Bacillus anthracis, the etiological agent of anthrax, is a Gram-positive, rod-shaped bacterium that forms spores that are highly resistant to heat, ultraviolet light, radiation, pressure, and chemical agents. The durability of these spores make this bacterium a potential bioweapon (1).

Figure 1

Materials: LFI and the isotopically labeled ISTD (Figure 1) were provided by the Medicinal Chemistry Department (Merck Research Laboratories, West Point, Pennsylvania) and the Labeled Compound Synthesis group in the Drug Metabolism and Pharmacokinetics Department (Merck Research Laboratories, Rahway, New Jersey), respectively. All solvents and reagents were of HPLC or analytical reagent grade and were purchased from Fisher Scientific (Fair Lawn, New Jersey). The drug-free heparinized human plasma was obtained from Biological Specialties (Colmar, Pennsylvania).

Instrumentation: The HPLC–MS-MS system consisted of an Applied Biosystems/MDS Sciex (Foster City, California) API 4000 tandem mass spectrometer equipped with an electrospray ionization interface, a Perkin-Elmer (Norwalk, Connecticut) Series 200 quaternary pump, and a Varian (Palo Alto, California) Pro Star Model 430 autosampler. Data were processed on a Dell Pentium 4 computer using Analyst 1.4 software (Sciex). A Tomtec Quadra 96 (Hamden, Connecticut) liquid-handling robot was used for sample preparation.

HPLC–MS-MS conditions: HPLC separation was performed on a 50 mm ×2.1 mm, 3.5-μm d_p Symmetry C8 column (Waters, Milford, Massachusetts) at ambient temperature. The mobile phase was a mixture of 0.1% acetic acid in water–acetonitrile (80:20, v/v) and was delivered at a flow rate of 0.300 mL/min. The retention time was 2.25 min.