Immunoaffinity/Gas Chromatography/High-Resolution Mass Spectrometry Method for the Detection of N²,3-Ethenoguanine

Abstract

Etheno adducts are formed after exposure to a number of carcinogens, including vinyl chloride, as well as endogenously as a result of lipid peroxidation. A sensitive and selective assay for N(2), 3-ethenoguanine (epsilonGua) was developed using immunoaffinity (IA) columns made with polyclonal antibodies to epsilonGua followed by gas chromatography/electron capture negative chemical ionization/high-resolution mass spectrometry (GC/ECNCI/HRMS) analysis of its pentafluorobenzyl derivative. These IA columns were specific for epsilonGua and did not bind guanine, deoxyguanosine, 1, N(6)-ethenoadenine, or 1,N(2)-ethenoguanine. The level of recovery of standards from the IA columns was 107 +/- 7% and throughout the entire method (using nucleoside enzymatic digestion) with or without DNA was 72 +/- 6%. Four different hydrolysis/digestion procedures were compared, nucleoside enzymatic (EZ), neutral thermal hydrolysis (NT), formic acid hydrolysis (FA), and HCl hydrolysis. All hydrolysis methods with subsequent IA chromatography produced linear standard curves with r(2) values of 0.999 or better. The level of epsilonGua in chloroethylene oxide-treated calf thymus DNA (CEO-ctDNA) was 38 +/- 2, 42 +/- 3, and 49 +/- 2 fmol of epsilonGua/microg of DNA using EZ, NT, and FA, respectively. These numbers remained consistent when the amount of DNA processed was doubled or tripled. These numbers were comparable to the previously published value of 55 +/- 8 fmol of epsilonGua/micrograms of DNA for the same DNA using HCl hydrolysis, cation exchange cleanup, and LC/MS analysis [Yen, T. Y., et al. (1996) J. Mass Spectrom. 31, 1271-1276]. Additionally, HCl hydrolysis of rat liver DNA from control and vinyl fluoride-exposed rats gave similar epsilonGua results when compared to those from enzymatic digestion using this method. This method gave a detection limit of 5 epsilonGua adducts/10(8) normal dGuo nucleosides in 150 micrograms of DNA using EZ and somewhat lower detection limits using NT and HCl hydrolysis. The method is more sensitive and selective than previously used methods for the quantitation of this adduct.

Analysis of Diepoxide-Specific Cyclic N -Terminal Globin Adducts in Mice and Rats after Inhalation Exposure to 1,3-Butadiene

“…Before the analysis of in vivo samples, the precision and accuracy of the method were determined using control globin from mice, rats, and humans spiked with the corresponding pyr- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11) N,N-(2,3-dihydroxy-1,4-butadiyl)-valine peptides of all three species with equal efficiency. The limits of detection (S/N Ͼ 3) were 10 fmol on column, and the limits of quantitation were 50 fmol on column.…”

“…The N-terminal ␣-chain peptides for mouse (pyr-VLSGEDKSNIK), rat (pyr-VLSADDKTNIK), and human (pyr-VLSPADKTNVK) derived from rac 1,2;3,4-diepoxybutane were synthesized as described by Jayaraj et al (8). For accurate quantitation, the corresponding stable isotope-labeled peptides were synthesized containing [ 2 H 3 ]L. The pyr-(1-7) peptide standards and internal standards were prepared from pyr- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11) peptides by trypsin hydrolysis. In addition to the previously described characterization (8), all standards were sequenced by LC-MS/MS on an LCQ-Deca ion trap mass analyzer (ThermoFinnigan, San Jose, CA; data not shown).…”

“…Four New Zealand White rabbits were immunized with the conjugate, and bleedings were withdrawn periodically. The sera were checked for pyr- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11) titer by enzyme-linked immunosorbent assay, and sera from the 2-month post-immunization bleed were selected to prepare immunoaffinity columns as described previously by Ham et al (9).…”

Rapid analysis of inflammatory cytokines in cerebrospinal fluid using chip‐based immunoaffinity electrophoresis

“…Immunoaffinity preanalysis cleanup is becoming a popular approach in many fields with antibody extraction columns being coupled with GC-MS [23,24], HPLC [25][26][27] and MS alone [28][29][30]. Preselection by immobilized antibodies or FAb fragments is a relatively simple approach to isolating single analytes from complex biological materials, removing unwanted materials prior to analysis.…”

Site Specific Synthesis and Polymerase Bypass of Oligonucleotides Containing a 6-Hydroxy-3,5,6,7-tetrahydro-9H-imidazo[1,2-a]purin-9-one Base, an Intermediate in the Formation of 1,N2-Etheno-2‘-deoxyguanosine

“…Alternatively, dGuo could be alkylated at N1 with (R)-glycidol to give the same N1-(2,3-dihydroxypropyl)-dGuo (14) as a single diastereomer. Periodate cleavage of the vicinal diol created the aldehyde, which was not observed because it spontaneously cyclized to HOEdGuo (11). NMR analysis of the product mixture showed the presence of approximately 20% of the ε-dGuo (7).…”