Four stereoisomers of anti benzo［a］pyrene diol epoxide deoxyguanosine adduct(anti-BPDE-N2-dG) were synthesized in vitro.By optimizing the reaction conditions，the yields of four isomers were two times higher than those of the reported synthesis methods.The synthesis provided standard substance for the quantitative detection of anti BPDE N2 dG in vivo.The reaction mixture of anti-BPDE-N2-dG stereoisomers were further separated and purified by reversed phase high performance liquid chromatography(HPLC)，in which a pentafluorophenyl silica gel column(PFP column，250 mm×4.6 mm，5 μm) was introduced for the first time.It was found that the four isomers could be separated completely on the PFP column within 45 min，using acetonitrile-0.1% formic acid water(225∶775) as mobile phase at a flow rate of 1.2 mL/min.Compared with the conventional C18 column(250 mm×4.6 mm，5 μm) taking 160 min or silicone polymer coating phenyl column(250 mm×4.6 mm，5 μm) needing 85-100 min for separation of the four isomers，the reported method greatly shortened the separation and purification time.The four stereoisomers of the anti BPDE N2 dG were identified and characterized by a combination of HPLC-MS/MS，UV and circular dichroism(CD) spectra.The order of the peaks in chromatograms were determined as trans(-)，trans(+)，cis(+) and cis(-)-anti-BPDE-N2-dG.In addition，in the analysis of HPLC-MS/MS in MRM mode，the four stereoisomers were rapidly and well separated on the PFPP column within 30 min using a mobile phase consisted of acetonitrile-0.1% formic acid(28∶72) at a flow rate of 0.5 mL/min.This work demonstrates that the introduction of the PFPP column is very helpful for the separation，purification and detection of anti-BPDE-N2-dG isomers.