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Quantification of meCCNU ‐induced dG‐dC crosslinks in oligonucleotide duplexes by liquid chromatography/electrospray ionization tandem mass spectrometry
Author(s) -
Bai Baoqing,
Zhao Lijiao,
Zhong Rugang
Publication year - 2011
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.5064
Subject(s) - chemistry , electrospray ionization , chromatography , detection limit , tandem mass spectrometry , selected reaction monitoring , mass spectrometry , electrospray , oligonucleotide , analytical chemistry (journal) , dna , biochemistry
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 15 N 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 © 2011 John Wiley & Sons, Ltd.