Quantifying an Anthrax LF Inhibitor in Human Plasma by HPLC-MS-MS

In 2001, an anthrax outbreak demonstrated the urgent need for agents to counter potential bioterrorism attacks involving anthrax spores. In response to this need, Merck Research Laboratories began developing a potent, selective anthrax Lethal Factor inhibitor (LFI), potentially for the Strategic National Stockpile, a repository for therapeutic agents for national emergencies. To provide bioanalytical support for impending human pharmacokinetic evaluation of this therapeutic, an analytical method with a limit of quantification (LOQ) of 5 ng/mL was required. High performance liquid chromatography (HPLC), coupled with tandem mass spectrometry (MS-MS) detection is ideal for highly selective trace-level quantitative determinations of analytes like LFI in biological matrices. High polarity complicated the development of sample preparation and chromatographic conditions. The final method consisted of a liquid-liquid extraction with ethyl acetate and reversed-phase chromatography with a highly aqueous mobile phase.