Bifunctionalized isotopologues as calibrants for standard-free quantitation of drug metabolites in plasma by liquid chromatography coupled with radiometry and mass spectrometry.

Abstract

The following method describes a novel workflow that eliminates the need of authentic reference standards for the quantitation of drug metabolites in biological samples using a single multi-isotopically labeled compound bearing both radio and stable isotopes. The resulting radio and stable bifunctionalized isotopologue (RADSTIL) of parent drug is employed as a substrate for in vitro biotransformation to targeted RADSTILs of metabolites as calibrants. Inclusion of a radio label enables both radiometric and mass spectrometric detection. Addition of stable labels ensures subsequent isotopic interference-free quantitation of unlabeled metabolites in preclinical and clinical samples. This affords a more accurate quantitation workflow compared to current semi-quantitation method, which utilizes isotopic interfering radio isotopologues of metabolites alone as calibrants. The proof-of-concept is illustrated with (14 C,13 C2 )-acetaminophen where in vitro biotransformation produced (14 C,13 C2 )-sulfate and (14 C,13 C2 )-glucuronide calibrants. Absolute quantitation of the acetaminophen metabolites was then achieved by liquid chromatography coupled with radiometry and mass spectrometry (LC-RAD/MS). Quantitative data obtained by this method fell within 82-86% of the values from conventional LC-MS/MS method.