A new mutagenic compound has been isolated from ground beef which was fried at 300 degrees C for 5.5 min on each side. The new mutagen was purified using an aqueous acid extraction, XAD-2 adsorption-solvent elution, a series of preparative and analytical h.p.l.c. purification steps, and monitored with the Ames/Salmonella assay. This study reveals a new mutagen member of the amino-imidazoazaarene class of aromatic amines, having a mol. w of 224, and a formula of C13H12N4 as determined by high-resolution mass spectrometry. N.m.r. spectrometry supports the structure, 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP), for the new mutagen. The 1-methyl and 3-methyl synthesized isomers of PhIP were compared to the purified mutagen. The two isomers had identical mass spectra to the purified compound, but only the 1-methyl isomer showed similar u.v. and n.m.r. spectra. The two synthetic isomers were separable by h.p.l.c. and the beef derived component co-eluted with the 1-methyl-PhIP isomer. PhIP has a specific activity in the Ames/Salmonella assay of 1950 revertants/microgram. Although it is not as mutagenic as other compounds isolated from fried beef (e.g. MeIQx, 58 000 revertants/microgram) it is the most abundant mutagenic compound by mass in fried beef. PhIP is present at approximately 15 p.p.b. of the original weight of uncooked beef (accounting for 75% of the mass of genotoxic material) and contributes 18% of the total mutagenicity of the fried beef.

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The purification of cooking mutagens depends on the extraordinary sensitivity of the Ames/Salmonella mutagenicity test and its usefulness for tracking the mutagens during the purification steps. Following aqueous/acid (pH 2) extraction of fried ground beef (cooked at 200, 250, or 3000C), XAD-2 column adsorption and elution with acetone, and acidic and basic liquid/liquid extractions, the samples are separated into six distinct peaks with preparative reverse-phase HPLC. A total of nine distinct mutagens can be separated after two additional HPLC steps. These compounds fall into a class of compounds called aminoimidazoa- the PhIP, which contributes the highest mass content to the cooked meat, but has the lowest mutagenic potency, might ultimately make a significant contribution to the carcinogenicity.

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