Nuclear magnetic resonance at the picomole level of a DNA adduct.

We investigate the limit of detection for obtaining NMR data of a DNA adduct using modern microscale NMR instrumentation, once the adduct has been isolated at the picomole level. Eighty nanograms (130 pmol) of a DNA adduct standard, N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene 5'-monophosphate (AAF-dGMP), in 1.5 µL of D2O with 10% methanol-d4, in a vial, was completely picked up as a droplet suspended in a fluorocarbon liquid and loaded efficiently into a microcoil probe. This work demonstrates a practical manual method of droplet microfluidic sample loading, previously demonstrated using automated equipment, which provides a severalfold advantage over conventional flow injection. Eliminating dilution during injection and confining the sample to the observed volume produce the full theoretical mass sensitivity of a microcoil, comparable to that of a microcryo probe. With 80 ng, an NMR spectrum acquired over 40 h showed all of the resonances seen in a standard spectrum of AAF-dGMP, with a signal-to-noise ratio of at least 10, despite broadening due to previously noted effects of conformational exchange. Even with this broadening to 5 Hz, a two-dimensional total correlation spectroscopy spectrum was acquired on 1.6 µg in 18 h. This work helps to define the utility of NMR in combination with other analytical methods for the structural characterization of a small amount of a DNA adduct.

Synthesis of [4,5,6,8-(13)C4]guanine, a reagent for the production of internal standards of guanyl DNA adducts.

Using readily available labeled compounds, [4,5,6,8-(13)C4]guanine was synthesized in high overall yield. Intermediates as well as the final product were characterized by 1H NMR, 13C NMR, and high resolution mass spectrometry. The labeled guanine was used to generate [13C4]-labeled analogs of the guanine adducts, N2,3-ethenoguanine and 7-(2-hydroxyethyl)guanine. The application of such adducts in isotope dilution mass spectrometry was illustrated with DNA samples from rats exposed to two different mutagenic compounds, vinyl chloride and ethylene oxide.