2-Phosphoglycolate and glycolate-electrophore detection, including detection of 87 zeptomoles of the latter by gas chromatography electron-capture mass spectrometry

As a first stage towards a goal of studying some aspects of oxidative damage to DNA and its subsequent repair, set up three techniques for the detection of 2-phosphoglycolate (PG). This compound is released as a metabolite from the DNA in certain cases of this process. We explored three techniques because we wanted to learn which one(s) would be most sensitive, given the anticipated availability of small biological samples for analysis. By employing indirect detection with fluorescein as the fluorophore in capillary electrophoresis, we detected 5 . 10(-6) M PG (corresponding to 5 pmol/mu l, projecting that a final sample volume of 1 mu l could be handled). The specificity of this technique can be enhanced by converting the PG to glycolate enzymatically. Flow injection analysis (FIA; 1.0-mu l injection volume) negative-ion electrospray mass spectrometry was similarly sensitive (1.1 . 10(-6) M). Based on our prior experience, substituting capillary HPLC for the FIA in this technique is anticipated to lower the detection limit by 20- to 150-fold. Gas chromatography-electron-capture mass spectrometry (1.0-mu l injection volume) was able to detect, as a standard, 87 zmol of O-2-pivalyl-3',5'-bis(trifluoromethyl)benzylglycolate, a product that can be obtained from PG via hydrolysis followed by derivatization. We pian to continue working with all three techniques since each is very sensitive, and has certain advantages.