Determination of embutramide in mammalian tissues

T-61 is a commonly used euthanasic agent containing N-[2-[m-methoxyphenyl)-2-ethyl-buty1(1)]-gamma-hydroxybutyramid e (embutramide), 4,4'methylenebis(cyclohexyl-trimethylammonium iodide), and tetracaine hydrochloride. In order to confirm the exposure of animals and man to T-61, a procedure was developed for identification and quantification of embutramide in tissues and body fluids. Tissue was homogenized in acetonitrile (CH3CN) and the drug partitioned into methylene chloride (CH2Cl2) from aqueous CH3CN at neutral pH and at pH 9. The drug was purified by gel permeation chromatography in cyclohexane/CH2Cl2 (85:15). Embutramide was identified by gas chromatography-mass spectrometry on a 0.25 mm x 15 m fused silica capillary column of 0.25 micron DB-5, programmed from 215 to 275 degrees C at 25 degrees/min with El ionization at 70 ev. Quantification was selected ion monitoring of m/z 135, 190 and 293. Embutramide was examined in brain, lung, liver, kidney, skeletal muscle, urine, bile, eye fluid and blood of bovine, canine, caprine, feline, ovine and porcine species.