Rapid and sensitive ultra-high-pressure liquid chromatography–tandem mass spectrometry analysis of the novel anticancer agent PR-104 and its major metabolites in human plasma: Application to a pharmacokinetic study

PR-104 is a dinitrobenzamide mustard currently in clinical trial as a hypoxia-activated prodrug. It is converted systemically to the corresponding alcohol, PR-104A, which is activated by nitroreduction to the hydroxylamine (PR-104H) and amine (PR-104M). PR-104A is also metabolised to the O-glucuronide (PR-104G), and by oxidative debromoethylation to the semi-mustard PR-104S. We now report a validated ultra-high-pressure liquid chromatography and tandem mass spectrometry (UHPLC–MS/MS) method for the determination of these metabolites in human plasma. Plasma proteins were precipitated with acidified methanol and the supernatant diluted into water. Aliquots were analysed by UHPLC–MS/MS using a Zorbax Eclipse XDB-C18 Rapid Resolution HT (50 mm × 2.1 mm, 1.8 μm) column and gradient of acetonitrile and 0.01% formic acid with a 6 min run time. The method had a linear range of 0.1–50 μM for PR-104, PR-104A and PR-104G, 0.05–5 μM for PR-104H, 0.025–2.5 μM for PR-104M and 0.01–1 μM for PR-104S. The intra-day and inter-day precision and accuracy were within 14%. The extraction recovery of all analytes was over 87%. The validated method was illustrated by using it to study the pharmacokinetics of PR-104 and its metabolites in a human patient.