High-Performance Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry for the Detection and Quantitation of Pyrrolizidine Alkaloid-Derived DNA Adducts in Vitro and in Vivo

• Published in: Chemical research in toxicology Vol. 23; no. 3; pp. 637 - 652
• Main Authors: Fu, Peter P, Chou, Ming W, Churchwell, Mona, Wang, Yuping, Zhao, Yuewei, Xia, Qingsu, Gamboa da Costa, Gonçalo, Marques, M. Matilde, Beland, Frederick A, Doerge, Daniel R
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 15-03-2010
• Subjects: Animals; Calibration; Cattle; Chromatography, High Pressure Liquid - methods; DNA - analysis; DNA - metabolism; DNA Adducts - analysis; DNA Adducts - metabolism; Female; Liver - metabolism; Pyrrolizidine Alkaloids - adverse effects; Rats; Rats, Inbred F344; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization - methods; Tandem Mass Spectrometry - methods
• ISSN: 0893-228X; 1520-5010
• DOI: 10.1021/tx900402x

Pyrrolizidine alkaloid-containing plants are widespread in the world and are probably the most common poisonous plants affecting livestock, wildlife, and humans. Pyrrolizidine alkaloids require metabolism to exert their genotoxicity and tumorigenicity. We have determined that the metabolism of a series of tumorigenic pyrrolizidine alkaloids in vitro or in vivo generates a common set of (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts that are responsible for tumor induction. The identification and quantitation of the DHP-derived DNA adducts formed in vivo and in vitro were accomplished previously by 32P-postlabeling/HPLC methodology. In this article, we report the development of a sensitive and specific liquid chromatography−electrospray ionization−tandem mass spectrometry (HPLC-ES-MS/MS) method to detect DHP-derived DNA adducts formed in vitro and in vivo. The method is used to quantify the levels of DHP-2′-deoxyguanosine (dG) and DHP-2′-deoxyadenosine (dA) adducts by multiple reaction monitoring (MRM) analysis in the presence of known quantities of isotopically labeled DHP-dG and DHP-dA internal standards. This HPLC-ES-MS/MS method is accurate and precise. When applied to liver samples from rats treated with the pyrrolizidine alkaloids riddelliine and monocrotaline, the method provided significant new information regarding the mechanism of DNA adduct formation.