Quantitative Analysis of the DNA AdductN2,3-Ethenoguanine Using Liquid Chromatography/Electrospray Ionization Mass Spectrometry

Abstract

The need for specificity and sensitivity in the analysis of DNA adducts has led the development of GC/MS methods. Such methods require chemical derivatization (i.e. silylation, electrophore labelling), which can also bring its own sets of problems, including the production of artifacts, interferences and sample to sample variability in derivatization. To obviate such problems, a liquid chromatographic/electrospray ionization mass spectrometric (LC/ESI‐MS) method was developed to quantify N2,3‐ethenoguanine (εGua), a promutagenic DNA adduct of vinyl chloride exposure. The response of εGua to isotopically labelled internal standard [13C4]εGua was linear (r2 = 0.999) and reproducible from 0.027 to 0.538 pmol μl‐1. We obtained an accuracy of 86±14% by analyzing chloroethylene oxide (CEO)‐treated calf thymus DNA enriched with authentic εGua. The analysis of CEO‐treated calf thymus DNA samples not enriched with authentic εGua provided a precision of 15%. The detection limits with a signal‐to‐noise ratio (S/N) 2.5:1 were obtained in the determination of authentic εGua at 5 fmol per injection. The detection limit obtained in the routine analysis of the biological samples was 50 fmol εGua with S/N = 3:1. The applicability of the method was established by determining εGua in rats treated with CEO by portal vein injection and an unexposed human liver. It was observed that the concentration of εGua in the rat livers increased with increase in dose and was inversely related to the time after, CEO exposure. This trend suggests rapid repair of the adduct in rat livers. In the human liver DNA sample, εGua was quantitated at 0.06±0.01 pmol mg‐1 DNA.

Quantitative analysis of 1,3-butadiene‐induced DNA adducts in vivo and in vitro using liquid chromatography electrospray ionization tandem mass spectrometry

“…THB adducts), production of artifacts during the derivatization step and sample to sample variability. 28 In addition, derivatization methods are usually analyte-speciÐc and cannot be applied to analyze simultaneously chemically diverse compounds in the same sample. LC/ESI`-MS makes it possible to analyze DNA adducts directly, without derivatization, since volatility is not necessary.…”

Analysis of nucleoside-estrogen adducts by LC-ESI-MS-MS†

“…It is also capable of providing extended structural information on adducts by means of tandem MS experiments, [19][20][21][22][23] especially at the very low levels encountered in biological samples. 24,25 The LC-ESI-MS-MS method developed in this work provides new data on the chemical reactivity of E-2,3-Q towards deoxyribonucleosides. Thus, the formation of depurinating adducts with dG and of a new adduct with dC could be demonstrated.…”

Absolute Quantification of RNA or DNA Using Acid Hydrolysis and Mass Spectrometry

“…Chemical approaches for RNA or DNA hydrolysis have been around for decades , and have evolved continuously with improvements in accuracy and specificity. Predominantly though, acid hydrolysis has been applied for atypical or modified nucleobase detection (DNA damage or carcinogenic adduction − ) rather than with the intent to quantify the intact oligonucleotide. Recently, chemical hydrolysis was demonstrated using double-stranded genomic DNA from bacteriophage lambda (∼48.5 kbp), showing that DNA can be accurately quantified using just such an approach .…”