The separation of the isomeric phenols of benzo( a)pyrene by high-performance liquid chromatography

A method has been developed to separate the 12 isomeric phenols of benzo( a)pyrene by using high-performance liquid chromatography with fluorescence detection and various sequences of methanol/water gradients. The elution of the 12 isomers by different concentrations of methanol/water was studied showing that many isomers have almost identical retention times. The effects of flow rate, temperature, and various types of gradients on the separation of the isomeric phenols, as well as the difficulty to separate some of the isomers, are discussed. It has been found that under the conditions normally employed to separate these phenols from other metabolites of benzo( a)pyrene, several of the phenols are not separated possibly leading to incorrect identification as well as quantitation. Detection of the phenols, as well as the parent benzo( a)pyrene, by fluorescence has increased the sensitivity of the method about 200 times when using the most sensitive setting as compared to uv detection. All the available pure diols and diones of benzo( a)pyrene are also separated by this procedure.