenate with acetonitrile/0.1% formic acid. RESULTS: The reconstituted dried extracts were chromatographed and then monitored using a triple quadrupole spectrometer operating in the positive ion spray ionization mode. The method was validated over the concentration range of 7.5 nM-5000 nM. Inter- and intra-assay precision and accuracy of PrC-210-NEM quantitation were better than 10%. The limit of PrC-210-NEM quantitation was 2.5 nM. CONCLUSIONS: The method was applied to measure plasma and tissue homogenate concentrations of PrC-210 in over 126 mice administered either oral, itraperitoneal, or subcutaneous PrC-210 doses.