The quantitative analysis of DNA-adducts represents a formidable challenge because of their low abundance together with the ease with which artifacts can be generated. Methodology based on LC/MS offers the advantage that the intact adduct containing the sugar moiety can be analyzed. In the case of urinary adducts, co-eluting contaminants from the urine can cause substantial signal suppression in electrospray ionization so we have explored the use of APCI, which is less prone to such suppression effects. Initially, we explored the use of electron capture methodology coupled with normal phase chromatography. PFB derivatives of the etheno-dGuo adducts were prepared and found to give high intensity signals. Unfortunately, the background noise was unacceptable, particularly for urine samples. Therefore, the use of more conventional positive ion APCI methodology coupled with reversed phase chromatography was explored. PFB derivatization of E-dGuo and H-dGuo caused the expected shift in retention times on the reversed-phase column (Figures 4 and 5). This caused an increase in sensitivity because the solvents had a higher organic content and so APCI was more efficient. CID and MS/MS analysis revealed one major product ion for each analyte and its relevant stable isotope internal standard. This made it possible to conduct relatively sensitive MRM analysis of the two analytes and heavy isotope internal standards (Figure 6). Excellent recovery was obtained for the two internal standards from the urine samples as evidenced in Figure 7. Signals from E-dGuo and H-dGuo were a little obscured by co-eluting endogenous interfering substances that were extracted from the urine. However, the LC/MS assay will be useful for analysis of the two etheno-dGuo adducts in settings of oxidative stress when levels are expected to be much higher.
|Number of pages||26|
|Journal||ACS Symposium Series|
|Publication status||Published - 2003|
ASJC Scopus subject areas
- Chemical Engineering(all)