Sensitive determination of a glyoxal–DNA adduct biomarker candidate by column switching capillary liquid chromatographyelectrospray ionization mass spectrometry

Abstract

A method based on column switching packed capillary liquid chromatography electrospray mass spectrometry has been developed for the determination of the adduct glyoxal–deoxyguanosine, a biomarker candidate for the assessment of glyoxal exposure, in DNA hydrolysate solutions. Microgram amounts of DNA were isolated and enzymatically hydrolyzed to deoxyribonucleosides, prior to ultrafiltration and subsequent dilution to a sample solution consisting of water–acetonitrile–formic acid (98 : 2 : 0.2, v/v). The sample solution was loaded onto a 1 mm I.D. × 5 mm Hypercarb (5 μm) porous graphitic carbon trap column for analyte enrichment using an injection volume of 200 μl, and was subsequently back-flushed onto a 0.30 mm I.D. × 150 mm Lichrospher diol (5 μm) analytical column. The samples were loaded with a flow rate of 40 μl min⁻¹ and glyoxal–deoxyguanosine was desorbed from the trap column and eluted with an isocratic mobile phase consisting of water–acetonitrile–formic acid (50 : 50 : 0.2, v/v) at a flow rate of 5 μl min⁻¹. Mass spectrometric determination of glyoxal–deoxyguanosine was obtained by multiple reaction monitoring of the transition [M + H]⁺m/z 326 → m/z 210. The method was evaluated over the concentration range 0.25–50 ng ml⁻¹ of glyoxal–deoxyguanosine in the hydrolysate of 5 μg DNA. The method was linear with a correlation coefficient of 0.9998 in this range. The within-day (n = 6) and between-day (n = 6) precisions were determined as 1.2–11% and 1.4–11% RSD, respectively, and the recovery was close to 100%. The mass limit of detection was 15 pg, corresponding to a concentration limit of detection of 75 fg μl⁻¹ DNA hydrolysate solution, corresponding to 48 adducts per 10⁶ normal nucleosides. The method was applied for the determination of glyoxal–deoxyguanosine in DNA hydrolysate solutions of calf thymus DNA and cell cultures after reaction or incubation with glyoxal.