Analytical and Bioanalytical Chemistry

, Volume 402, Issue 3, pp 1199–1208 | Cite as

Simultaneous quantification of methylated purines in DNA by isotope dilution LC-MS/MS coupled with automated solid-phase extraction

  • Chiung-Wen Hu
  • Chih-Ming Chen
  • Hsin Hui Ho
  • Mu-Rong Chao
Original Paper
First Online:
Received:
Revised:
Accepted:

Abstract

Since methylation at the N-7 and O6 positions of guanine and the N-3 position of adenine in DNA are the predominant reaction sites, N 7-methylguanine (N 7-MeG), O 6-methylguanine (O 6-MeG), and N 3-methyladenine (N 3-MeA) have been suggested as good biomarkers for assessing exposure to methylating agents. Here, we report the development of a sensitive and selective assay based on liquid chromatography–tandem mass spectrometry (LC-MS/MS) to simultaneously measure N 7-MeG, O 6-MeG, and N 3-MeA in DNA hydrolysates. With the use of isotope internal standards (15N5-N 7-MeG, d 3-O 6-MeG, and d 3-N 3-MeA) and online solid-phase extraction, DNA hydrolysates can be directly analyzed within 12 min without prior sample purification. The limits of detection were 0.02, 0.002, and 0.01 ng/mL on-column (6.1, 0.6, and 3.4 fmol) for N 7-MeG, O 6-MeG, and N 3-MeA, respectively. Inter- and intraday imprecision (CV) were 3.6–9.6% and 2.7–13.6%, respectively. Mean recoveries were 96–109%. This method was then applied to quantitate the amounts of methylated purines in calf thymus DNA treated with methyl methanesulfonate (MMS). The levels of N 7-MeG, O 6-MeG, and N 3-MeA in calf thymus DNA increase with MMS concentration and incubation time. The ratio of relative yields of N 7-MeG, O 6-MeG, and N 3-MeA in MMS-treated DNA was found to be 1.00:0.0032:0.119, respectively. This LC-MS/MS assay provides the sensitivity and high throughput required to evaluate the extent of methylated lesions in DNA induced by methylating agents.

Keywords

Online solid-phase extraction LC-MS/MS Methylated DNA Methyl methanesulfonate 
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Notes

Acknowledgments

This study was supported by a grant from the National Science Council, Republic of China (grants NSC 97-2314-B-040-015-MY3 and NSC 100-2628-B-040-001-MY4).

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Chiung-Wen Hu
    • 1
    • 2
  • Chih-Ming Chen
    • 3
  • Hsin Hui Ho
    • 3
  • Mu-Rong Chao
    • 3
  1. 1.Department of Public HealthChung Shan Medical UniversityTaichungTaiwan
  2. 2.Department of Family and Community MedicineChung Shan Medical University HospitalTaichungTaiwan
  3. 3.Department of Occupational Safety and HealthChung Shan Medical UniversityTaichungTaiwan

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