Developing a GC-EI-MS/MS Method for Quantifying Warfarin and Five Hydroxylated Metabolites Generated by the Fenton Reaction.

Abstract

Abstract As a drug and rodenticide, warfarin is used globally. Extensive research has shown that warfarin and other rodenticides are recoverable in the environment and food chain (Fernandez, Santos, Cancela, Laize, & Gavaia, 2014; Gomez-Canela, Barata, & Lacorte, 2014; Nakayama, Morita, Ikenaka, Mizukawa, & Ishizuka, 2019; Rattner, Lazarus, Elliott, Shore, & van den Brink, 2014; Saito-Shida, Nemoto, Matsuda, & Akiyama, 2016; Waddell, Poppenga, & Drobatz, 2013). Current methods for their analytical determination focus on the target component rather than on metabolites or transformation products. Warfarin's phase 1 metabolism is extensively studied. Mono-hydroxylated metabolites are primarily formed (Watanabe et al., 2015; Wong & Davis, 1989). In this study, the use of the Fenton reaction aimed to generate mono-hydroxylated transformation products in the laboratory. A GC-MS/MS method was designed to quantify the expected derivatized metabolites by multiple reaction monitoring. Warfarin, 4'-, 5-, 6-, 7-, and 8-hydroxy warfarin, and isotopically labeled reference compounds were used for this approach. The method has a linear working range of 30 to 1800 pg/µL. Detection limits obtained range between 18.7 to 67.0 pg/µL. In addition, a C18-SPE step was used to enrich the analytes, and the recovery for each compound was calculated. The derivatization yield of warfarin for in-liner derivatization with m-TFPTAH was determined as well. The method was used successfully to show that 4'-O-Me-WAR is preferentially formed under the given experimental conditions. Furthermore, the Fenton reaction has generated all preselected mono-hydroxylated transformation products. The maximum intensities of all target analytes were reached at around 4 minutes and immediately decreased after that. The applicability of GC-MS for determining known metabolites of warfarin was demonstrated. With SPE clean-up, the method is also suitable for detecting metabolites in more complex matrices, for example, environmental water samples in the future.