Analytical Sciences

Preparation of N²-Ethyl-2′-deoxyguanosine-d₄ as an Internal Standard for the Electrospray Ionization–Tandem Mass Spectrometric Determination of DNA Damage by Acetaldehyde

Yukihiro ESAKA,*,** Hiromitsu ARUGA,* Saki KUNISHIMA,* Takuhei YAMAMOTO,* Hiroya MURAKAMI,*** Yoshinari SAWAMA,* Hironao SAJIKI,* and Bunji UNO*,**

*Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan
**United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan
***Department of Applied Chemistry, Aichi Institute of Technology, 1247 Yachigusa, Yakusa-cho, Toyota 470-0392, Japan

The deuteration of N²-ethyl-2′-deoxyguanosine (Et-dG), which is a DNA adduct generated from acetaldehyde, was studied by the addition reaction of acetaldehyde-d₄ to 2′-deoxyguanosine (dG) in deuterium oxide (D₂O), with the aim to obtain an isotope internal standard for the liquid chromatography/tandem mass spectrometry (LC/MS/MS) quantitation of Et-dG. The replacement of the dG C-8 hydrogen atom by a deuteron atom took place at 50°C in D₂O and afforded a mixture of Et-dG-d₄ and Et-dG-d₅. Et-dG-d₄, which was stable in aqueous solutions, was prepared by incubating the mixture in H₂O at 60°C for 48 h. The calibration curve was obtained by multiple reaction monitoring (MRM) measurements using a hydrophilic interaction chromatography–electrospray ionization–tandem mass spectrometric (HILIC/ESI-MS/MS) system between the Et-dG concentration, ranging from 1.0 × 10⁻¹⁰ to 4.0 × 10⁻⁹ M in the sample solutions, and the relative peak areas of Et-dG (m/z: 296.1 → 180.1) to the value of Et-dG-d₄ (m/z: 300.2 → 184.2), with an internal standard showing good linearity (R² = 0.995, n = 5).

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