Chloroethynitrosoureas　(CENUs)　are　important　alkylating　agents　widely　used　in　the　treatment　of　cancers.　Decomposition　of　CENUs　generates　active　electrophilic　ions　that　damage　DNA,　including　the　formation　of　dG-dC　crosslinks　which　represents　the　most　important　cytotoxic　mechanism　of　CENUs.　In　this　work,　a　high-performance　liquid　chromatography/electrospray　ionization　tandem　mass　spectrometry　(HPLC/ESI-MS/MS)　method　was　employed　to　analyze　the　dG-dC　crosslinks　induced　by　1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea　(meCCNU,　Semustine).　The　direct　quantitation　of　dG-dC　crosslinks　in　oligonucleotide　duplexes　was　achieved　by　the　selected　reaction　monitoring　(SRM)　mode　using　synthesized　N-15(3)-labeled　dG-dC　as　an　internal　standard.　Methods　of　enzymatic　digestion　and　HPLC　separation　were　developed　for　obtaining　separation　and　reproducibility　of　the　dG-dC　peak　in　chromatograms.　The　limit-of-detection　(LOD)　was　determined　to　be　0.08　nM　and　the　limit-of-quantification　(LOQ)　was　determined　to　be　0.16　nM.　The　linearity　of　the　calibration　curve　was　0.9997　over　the　range　of　0.08　to　32　nM.　The　precision　and　accuracy　of　the　method　ranged　from　1.1　to　6.6%　and　96　to　109%,　respectively.　The　recovery　of　the　dG-dC　crosslink　in　the　enzymatic　hydrolysates　from　the　oligonucleotide　duplex　was　determined　to　be　from　91　to　106%.　The　results　of　the　validation　study　indicate　that　the　method　is　suitable　for　quantifying　dG-dC　crosslinks　in　DNA.　Consequently,　this　method　was　used　to　determine　meCCNU-induced　dG-dC　crosslinks　in　four　duplexes　with　different　GC　contents.　The　results　showed　that　the　crosslinking　fraction　(CF)　increased　as　the　GC　content　in　the　duplex　increased,　and　a　relatively　low　CF　was　observed　in　the　early　period　of　the　reaction.　Copyright　(C)　2011　John　Wiley　&　Sons,　Ltd.