Stable Isotope Dilution LC-Nanospray Ionization Tandem Mass Spectrometric Analyses for Simultaneous Quantification of (1) Three Etheno DNA Adducts in Human Blood and (2) Ethylpurine Adducts in Human Urine

• Main Authors: Guan-Jih Lin, 林冠誌
• Other Authors: Hauh-Jyun Candy Chen
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/67813177758754924904

碩士 === 國立中正大學 === 化學所 === 97 === (1)The promutagenic etheno DNA adducts are derived from exogenous as well as endogenous sources, such as lipid peroxidation. The levels of etheno DNA adducts are elevated in cancer-prone tissues. In this study, we developed a highly specific and sensitive method for simultaneous quantification of etheno DNA adducts: 1,N6-etheno-2´-deoxyadenosine (εdAdo), 3,N4-etheno-2´-deoxycytidine (εdCyd), and 1,N2-etheno-2´-deoxyguanosine (εdGuo) in human blood by stable isotope dilution nanoflow LC-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) under the highly selective reaction monitoring (H-SRM) mode. We extracted human white blood cell (WBC) DNA from whole blood and digested DNA into nucleosides by enzyme hydrolysis. A C18 solid phase extraction column was used to enrich the etheno DNA adducts, which were analyzed by nanoLC-NSI/MS/MS on a triple quadrupole instrument. The detection limit of εdAdo, εdCyd and εdGuo injected on-column was 1.8, 20 and 86 amol, respectively. The levels of εdAdo, εdCyd and εdGuo in 7 human WBC DNA samples were 4.6 ± 1.5, 6.1 ± 2.7, and 4.9 ± 4.1 (mean ± S.D.) adducts per 107 parent nucleosides, respectively. With minimum amount of DNA (6 μg), this nanoLC-NSI/MS/MS method provides a useful assay in measuring etheno DNA adducts as noninvasive biomarkers in cancer risk assessment.(2)Tobacco smoke contains over 4,000 compounds, which includes alkylating agents. According to the literature, exposure to tobacco smoke results in increased levels of 3-ethyladenine (3-EtA) and 7-ethylguanine (7-EtG) and their levels in smokers’ urine are higher than those in non-smokers’. In this study, we attempted to analyze 3-EtA and 7-EtG in human urine simultaneously. Urine was centrifuged and the adducts were enriched by a C18-OH SPE column. The fractions containing 3-EtA and 7-EtG were analyzed by capillary liquid chromatography nanospray ionization tandem mass spectrometry (capLC-NSI/MS/MS) under the highly selected monitoring mode. Because urine contains many impurities, we use on-line sample purification with a trap column before MS analysis. The detection limit of 3-EtA and 7-EtG injected on-column was 0.3 and 0.55 fmol, respectively. In order to remove the interference in the urine samples, so we tried to use different pretreatment procedure such as NH2 SPE column or SCX SPE column. The NH2 SPE column did not remove the interference in the urine samples. Because 3-EtA and 7-EtG retained in SCX SPE column, it’s possible to use SCX SPE column for adduct enrichment and sample clean-up. We hoped this assay can be useful in evaluation of urinary 3-EtA and 7-EtG as non-invasive biomarkers for measuring DNA damage following exposure to ethylating carcinogens.